title: Differentiated embryonic neurospheres from familial Alzheimer’s disease model show Innate Immune and Glial Cell Responses

city: São Paulo

first: Eduardo

middle: Moraes

last: Reis

state: SP

address: Av. Prof. Lineu Prestes, 748

department: Biochemistry

country: Brazil

institute: University of São Paulo

summary: Proteins involved in the Alzheimer’s disease (AD), such as amyloid precursor protein (APP) and presenilin-1 (PS1), play critical roles in early development of the central nervous system (CNS), as well as in innate immune and glial cell responses. However, it is unknown whether these alterations start on development of the CNS of carriers of APPswe and PS1dE9 mutations , associated with familial AD. Using microarray technology we studied the genome-wide gene expression profiles in the differentiated neural progenitor cells (NPCs) from wild-type (WT) and APPswe/PS1dE9 embryo telencephalon. Differential gene expression analysis indicated the occurrence of strong innate immune and glial cell responses in APPswe/PS1dE9 neurospheres, involving mainly microglial cells, inflammatory mediators and chemokines. APPswe/PS1dE9 cells expressed up to 100-fold more CCL12, CCL5, CCL3, C3, CX3CR1, TLR2 and TNF-alpha, which was confirmed by real time PCR. The expression of activated microglia marker Iba-1 was also 20-fold upregulated in APPswe/PS1dE9 cells. The secretome of APPswe/PSldE9 differentiated NPCs increased the chemoattraction of peripheral blood mononuclear cells (PBMCs) compared to the secretome of WT cells. Taken together, our results demonstrated that neurogenic niche obtained from differentiation of embryonic APPswe/PS1dE9 neurospheres is an excellent model for AD in vitro, since this culture spontaneously presents several alterations observed in the adult-brain with this pathology. Moreover, our data strengthen pathophysiological hypotheses that propose an early neurodevelopmental origin for familial AD.

overall_design: Two-condition experiment, APPswe/PS1dE9 vs. WT cells. Biological replicates: 4 alzheimer, 4 WT replicates.

first: Micheli

last: Pillat

first: Ana

last: Ayupe

first: Guilherme

last: Juvenal

first: Talita

last: Glaser

first: Carine

last: Mainers

first: Diogo

last: Pellegrina

first: Alexander

middle: H

last: Ulrich


title: Efficient control of tumor progression by CD4 T cell epitope-based prime-boost therapeutic vaccination

status: Public on Sep 30 2023

city: Chicago

first: Jun

last: Huang

state: IL

address: Eckhardt, 5640 S Ellis Ave

department: Institute for Molecular Engineering

country: USA

institute: University of Chicago

type: Expression profiling by high throughput sequencing

summary: We reported the differences of transcriptomic landscapes and clonal TCRs in CD4 and CD8 T cells among the control group, vaccination group and naïve group.

overall_design: Examination of single-cell RNA-seq and single-cell TCR-seq  in CD4 and CD8 T cells obtained from naïve and tumor-bearing mice with or without vaccination.

first: Lilin

last: Ye

first: Guoshuai

last: Cao

first: Luoyingzi

last: Xie


title: Microenvironment-dependent growth of Sezary cells in humanized IL-15 mice [scRNA/TCR-seq]

status: Public on Sep 29 2023

city: Tarrytown

first: Gabrielle

last: Choonoo

state: NY

address: 777 Old Saw Mill River Rd

institute: Regeneron Pharmaceuticals, Inc

summary: Sezary syndrome (SS) is a rare, aggressive leukemic variant of cutaneous T cell lymphoma (CTCL) that lacks adequate therapeutic options and representative small animal models. Here we demonstrate that IL-15 is a critical CTCL growth and survival factor. Importantly, a genetically engineered immunodeficient mouse model expressing human IL-15 uniquely supported the growth of patient derived Sezary cells relative to conventional immunodeficient mouse strains. Patient derived xenograft (PDX) SS models recapacitated the pathologic features of the human disease, including skin infiltration and spread of leukemic cells to the periphery, and maintained the dependence on human IL-15 upon serial in vivo passaging. Detailed molecular characterization of the engrafted cells by single cell transcriptome analysis revealed tissue specific regulation of gene expression and distinct clonal engraftment patterns. Overall, we document an important dependence of Sezary cell survival and proliferation on IL-15 signaling and the utility of the humanized IL-15, immunodeficient mice for SS PDX model generation. Furthermore, these studies advocate for the thorough molecular understanding of the resultant PDX models to maximize their translational impact.

overall_design:
Data inludes 1 primary Sezary syndrome patient PBMC, 1 healthy donor PBMC. Samples were enriched for CD4+ cells and subjected to single cell RNA-seq.
***Submitters declare that the raw data will not be deposited in a repository due to their data privacy policy****

first: Gao

last: Jie

first: Ren

last: Shumei

first: Choonoo

last: Gabrielle

first: Frleta

last: Davor

first: Zhong

last: Jun

first: Gupta

last: Namita

first: Atwal

last: Mickey

first: Macdonald

last: Lynn

first: Murphy

last: Andrew

first: Kuhnert

last: Frank


title: Microenvironment-dependent growth of Sezary cells in humanized IL-15 mice

overall_design: Data inludes 7 Sezary patient-derived xenograft (PDX) P3 mice samples. 4 come from spleen and 3 come from skin. These are SRG15 mice modeling humanization of IL15 allele on an SRG immunodeficient background. Samples were enriched for CD4+ cells and subjected to single cell RNA-seq.

first: Jie

last: Gao

first: Shumei

last: Ren

first: Davor

last: Frleta

last: Zhong

first: Namita

last: Gupta

first: Mickey

last: Atwal

first: Lynn

last: Macdonald

first: Andrew

last: Murphy

first: Frank

last: Kuhnert


title: Deciphering m6A dynamics at single-base level during planarian anterior-posterior (AP) axis specification [mRNA]

city: Guangzhou

first: Zixin

last: Chen

state: Guangdong

address: No. 106, Zhongshan Second Road

department: Guangdong Cardiovascular Institute Medical Research Center

country: China

institute: Guangdong Provincial People's Hospital

summary: We accomplished de novo transcriptome assembly for planarian Dugesia japonica.

overall_design: 10-20 planarians were cut and separated into head, trunk, and tail, then mixed to extract total RNA, and then enriched mRNA by polyT for library construction.We conducted the library in the six time-points samples (day 0 to day 10: D0, D1, D3, D5, D7 and D10) from head, trunk and tail, respectively.

first: Liqian

first: Hui

last: Zhen


title: Using Zinc-based probes to generate pseudoislets from purified human alpha-cells for functional analyses

city: Boston

first: Jonathan

middle: M

last: Dreyfuss

state: MA

address: 1 Joslin Pl

department: Bioinformatics & Biostatistics Core

institute: Joslin Diabetes Center

summary: Investigation of the function of human alpha-cells remains a challenge due to the lack of simple and cost-effective purification methods to isolate high-quality alpha-cells from islets. Here, we introduce a novel and simple method for enriching live alpha-cells from dissociated human islet cells by using the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) with > 97% purity.

overall_design: Sorted islet cells from four islet donors were seeded in U-bottom low attachment 96 well plates (1000 cells/200 ul/well). Half of the medium was refreshed every other day for 10 days.

first: Sevim

last: Kahraman

first: Rohit

middle: N

last: Kulkarni


title: DeltaNp63 silencing, DNA methylation shifts and epithelial mesenchymal transition resulted from TAp63 genome editing in squamous cell carcinoma [RRBS]

city: Yokosuka

first: Iyoko

last: Katoh

address: 82 Inaoka-cho

country: Japan

institute: Kanagawa Dental University

type: Methylation profiling by high throughput sequencing

summary: TP63 (p63) is strongly expressed in lower-grade carcinomas of head-and-neck, skin, breast, urothelium, etc. to maintain the well-differentiated phenotype. TP63 has two transcription start sites at exon 1 and exon 3’ to produce TAp63 and DeltaNp63 isoforms, respectively. The major protein, DeltaNp63alpha, functions as a core factor to organize super enhancers of genes essential for epidermal/craniofacial differentiation and for self-activation of DeltaNp63. To examine whether very weakly expressed TAp63 has a specific role, we disrupted exon 1 by CRISPR-Cas9 homology directed repair (HDR) in a head and neck squamous cell carcinoma (SCC) line. Surprisingly, TAp63 knockout cells, with either ‘monoallelic HDR and a frameshift deletion on the other allele’ or ‘biallelic HDR’, caused  DeltaNp63 silencing. Loss of keratinocyte-specific gene expression, replacement of KRT5 with VIM, and transcriptional suppression of cell-cell and cell-matrix adhesion components indicated core events of epithelial mesenchymal transition. Most of the positively and negatively impacted genes including DeltaNp63 displayed local CpG methylation changes. Furthermore, DeltaNp63 expression was partially rescued by transfection of TAp63alpha, followed by incubation with DNA methyltransferase inhibitor Zebularine. This study suggests that TAp63 is indispensable for DeltaNp63 expression by which keratinocyte-specific epigenome is maintained in SCC.

overall_design: Comparison of genome DNA methylation by RRBS between parental (control) FaDu squamous cell carcinoma line and TAp63 knockout cell line derived from FaDu

first: Shun-ichi

last: Kurata


title: DeltaNp63 silencing, DNA methylation shifts and epithelial mesenchymal transition resulted from TAp63 genome editing in squamous cell carcinoma

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

overall_design: Refer to individual Series


title: Mitigating a TDP-43 proteinopathy by targeting ataxin-2 using RNA-targeting CRISPR effector proteins

status: Public on Sep 28 2023

city: Urbana

first: Thomas

last: Gaj

address: 1406 W. Green St.

department: Bioengineering

institute: University of Illinois Urbana-Champaign

summary: The TDP-43 proteinopathies, which include amyotrophic lateral sclerosis and frontotemporal dementia, are a devastating group of neurodegenerative disorders that are characterized by the mislocalization and aggregation of TDP-43. Here we demonstrate that RNA-targeting CRISPR effector proteins, a programmable class of gene silencing agents that includes the Cas13 family of enzymes and Cas7-11, can be used to mitigate TDP-43 pathology when programmed to target ataxin-2, a modifier of TDP-43-associated toxicity. In addition to inhibiting the aggregation and transit of TDP-43 to stress granules, we find that the in vivo delivery of an ataxin-2-targeting Cas13 system to a mouse model of TDP-43 proteinopathy improved functional deficits, extended survival, and reduced the severity of neuropathological hallmarks. Further, we benchmark RNA-targeting CRISPR platforms against ataxin-2 and find that high-fidelity forms of Cas13 possess improved transcriptome-wide specificity compared to Cas7-11 and a first-generation effector. Our results demonstrate the potential of CRISPR technology for TDP-43 proteinopathies.

overall_design: Analyzed the differentially expressed genes for RfxCas13d, a high-fidelity RfxCas13d variant and DiCas7-11 when programmed to target the mouse ataxin-2 mRNA. Experiments were conducted in Neuro2A cells and done in triplicate. Each replicate is an independent biological replicate.

first: Maria

middle: A

last: Zeballos C.

first: Hayden

middle: J

last: Moore

first: Tyler

last: Smith

first: Jackson

middle: E

last: Powell

first: Najah

middle: S

last: Ahsan

first: Sijia

last: Zhang


title: SUN1/2 Controls Macrophage Polarization via Modulating Nuclear Size and Stiffness

city: Shanghai

first: qiudong

last: zhao

address: 225 Changhai Road

department: Tumor Immunology and Gene Therapy Center

institute: Shanghai Eastern Hepatobiliary Surgery Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Alteration of the size and stiffness of the nucleus triggered by environmental cues are thought to be important for eukaryotic cell fate and function. Here we identify the nuclear envelope proteins SUN1/2 as mechano-regulators of the nucleus during M1 polarization of the macrophage. Specifically, we show that LPS treatment decreases the protein levels of SUN1/2 in a CK2-bTrCP-dependent manner to shrink and soften the nucleus, therefore altering the chromatin accessibility for M1-associated gene expression.

overall_design: Chromatin Immunoprecipitation DNA-sequencing (ChIP-seq) for histone modification H3K4me3 in macrophages.

first: Shi

last: Jiao

first: Zhaocai

last: Zhou


title: CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses

city: Helsinki

last: Sokolova

address: Viikinkaari 5

department: Institute of Biotechnology

country: Finland

institute: University of Helsinki

sample_organism:
Drosophila melanogaster
Mus musculus

platform_organism:
Drosophila melanogaster
Mus musculus


title: Deciphering m6A dynamics at single-base level during planarian anterior-posterior (AP) axis specification [m6A]

first: Zixin

last: Chen

summary: We accomplished the m6A landscapes at single-base resolution with multiple time-points during AP axis regeneration for planarian Dugesia japonica.

overall_design: 10-20 planarians were cut and separated into head, trunk, and tail, then mixed to extract total RNA, and then enriched mRNA by polyT for library construction. We conducted the m6A-REF-seq analysis with IVT library control in the six time-points samples (day 0 to day 10: D0, D1, D3, D5, D7 and D10) from head, trunk and tail, respectively

first: Liqian

last: Zhen


title: Epigenetic aging of Māui and Hector's dolphins

city: Newport

first: Keith

last: Hernandez

state: OR

address: 2030 SE Marine Science Drive

department: Marine Mammal Institute

institute: Oregon State University

type: Methylation profiling by genome tiling array

summary: An Infinium microarray platform (GPL28271, Horvath MammalMethylChip40) was used to generate DNA methylation data from skin samples of Hector's and Māui dolphins.

sample_organism:
Cephalorhynchus hectori hectori
Cephalorhynchus hectori maui

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: N = 141 skin samples, n = 97 originating from Māui dolphins and 44 from Hector's dolphins. Profiled on the mammalian methylation array platform (GPL28271).

first: Steve

last: Horvath

first: C

last: Baker


title: Effect of depletion of MSIL2 and MSIL4 RNA-binding proteins on gene expression in the Arabidopsis inflorescence stem

city: Perpignan

first: Thierry

last: Lagrange

address: 58 Avenue Paul Alduy

country: France

institute: CNRS

summary: The goals of this study are to compare the gene expression in Wild-type (Col-0) and msil2/4 double mutant inflorescence stems.

overall_design: RNA-seq

first: Natacha

last: Bies-Etheve


title: Deciphering m6A dynamics at single-base level during planarian anterior-posterior (AP) axis specification

summary:
Background: The establishment of the anterior-posterior (A-P) axis is a crucial step during tissue repair and regeneration. Despite the association reported recently of N6-methyladenosine (m6A) with regeneration, the mechanism underlying the regulation of m6A in A-P axis specification during regeneration remains unknown.
Herein, we deciphered the m6A landscape at a single-base resolution at multiple time points during A-P axis regeneration and constructed the de novo transcriptome assembly of the Dugesia japonica planarian.

overall_design: 18 samples from Header, Trunk and Tail were selected and performed RNA-seq and m6A-REF-seq.


title: Essential role of the amino-terminal region of Drosha for the Microprocessor function

city: san francisco

first: Akiko

last: Hata

state: CALIFORNIA

address: 555 Mission Bay Blvd. South, CVRI, ucsf, Room 252T

department: Cardiovascular Research Institute

institute: University of California San Francisco

type: Non-coding RNA profiling by high throughput sequencing

summary:
To investigate the role of amino terminal region (NTR) of Drosha in microprocessor function, we generated amino terminus lacking Drosha (∆N-Drosha) HEK293T cell line by CRISPR-Cas9 genome editing.
We then performed miRNA expression profile analysis using data obtained from miRNA-seq of WT-Drosha and ∆N-Drosha containing HEK293T cells.

overall_design: Comparative miRNA expression profiling analysis of miRNA-seq data for HEK293T cell line with WT-Drosha and ∆N-Drosha alleles.

first: Amit

last: Prabhakar

first: Song

last: Hu

first: Jin

last: Tang

first: Prajakta

last: Ghatpande

first: Giorgio

last: Lagna

first: Xuan

last: Jiang


title: Spatially and temporally distinct patterns of expression for VPS10P domain receptors in human cerebral organoids

status: Public on Sep 27 2023

city: Aarhus N

first: Morten

middle: T

last: Veno

state: DK

address: Aabogade 15

country: Denmark

institute: Omiics ApS

summary: Vacuolar protein sorting 10 protein (VPS10P) domain receptors are a unique class of intracellular sorting receptors that emerge as major risk factors associated with psychiatric and neurodegenerative diseases, including bipolar disorders, autism, schizophrenia, as well as Alzheimer’ disease and frontotemporal dementia. Yet, the lack of suitable experimental models to study receptor functions in the human brain have hampered elucidation of receptor actions in brain disease. Here, we have adapted protocols to the study of VPS10P domain receptors in human cerebral organoids and applied these to the detailed characterization of receptor expression during development and in mature tissue, including single-cell RNA sequencing. These studies uncovered spatial and temporal patterns of expression unique to individual receptor species in the human brain. While SORL1 expression is abundant in stem cells and SORCS1 peaks at onset of neurogenesis, SORT1 and SORCS2 show increasing expression coinciding with maturation of neuronal and non-neuronal cell types, arguing for distinct functions in development versus the adult brain. In neurons, subcellular localization also distinguishes between two types of receptors, either mainly localized to the cell soma (SORL1 and SORT1) or also to neuronal projections (SORCS1 and SORCS2), suggesting divergent functions in protein sorting between Golgi and the endo-lysosomal system or along axonal and dendritic tracks. Taken together, our findings provide an important resource concerning temporal, spatial, and subcellular patterns of VPS10P domain receptor expression in cerebral organoids for further elucidation of receptor (dys)functions causative of behavioral and cognitive defects of the human brain.

overall_design: Cerebral organoids derived from hiPSCs (human induced pluripotent stem cells) were obtained  using adapted published protocols (Lancaster et al., 2013; Giandomenico et al., 2021). At day 21, 9 organoids were collected and made into single cells. Libraries were generated using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 Dual Index (10X Genomics Inc., USA). Barcoded cells were sequenced on a NovaSeq 6000 instrument (Illumina, USA). Barcode processing and single-cell 3′ gene quantification was done using the Cell Ranger Single-Cell Software Suite (v 3.1.0) using the GRCh38 reference genome.

first: Fabia

last: Febbraro

first: Meagan

last: Kitt

first: Helena

last: Hørdum Breum Andersen

first: Thomas

last: Willnow


title: Tumor-infiltrating CCR2+ inflammatory monocytes counteract specific immunotherapy

city: Mainz

first: Matthias

last: Klein

address: Langenbeckstrasse 1

department: Inst. for Immunology

country: Germany

institute: University Medical Center Mainz

summary: We used Single-cell RNA-sequencing to analyze the composition of the tumor microenvironment of MC38mOVA tumors upon transcutaneous immunization with our immunization method DIVA.

overall_design: MC38mOVA tumor cells were inoculated s.c. into WT mice. Mice were treated by transcutaneous immunization at day 6/7 and 13/14 after inoculation or left untreated. At day 16, respectively day 20, single cell suspensions were prepared from excised whole tumors, and CD45+ cells were enriched by magnetic bead separation. Afterwards Single-cell RNA-sequencing of these CD45+ cells was performed.

first: Ann-Kathrin

last: Hartmann

first: Danielle

last: Arnold-Schild

first: Lara

last: Stein

first: Federico

last: Marini


title: Tracking cell-type-specific temporal dynamics in human and mouse brains

city: New York

first: Ziyu

last: Lu

address: 1230 York Avenue

institute: The Rockefeller University

summary: Progenitor cells play fundamental roles in preserving optimal organismal functions under normal, aging, and disease conditions. However, progenitor cells are incompletely characterized, especially in the brain, partly because conventional methods are restricted by inadequate throughput and resolution for deciphering cell-type-specific proliferation and differentiation dynamics in vivo. Here, we developed TrackerSci, a new technique that combines in vivo labeling of newborn cells with single-cell combinatorial indexing to profile the single-cell chromatin landscape and transcriptome of rare progenitor cells and track cellular differentiation trajectories in vivo. We applied TrackerSci to analyze the epigenetic and gene expression dynamics of newborn cells across entire mouse brains spanning three age stages and in a mouse model of Alzheimer's disease. Leveraging the dataset, we identified diverse progenitor cell types less-characterized in conventional single cell analysis, and recovered their unique epigenetic signatures. We further quantified the cell-type-specific proliferation and differentiation potentials of progenitor cells, and identified the molecular programs underlying their aging-associated changes (e.g., reduced neurogenesis/oligodendrogenesis). Finally, we expanded our analysis to study progenitor cells in the aged human brain through profiling ~800,000 single-cell transcriptomes across five anatomical regions from six aged human brains. We further explored the transcriptome signatures that are shared or divergent between human and mouse oligodendrogenesis, as well as the region-specific down-regulation of oligodendrogenesis in the human cerebellum. Together, the data provide an in-depth view of rare progenitor cells in mammalian brains. We anticipate TrackerSci will be broadly applicable to characterize cell-type-specific temporal dynamics in diverse systems.

overall_design: single nucleus RNA-seq and single nucleus ATAC-seq profiling for rare newborn cells in mouse brains spanning three age stages and in a 5XFAD mutant.

first: Melissa

last: Zhang

first: Jasper

last: Lee

first: Andras

last: Sziraki

first: Sonya

last: Anderson

first: Shaoyu

last: Ge

first: Peter

last: Nelson

first: Wei

last: Zhou

first: Junyue

last: Cao


title: Spatially and temporally distinct patterns of expression for VPS10P domain receptors in human cerebral organoids.

first: Morten

last: Veno

overall_design: Cerebral organoids derived from hiPSCs (human induced pluripotent stem cells) were obtained  using adapted published protocols (Lancaster et al., 2013; Giandomenico et al., 2021). At day 100, 5-6 organoids were collected and made into single cell. Libraries were generated using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 Dual Index (10X Genomics Inc., USA). Barcoded cells were sequenced on a NovaSeq 6000 instrument (Illumina, USA). Barcode processing and single-cell 3′ gene quantification was done using the Cell Ranger Single-Cell Software Suite (v 3.1.0) using the GRCh38 reference genome.

first: Fabia

last: Febbraro


title: Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration [Mate-Pair]

city: Cambridge

first: Vishnu

last: Dileep

address: 43 Vassar St

department: Brain and Cognitive Sciences

institute: Massachusetts Institute of Technology

summary: Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer’s Disease (AD) with the potential to disrupt genome integrity. We show increased mosaic structural variations and gene fusions in neurons burdened with DSBs in the CK-p25 inducible mouse model of neurodegeneration. Next, we used full-transcript single-nucleus RNA-seq across 47 human post-mortem prefrontal cortex and find that excitatory neurons in AD are enriched for mosaic gene fusions. In addition, gene fusions are particularly enriched in excitatory neurons with senescence and DNA repair gene signatures. Neurons enriched for DSBs also have elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic and neuronal development genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases.

overall_design:
Mate-Pair library preparation was performed using the Nextera® Mate Pair Library Preparation (FC-132-1001). Briefly, formaldehyde-fixed FANS sorted nuclei were reverse crosslinked and genomic DNA was purified. Genomic DNA was tagmented to a size range of 1-5kb using mate pair transposome containing biotinylated adaptors followed by strand displacement to repair single-stranded gaps. Tagmented genomic DNA was circularized and then fragmented using sonication. The junctions were purified using streptavidin beads.

Sequencing libraries were prepared using TruSeq DNA LT Sample Prep (15027084) provided with the mate pair kit or NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645S) and NEBNext Multiplex Oligos for Illumina (E6440S). Illumina libraries were sequenced in 75bp or 40bp paired-end mode using Nextseq500.

FASTQ files were trimmed to remove Illumina adaptors and paired-end reads were mapped separately using Bowtie2. Mapped sam files were converted to bam files using samtools view. Bam files were sorted and PCR duplicates were removed using samtools rmdup. Bam files were converted to bed files using bedtools bamtobed. BED files from corresponding paired reads were merged based on FASTQ ID to create the matepair.txt files for each sample.

first: Carles

last: Boix

first: Hansruedi

last: Mathys

first: Asaf

last: Marco

first: Gwyneth

last: Welch

first: Hiruy

last: Meharena

first: Anjanet

last: Loon

first: Ritika

last: Jeloka

first: Zhuyu

last: Peng

first: David

last: Bennett

first: Manolis

last: Kellis

first: Li-Huei

last: Tsai


title: Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration


title: Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration [Hi-C]

summary: Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer’s Disease (AD), with the potential to disrupt genome integrity. We used single-nucleus RNA-seq in human post-mortem prefrontal cortex samples and found that excitatory neurons in AD were enriched for somatic mosaic gene fusions. Gene fusions were particularly enriched in excitatory neurons with DNA damage repair and senescence gene signatures. In addition, somatic genome structural variations and gene fusions were enriched in neurons burdened with DSBs in the CK-p25 mouse model of neurodegeneration. Neurons enriched for DSBs also had elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic, neuronal development, and histone genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases.

overall_design:
In situ Hi-C was performed as previously described in Rao et al., 2014. 80-120k FANS isolated nuclei were used for CK-p25. Nuclei were permeabilized. DNA was digested with 100 units of MboI, and the ends of restriction fragments were labeled using biotinylated nucleotides and ligated in a small volume. After reversal of crosslinks, ligated DNA was purified and sheared to a length of ∼400 bp, at which point ligation junctions were pulled down with streptavidin beads and prepped for Illumina sequencing. Illumina libraries were sequenced in 38 bp paired-end mode. Three replicates were performed, where each replicate was a pool of 3 CK-p25 mice forebrains. P301S Tau mice and primary neuronal culture Hi-C were performed using 120-170k FANS isolated nuclei with the Dovetail Hi-C kit (SKU: 21004) according to the manufacturer’s instructions.

Hi-C was analyzed using the HOMER Hi-C analysis pipeline (http://homer.ucsd.edu/homer/interactions2/index.html) and Juicer.


title: Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration [BLISS]

overall_design:
BLISS was performed as previously described in Yan et al., 2017. Sorted neurons were attached to a MatTek 35 mm Dish | No. 1.0 Coverslip | 14 mm Glass Diameter | Poly-D-Lysine Coated (P35GC-1.0-14-C) by adding sorted cells to the center coverslip in 4% formaldehyde in PBS and spinning at 2000xg for 2 minutes. The attached nuclei were then end-repaired and A-tailed. Next BLISS adaptors were ligated overnight at 16°C. Excess BLISS adaptors were washed off and genomic DNA was isolated. Next DNA was sonicated to an average size of 500 bp and in-vitro transcribed. Transcribed RNA was isolated, and reverse transcribed to cDNA to prepare Illumina sequencing libraries. Custom BLISS adaptors containing RA3 and RA5 sequences were amplified using RP1 and selected RPI Illumina primers as described in the Illumina TruSeq Small RNA Library Preparation Kit.	Illumina libraries were sequenced in 75 bp single-end mode.

FASTQ files were demultiplexed based on sample barcodes. PCR duplicates were removed based on the UMI. Reads after removing PCR duplicates were mapped to mm10 genome build using Bowtie2. Mapped sam files were converted to bam files using samtools view and to bed files using bedtools bamtobed.


title: Interferon-γ couples CD8+ T cell avidity and differentiation during infection

city: Oxford

first: Audrey

last: Gerard

address: Roosevelt Dr.

country: United Kingdom

institute: The Kennedy Institute of Rheumatology

summary: Expression and relevance of IFNg signaling in CD8 T cells during infection

overall_design: Expression profiling by high throughput sequencing. For 24hrs experiment, C57 Bl6 mice were left naive or infected with Listeria-OVA infected 24 hrs . CD3+ CD8+ T cells were isolated by FACS from the spleen and subjected to scRNAseq. For day 9 experiment, C57Bl6 WT mice and CD8-IFNgRKO mice were infected with LM-OVA. CD3+ CD8+ OVA-tetramer+ T cells were isolated by Fluorescence-activated cell sorting (FACS) from the spleen after 9 days and subjected to scRNA and scTCR sequencing.

last: Gérard


title: DeltaNp63 silencing, DNA methylation shifts and epithelial mesenchymal transition resulted from TAp63 genome editing in squamous cell carcinoma [microarray]

first: Iyoko

last: Katoh

summary: TP63 (p63) is strongly expressed in lower-grade carcinomas of head-and-neck, skin, breast, urothelium, etc. to maintain the well-differentiated phenotype. TP63 has two transcription start sites at exon 1 and exon 3’ to produce TAp63 and DeltaNp63 isoforms, respectively. The major protein, DeltaNp63alpha, epigenetically activates genes essential for epidermal/craniofacial differentiation, including DeltaNp63 itself. To examine whether weakly expressed TAp63 has a specific role, we disrupted exon 1 by CRISPR-Cas9 homology directed repair in a head and neck squamous cell carcinoma (SCC) line. Surprisingly, TAp63 knockout cells, either by monoallelic GFP cassette insertion paired with a frameshift deletion allele or by biallelic GFP cassette insertion, caused DeltaNp63 silencing. Loss of keratinocyte-specific gene expression, switching of intermediate filaments from KRT(s) to VIM, and suppression of cell-cell and cell-matrix adhesion components indicated core events of epithelial mesenchymal transition. Most of the positively and negatively impacted genes including DeltaNp63 displayed local DNA methylation changes. Furthermore, DeltaNp63 expression was partially rescued by transfection of TAp63alpha followed by incubation with DNA methyltransferase inhibitor Zebularine. TAp63, as a minor part of TP63 gene, may possibly be involved in the auto-activation mechanism of DeltaNp63 by which keratinocyte-specific epigenome is maintained in SCC.

overall_design: Comparison of genome-wide gene expression between parental (control) FaDu squamous cell carcinoma line and two TAp63 knockout cell lines derived from FaDu

first: Shun-ichi

last: Kurata


title: Unravelling Chilling-stress Resistance Mechanisms in Endangered Mangrove Plant Lumnitzera littorea (Jack) Voigt

first: Yuchen

last: Yang

address: Xingangxi Road 135

institute: Sun Yat-sen University

summary: Mangrove plants are the demonstrated woody plants in tropical and subtropical coastal area with great ecological and economic importance. However, due to the extreme global climate change and increased human activities, mangroves is suffering a dramatic declining worldwide. Lumnitzera littorea (Jack) Voigt is one of the most endangered mangrove species in China. A comprehensive understanding on its resistance mechanisms to low ambient temperature help us to better conserve and restore L. littorea. In the current study, we performed comparative transcriptome analysis to investigate the genome-wide changes of gene expression profile in L. littorea under chilling stress (8°C day/5°C night) over normal condition (25°C day/23°C night). The low temperature suppressed fatty acid elongation and protein phosphorylation in L. littorea, while induced calcium ion binding process and signaling transduction, suggesting an activation of cold-stress sensing and signaling in L. littorea. Combining our analysis with our previous physiological assays, we showed a substantial photoinhibition occurring in the seedlings of L. littorea with the decrease of ambient temperature, and the synthesis of photosystem II reaction center protein D1 and peroxidase-involved scavenging of reactive oxygen species (ROS) were enhanced accordingly to combat the adverse impacts. Finally, we highlighted the biological significance of post-transcriptional regulation, including RNA binding and surveillance, in coping with cold stress in L. littorea. Collectively, our findings provide a global view to the resistance mechanisms of chilling stress in L. littorea, and valuable genetic resources to assist the protection and restoration of L. littorea.

overall_design: To investigate the molecular mechanisms underlying cold-stress resistance in L. littorea, we conducted comparative transcriptome analysis for the seedlings grown under normal condition (the growth temperature of 25°C day/23°C night, CK) and chilling stress (8°C day/5°C night, CT).

first: Lulu

last: Hao

first: Ying

last: Zhang


title: Integrated chromatin accessibility and gene expression landscape of human triple-negative breast cancer cell lines reveals variation by patient donor ancestry [RNA-seq]

city: Bethesda

first: Stefan

last: Ambs

state: MD

address: Building 37, Room 3050B

department: CCR

institute: NCI

summary: African American (AA) women are at increased risk of developing and dying from Triple-Negative Breast Cancer (TNBC), an aggressive breast cancer subtype, compared to European American (EA) women in the United States. In addition to social determinants, further investigation into biologic factors that contribute to these disparities is needed to fully understand this multi-factorial problem. In particular, the epigenetics of racial/population diversity and its influence on breast cancer incidence and outcomes remains underexplored. Using ATAC-sequencing and RNA-sequencing, we characterized differences in chromatin accessibility and gene expression between EA-derived versus AA-derived TNBC cell lines (N=9). Our analyses revealed significant differences in transcription factor binding and downstream gene expression associated with cancer stemness, resistance, and epithelial to mesenchymal transition. Differences were exacerbated under conditions of hypoxia. Together, these data suggest a differential chromatin and transcriptomic landscape that may contribute to worsened TNBC biology in women of African ancestry. Additionally, as many of these cell lines are used routinely in biomedical research, these findings also indicate that the ancestral origin of patient derived cell lines matters and may contribute to biologic variation in experimental data, suggesting that inclusion of diversely sourced cell lines should be considered in experimental design.

overall_design: RNA was isolated from breast cancer cell lines (n = 8 normoxia and n = 8 hypoxia) using TRIzol method. The mRNA profiles of the above breast cancel cell lines were generated by deep sequencing using Illumina  NextSeq 2000  P2.

first: Alexandra

middle: R

last: Harris

first: Gatikrushna

last: Panigrahi

first: Huaitian

last: Liu

first: Vishal

last: Koparde

first: Maeve

last: Bailey-Whyte

first: Tiffany

last: Dorsey

first: Clayton

last: Yates


title: Integrated chromatin accessibility and gene expression landscape of human triple-negative breast cancer cell lines reveals variation by patient donor ancestry [Bulk ATAC-seq]

overall_design: Cell revival, cell lysis, transposition, and DNA extraction were done using a published method by Corces et al., 2017 (PMID: 28846090) with some modifications for cryopreserved cell processing (Caravaca et al., 2022). The peaks (open chromatin regions) of the above breast cancel cell lines were generated by sequencing using Illumina  NextSeq 2000  P2.


title: The cyclooxygenase-expressing mesenchyme resists intestinal epithelial injury byparacrine signaling

status: Public on Sep 26 2023

city: Beijing

first: Ye-guang

last: Chen

address: Tsinghua Road, Medical Building

department: School of life science

institute: Tsinghua University

summary: Paracrine signals play pivotal roles in organ homeostasis. Mesenchymal stromal cells (MSCs) play a key role in regulating epithelium homeostasis in the intestine while their paracrine effects are poorly characterized. Here, we identified prostaglandin E2 (PGE2) secreted by cyclooxygenase (COX)-expressing MSCs as a vital factor to maintain the intestinal mucosal barrier. We found that MSCs-induced organoid swelling through paracrine effect in vitro, a process due to enhanced water adsorption and is mediated by the COX-PGE2-EP4 axis. To further explore the regulatory effect of this axis on the intestinal epithelial barrier in vivo, we established the conditional knockout mouse model to specifically delete COX in MSCs and found that PGE2 reduction downregulated the gene Muc2 and induced a gastric metaplasia-like phenotype. Moreover, PGE2 defects increased the susceptibility of intestinal epithelium to colitis. Our study uncovers the paracrine signaling of COX-expressing MSCs in intestinal mucosal barrier maintenance, providing a basis for understanding the role of mesenchymal cells in the psychophysiology function of the intestine.

overall_design: One month after tamoxifen administration, the crypt tissue/villus tissue of mouse small intestine from Gli1-CreERT2; Rosa26-tdTomato mice or Gli1-PtgscKO mice were sorted. Then their RNA was collected and performed RNA sequencing.

first: Siting

last: Wei

first: Meng

last: Li

first: Wanlu

last: Song


title: Integrated chromatin accessibility and gene expression landscape of human triple-negative breast cancer cell lines reveals variation by patient donor ancestry

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

overall_design: Refer to individual Series


title: Transcriptional dynamics of the cycling mouse uterus

city: Austin

first: Elle

last: Roberson

state: Texas

address: 2401 Speedway, PAT 216

department: Molecular Biosciences

institute: University of Texas at Austin

summary: The adult uterus regenerates in the human during the menstrual cycle, and remodels in the mouse during the estrous cycle. Decades of work has demonstrated that this process is controlled by cycling steroid hormones, estrogen and progesterone. However, downstream signaling pathways that link hormonal action to this regeneration and remodeling are yet to be identified in the cycling uterus. We set out to identify these pathways, with the overarching hypothesis that developmental signaling pathways are redeployed in the adult uterus to control remodeling in the mouse.

overall_design: Mouse uteri from 8-12 week-old estrous-cycle-staged Swiss Webster female mice were collected at each of the four estrous cycle stages. RNA was extracted from the whole uterus: Proestrus n=3, Estrus n=3, Metestrus n=3, Diestrus n=3.

middle: C

first: Ngan

middle: K

last: Tran

first: Anushka

last: Godambe

first: Trinity

last: Rust

first: Michelle

last: Nguimtsop

first: Harrison

last: Mark

first: Rebecca

middle: D

last: Fitch

first: John

middle: B

last: Wallingford


title: RIP-seq of NAT10 interacted cellular and KSHV transcripts in iSLK-KSHV cells

city: Nanjing

first: Liuxue

last: Sheng

state: Jiangsu

address: Tian Yuan East Road

institute: Nanjing Medical University

summary: Flag-RIP-seq was performed in NAT10-Flag overexpressing  iSLK-KSHV cells.

sample_organism:
Homo sapiens
Human gammaherpesvirus 8

platform_organism:
Homo sapiens
Human gammaherpesvirus 8

overall_design: NAT10-Flag and its control RFP-Flag was overexpressed in iSLK-KSHV cells, the Flag-RIP-seq was performed.

first: Qin

last: Yan

first: Jing

last: Zhou

first: Ziyu

last: Wang

first: Xiangya

last: Ding

first: Xinyue

last: Ma

first: Wan

first: Xuemei

last: Jia

first: Shou-Jiang

last: Gao

first: Chun

last: Lu


title: Reliability of human retinal organoid generation from hiPSC-derived neuroepithelial cysts

city: Leipzig

first: Sebastian

last: Canzler

address: Permoserstraße 15

department: Deparment Computational Biology

institute: Helmholtz-Centre for Environmental Research - UFZ

summary: The application potential of human induced pluripotent stem cells (hiPSC) derived human retinal organoids (HRO) relies on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare and optimize organoid protocols have been started to be established, but are not completed yet. To advance this, we explored the efficiency and reliability of a differentiation protocol that facilitates retina generation by formation of neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines, which reproducibly generated HROs. Histologic and ultrastructural analyses support regulated HRO differentiation and maturation. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Whereas previous reports showed that HRO in several other protocols contain a rather low numbers of cones compared to rods, HROs derived by the cyst protocol consistently are cone-richer and with an comparable ratio of cones, rods, and Müller glia. Additionally, we devised a potential strategy to systematically evaluate different protocols side-by-side through parallel differentiation from the same hiPSC batches: The cyst-protocol was compared to a conceptually different protocol based on cell aggregate formation from single hiPSCs. Comparison of four hIPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the cyst-protocol provided a higher HRO yield. Further, while cyst-derived HROs maintained stable at least up to date 250, whereas single hiPSC-derived HROs showed spontaneous pathologic changes already by day 200. Overall, our data provide insight into the efficiency, reproducibility, and stability of the cyst-protocol for HRO generation, which will be useful for further organoid system optimization, as well as basic and translational research applications.

overall_design: Dissociated cells (n = 5370) of one human retinal organoid

first: Manuela

last: Völkner

first: Lisa

last: Steinheuer

first: Madalena

last: Carido

first: Jörg

last: Hackermüller

first: Mike

middle: O

last: Karl


title: The role of ALKBH5 in maternal RNA decay during mammalian oocyte meiosis [RNA-seq]

city: Hangzhou

first: Long

last: Bai

state: Zhejiang

address: No. 268, Kaixuan Road

institute: Zhejiang University

summary: To investigate the function of ALKBH5 in the regulation of maternal RNA decay during mice oocyte meiosis, we established Alkbh5 knockout mice and analyzed the transcriptome changes between control and Alkbh5 knockout oocyte at different meiotic maturation stages

overall_design: Comparative gene expression profiling analysis of RNA-seq data for control and Alkbh5 knockout oocytes at different stages

first: Yu

last: Xiang

first: Yimin

last: Zhu


title: The role of ALKBH5 in maternal RNA decay during mammalian oocyte meiosis

summary: To investigate the function of ALKBH5 in the regulation of maternal RNA decay during mice oocyte meiosis, we established Alkbh5 knockout mice and analyzed the m6A level changes between control and Alkbh5 knockout oocyte at GV stage

overall_design: Comparative m6A peaks analysis of RNA-seq data for control and Alkbh5 knockout oocytes at GV stage


title: ac4C-seq of cellular and KSHV transcripts in iSLK-Puro, iSLK-KSHV (i.e. iSLK-RGB), iSLK-KSHV (WT), iSLK-KSHV (ΔNAT10) cells

first: Liuxue

last: Sheng

summary: We used ac4C-seq to detedc cellular and KSHV transcripts in iSLK-Puro, iSLK-KSHV, iSLK-KSHV (WT), iSLK-KSHV (ΔNAT10) cells after after doxycycline and sodium butyrate treatment.

overall_design: Examination of NAT10 dependent KSHV ac4C transcripts in 4 different iSLK cells.

first: Qin

last: Yan

first: Jing

last: Li


title: Phytocannabinoids reduce inflammation of primed macrophages and enteric glial cells Critical review of the effect of phytocannabinoids on gut immune cells

city: Rehovot

first: Ofer

last: Gover

address: Hertzel 229

department: Biochemistry and Nutrition

country: Israel

institute: Hebrew University Jerusalem

summary: In vitro study analyzed the putative-anti-inflammatory effect of nine-selected pure cannabinoids on enteric glial cells (EGC’s) triggered to undergo inflammation with lipopolysaccharide (LPS) . Our results demonstrate that THC at the lower concentrations tested exerted the most effective anti- inflammatory effect in EGC’s compared to other phytocannabinoids tested herein.  We then performed RNA-seq analysis of EGC’s exposed to LPS in the presence or absence of THC or THC-COOH. Transcriptomic analysis of these EGC’s revealed 23 differentially expressed genes (DEG) compared to treatment with only LPS. Pretreatment with THC resulted in 26 DEG and THC-COOH 25 DEG. To evaluate biological pathways affected by the different phytocannabinoid treatments we used the Ingenuity platform. We show that THC treatment but not THC-COOH affected

overall_design: Enteric glial cell lines (EGC/PK060399egfr) were grown in 75 mm2 flasks to 70% confluence in DMEM medium containing 10% (Sigma Aldrich, USA) fetal bovine serum (FBS) (Biological industries), 0.5% penicillin-streptomycin (Bio-logical industries), at 37°C under 5% CO2. Cells were trypsinized (using 0.25%) (Invitro-gen, Carlsbad CA, USA) and transferred every 2-3 days. Cells were incubeted for 1h without treatment, with 0.1hg/mL pure THC, 10nM pure THC-COOH, 10ug/L Sparstolonin B (Ssnb) (positive control). After 1h 1ug/mL LPS (E. coli 0111:B4) was added for 24h. After 24h media was removed and RNA was extracted using MN NucleoSpin RNA Kit.

first: Gal

last: Cohen

first: Betty

last: Schwartz


title: Cellulose biosynthesis inhibitor isoxaben causes nutrient-dependent and tissue-specific Arabidopsis phenotypes

city: Madison

first: Sarah

last: Whitcomb

state: WI

address: 502 Walnut St

department: Cereal Crops Research Unit

institute: USDA-ARS

summary: n/a - Letter to the Editor

overall_design: RNA-seq data from Arabidopsis thaliana Col-0 roots harvested 6 days after stratification

first: Michael

last: Ogden

first: Ghazanfar

last: Khan

first: Ute

last: Roessner

first: Rainer

last: Hoefgen

first: Staffan

last: Persson


title: Influence of metaphylaxis for bovine respiratory disease on host transcriptome responses

city: Canyon

first: Matthew

middle: Adam

last: Scott

state: TX

address: 3201 Russell Long Blvd

department: Large Animal Clinical Sciences

institute: Texas A&M University

summary: To evaluate how commonly-utilized antimicrobials affect the host transcriptome of commercial beef cattle overtime, we enrolled 105 feedlot beef steers randomly into seven different treatment groups (negative control, tulathromycin, tildipirosin, enrofloxacin, florfenicol, ceftiofur, oxytetracycline) to receive a one-time label dose of a commercial antimicrobial or not (negative control), and collected jugular whole blood into PAXgene RNA blood tubes at six time points: Day 0 (baseline), 3, 7, 14, 21, and 56.

overall_design: Whole blood from six randomly selected individuals per treatment group (n=7; negative control, tulathromycin, tildipirosin, enrofloxacin, florfenicol, ceftiofur, oxytetracycline) were used for gene expression profiling and differential analysis across six time points (Day 0, 3, 7, 14, 21, 56).

first: Robert

last: Valeris-Chacin

first: Paul

last: Morley

first: John

last: Richeson


title: Tau Protein Modulates an Epigenetic Mechanism of Cellular Senescence in Human SH-SY5Y Neuroblastoma Cells

status: Public on Sep 25 2023

city: Bellinzona

first: Luciano

last: Cascione

state: Ticino

address: Via Chiesa 5

department: Lymphoma & Genomics Research Program

country: Switzerland

institute: IOR

summary: Progressive Tau deposition in neurofibrillary tangles and neuropil threads is the hallmark of tauopathies, a disorder group that includes Alzheimer’s disease. Since Tau is a microtubule-associated protein, a prevalent concept to explain the pathogenesis of tauopathies is that abnormal Tau modification contributes to dissociation from microtubules, assembly into multimeric β-sheets, proteotoxicity, neuronal dysfunction and cell loss. Tau also localizes in the cell nucleus and evidence supports an emerging function of Tau in DNA stability and epigenetic modulation. To better characterize the possible role of Tau in regulation of chromatin compaction and subsequent gene expression, we performed a bioinformatics analysis of transcriptome data obtained from Tau-depleted human neuroblastoma cells. Among the transcripts deregulated in a Tau-dependent manner, we found an enrichment of target genes for the polycomb repressive complex 2. We further describe decreased cellular amounts of the core components of the polycomb repressive complex 2 and lower histone 3 trimethylation in Tau deficient cells. Among the de-repressed polycomb repressive complex 2 target gene products, IGFBP3 protein was found to be linked to increased senescence induction in Tau-deficient cells. Our findings propose a mechanism for Tau-dependent epigenetic modulation of cell senescence, a key event in pathologic aging.

overall_design: Chromatin immunoprecipitation for histone modification H3K27me3 in neuroblastoma cell lines between two Tau-KO SH-SY5Y (two different AA, 231K and 232P) cell lines when compared to normal Tau-expressing cells (wild type)

first: Paolo

last: Paganeti

first: Claudia

last: Magrin

first: Stephanie

last: Papin


title: TNFα/TNFR2 axis mediates natural killer cell proliferation by enhancing their glycolytic plasticity

city: Ottawa

first: Seung-Hwan

last: Lee

state: Ontario

address: 451 Smyth Road

department: Biochemistry, Immunology and Microbiology

country: Canada

institute: University of Ottawa

summary: Natural Killer (NK) cells are predominant innate lymphocytes that provide the early response during infection. NK cells undergo metabolic switch to fuel augmented proliferation and activation following infection. TNFα is a well-known inammatory cytokine that enhances NK cell function, however, a mechanism for stimulation is not well established. Here, we demonstrated that upon infection/inammation, NK cells upregulate the expression of TNF receptor 2 (TNFR2), which is associated with increased proliferation, metabolic activity and effector function. Notably, IL-18 can induce TNFR2 on NK cells, augmenting their sensitivity towards TNFα. Mechanistically, TNFα-TNFR2 signaling induces CD25 (IL-2Rα) expression on NK cells predominantly by autocrine mode, leading to a metabolic switch towards aerobic glycolysis. Accordingly, genetic ablation of TNFR2 curtails the CD25 upregulation and TNFα-induced glycolysis, leading to impaired NK cell proliferation during MCMV infection in vivo. Collectively, our results delineate the crucial role of the TNFα-TNFR2 axis in NK cells for proliferation, glycolysis, and effector function via CD25 induction.

overall_design: Ly49H+ natural killer (NK) cells from CD45.1-WT and CD45.2-TNFR2 knockout (KO) mice were co-transferred into NSG mice. The mice were then infected with murine cytomegalovirus (MCMV). On day 3.5 post-infection, Ly49H+ cells (CD45.1 or CD45.2) were isolated using flow sorting and analyzed using bulk RNA-seq.

first: Abrar

first: Bryan

last: Marr


title: Sigma 28-dependent small RNAs regulate timing of flagella biosynthesis

first: Ryan

last: Dale

address: Rm 10D39, 10 Center Drive

department: National Institute of Child Health and Human Development

institute: National Institutes of Health

summary: Synthesis of flagella is very energy intensive and, while extensive transcriptional regulation has been described, little is known about the post-transcriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators; most base pairing with mRNAs to affect their stability and/or translation. Here we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor, sigma 28 in Escherichia coli. UhpU, MotR and FliX have distinct impacts on flagellin levels, flagella number and swimming, with MotR accelerating flagella synthesis and FliX decelerating flagella synthesis. Interestingly, MotR and FliX are noncanonical in that they base pair within the coding sequences of target genes. We characterize their respective positive and negative effects on genes encoding ribosomal proteins.

overall_design: Total RNA sequencing of E. coli strains carrying a control vector or overexpressing MotR* or FliX-S

first: Sahar

last: Melamed

first: Aixia

last: Zhang

first: Joshua

last: Mills

first: Michal

last: Jarnik

first: Hongen

first: Gisela

last: Storz


title: Hyperactive KRAS/MAPK signaling disrupts normal lymphatic vessel architecture and function

city: Dallas

first: Michael

last: Dellinger

address: 5323 Harry Hines Blvd

institute: University of Texas Southwestern Medical Centre

summary: Complex lymphatic anomalies (CLAs) are sporadically occurring diseases caused by the maldevelopment of lymphatic vessels. We and others recently reported that somatic activating mutations in KRAS can cause CLAs. However, the mechanisms by which activating KRAS mutations cause CLAs are poorly understood. Here we show that KRASG12D expression in lymphatic endothelial cells (LECs) during embryonic development impairs the formation of lymphovenous valves and causes the enlargement of lymphatic vessels. We demonstrate that KRASG12D expression in primary human LECs induces cell spindling, proliferation, and migration. It also increases AKT and ERK1/2 phosphorylation and decreases the expression of genes that regulate lymphatic vessel maturation. We show that MEK1/2 inhibition with the FDA-approved drug trametinib suppresses KRASG12D-induced morphological changes, proliferation, and migration. Trametinib also decreases ERK1/2 phosphorylation and increases the expression of genes that regulate the maturation of lymphatic vessels. We also show that trametinib and Cre-mediated expression of a dominant-negative form of MEK1 (Map2k1K97M) suppresses KRASG12D-induced lymphatic vessel hyperplasia in embryos. Last, we demonstrate that conditional knockout of wild-type Kras in LECs does not affect the formation or function of lymphatic vessels. Together, our data indicate that KRAS/MAPK signaling must be tightly regulated during embryonic development for the proper development of lymphatic vessels and further support the testing of MEK1/2 inhibitors for treating CLAs.

overall_design: To examine the effect of hyperactive KRAS signaling on gene expression, primary human LECs were infected with KRASG12D or GFP expressing lentivirus particles. RNA was isolated 72 hours after infecting the cells. To assess the impact of trametinib on KRASG12D-LECs, primary human LECs were infected with lentivirus particles, and the media was replaced with normal growth media 24 hours later. Forty-eight hours after infecting cells, they were treated with DMSO or trametinib (10 nM) for approximately 16 hours.

first: Lorenzo

last: Fernandes

first: Jeffrey

last: Tresemer

first: Jonathan

last: Rios


title: KDM2B regulates hippocampal morphogenesis by transcriptionally silencing Wnt signaling in neural progenitors (ChIP-Seq)

city: wuhan

first: zhao

last: chen

state: Hubei

address: shuiguohu

department: BIOLOGY

institute: wuhan university

summary: The hippocampus plays major roles in learning and memory. Similar to other parts of the brain, the development of hippocampus requires precise coordination of patterning, cell proliferation, differentiation, and migration, with both cell-intrinsic and extrinsic mechanisms involved. Here we genetically removed the chromatin-association capability of KDM2B - a key component of the variant Polycomb repressive complex 1 (PRC1) - in the developing dorsal telencephalon (Kdm2bEmx1-∆CxxC) to surprisingly discover that the size of Kdm2bEmx1-∆CxxC hippocampus, particularly the dentate gyrus, became drastically smaller with disorganized cellular components and structure. Kdm2bEmx1-∆CxxC mice displayed prominent defects in spatial memory, motor learning and fear conditioning. The differentiation path of the developing Kdm2bEmx1-∆CxxC hippocampus was greatly delayed, with significant amount of TBR2-expressing intermediate progenitors being stuck along the migratory path. Transcriptome and chromatin immunoprecipitation studies of neonatal hippocampi and their progenitors indicated that genes implicated in stemness maintenance, especially components of canonical Wnt signaling, could not be properly silenced by PRC1 and PRC2. Activating Wnt signaling disturbed hippocampal neurogenesis, recapitulating the effect of KDM2B loss. Together, we unveiled a previously unappreciated gene repressive program mediated by KDM2B that controls progressive fate specifications and cell migration, hence morphogenesis of hippocampus during development.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone H2AK119ub1, H3K27me3 as well as the histone modifications H3K36me2 in P0 hippocampus and neurosphere. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) for P0 hippocampus.

first: Bo

first: Chen

last: Zhao

first: Wenchen

last: Shen

first: Wei

last: Li

first: Yue

last: Zheng

first: Xiangfei

last: Kong

first: Junbao

last: Wang

first: Xudong

last: Wu

first: Ying

last: Liu

first: Yan

last: Zhou


title: KDM2B regulates hippocampal morphogenesis by transcriptionally silencing Wnt signaling in neural progenitors (ATAC-Seq)

last: Zhang


title: KDM2B regulates hippocampal morphogenesis by transcriptionally silencing Wnt signaling in neural progenitors


title: Fibroblast growth factor 18 stimulates the proliferation of hepatic stellate cells, thereby inducing liver fibrosis

city: Miyama, Funabashi-shi

first: Yuichi

last: Tsuchiya

state: Chiba

department: Pharmaceutical Sciences

institute: Toho University

summary: Liver fibrosis results from chronic liver injury triggered by factors such as viral infection, excess alcohol intake, and lipid accumulation. However, the mechanisms underlying liver fibrosis are not fully understood. Here, we demonstrate that expression of Fibroblast growth factor 18 (Fgf18) is elevated in the livers of mice following induction of chronic liver fibrosis models. Deletion and overexpression of Fgf18 in hepatocytes attenuate and promote liver fibrosis, respectively. Single-cell RNA-sequencing revealed that Lrat+ quiescent hepatic stellate cells (HSCs) are increased in the livers of Fgf18 transgenic mice. Mechanistically, FGF18 stimulates the proliferation of quiescent HSCs by inducing the expression of Ccnd1, resulting in the development of liver fibrosis. Moreover, the expression of FGF18 is correlated with the expression of profibrotic genes, such as COL1A1 and ACTA2, in human liver biopsy samples. Thus, FGF18 promotes liver fibrosis and could serve as a therapeutic target to treat liver fibrosis.

overall_design: Comparisons of global gene expression patterns between control and conditional knockout mice / transgenic mice by bulk RNA-seq (N=3 each).

first: Takao

last: Seki

first: Kenta

last: Kobayashi

first: Sachiko

last: Komazawa-Sakon

first: Shigeyuki

last: Shichino

first: Takashi

last: Nishina

first: Kyoko

last: Fukuhara

first: Kenichi

last: Ikejima

first: Hidenari

last: Nagai

first: Yoshinori

last: Igarashi

first: Satoshi

last: Ueha

first: Akira

last: Oikawa

first: Shinya

last: Tsurusaki

first: Soh

last: Yamazaki

first: Chiharu

last: Nishiyama

first: Tetuo

last: Mikami

first: Hideo

last: Yagita

first: Ko

last: Okumura

first: Taketomo

last: Kido

first: Atsushi

last: Miyajima

first: Kouji

last: Matsushima

first: Mai

last: Imasaka

first: Kimi

last: Araki

first: Toru

last: Imamura

first: Masaki

last: Ohmuraya

first: Minoru

last: Tanaka

first: Hiroyasu

last: Nakano


title: Endothelial deletion of EPH receptor A4 alters single-cell profile and Tie2/Akap12 signaling to preserve blood-brain barrier integrity

status: Public on Sep 24 2023

city: Blacksburg

first: Michelle

last: Theus

state: VA

address: 234 Craig Drive

institute: Virginia Polytechnic Institute and State University

summary: Neurobiological consequences of traumatic brain injury (TBI) result from a complex interplay of secondary injury responses and sequela that mediates chronic disability. Endothelial cells are important regulators of the cerebrovascular response to TBI. Our work demonstrates that genetic deletion of endothelial cell (EC)-specific EPH receptor A4 (EphA4) using conditional EphA4f/f/Tie2-Cre and EphA4f/f/VE-Cadherin-CreERT2 knockout (KO) mice, promotes blood-brain barrier (BBB) integrity and tissue protection, which correlates with improved motor function and cerebral blood flow recovery following controlled cortical impact (CCI) injury. scRNAseq of capillary-derived KO ECs showed increased differential gene expression of BBB-related junctional and actin cytoskeletal regulators, namely, A-kinase anchor protein 12, Akap12, whose presence at Tie2 clustering domains is enhanced in KO microvessels. Transcript and protein analysis of CCI-injured whole cortical tissue or cortical-derived ECs suggests EphA4 limits the expression of Cldn5, Akt, and Akap12 and promotes Ang2. Blocking the Tie2 receptor using sTie2-Fc, attenuated BBB and tissue protection and reversed Akap12 mRNA and protein levels in KO compared to WT cortical-derived ECs. Conversely, direct stimulation of Tie2 using Vasculotide, an angiopoietin-1 memetic peptide, phenocopied the neuroprotection. Finally, we report a noteworthy rise in soluble Ang2 in the sera of individuals with acute TBI, highlighting its promising role as a vascular biomarker for early detection of BBB disruption. Overall, we describe a novel contribution of the axon guidance molecule, EphA4, in mediating TBI microvascular dysfunction through negative regulation of Tie2/Akap12 signaling.

overall_design: cortical samples from EphA4f/f-WT and EphA4f/f-VeCadCreERT2 KO mice, at 1 day post-CCI injury to right parietal cortex of mice, were processed using CD45 and CD31 microbeads. The CD45 cells and flow through were collected and placed on Trizol and RNA extracted for sequencing.

first: Alison

last: Cash

first: Xie

last: Hehunag


title: Multi-omics analysis identifies coordination and hierarchy of transcription factors controlling specific epithelial cell fates in corneal epithelium [Bulk RNA-seq]

status: Public on Sep 22 2023

city: Nijmegen

first: Jo Huiqing

last: Zhou

address: Geert Grooteplein 26/28

country: Netherlands

institute: Radboud University

summary: The homeostasis of the transparent corneal epithelium in the eye is maintained by the proliferation and differentiation of limbal stem cells that possess the proper cell fate. A potential disease mechanism underlying corneal opacity has been proposed to be limbal stem cells acquiring the cell fate of keratinocytes in the non-transparent epidermis. In this study, we performed a multi-omics analysis of human limbal stem cells derived from the cornea and keratinocytes from the epidermal stratum basale and characterized the similar yet distinct molecular signatures of these cells. By gene regulatory network analysis, we identified cell fate defining transcription factors and their regulatory hierarchy that are shared by the two cell types and those that regulate specific epithelial programs. Our findings indicate that shared transcription factors often regulate limbal stem cell-specific transcription factors. Single-cell RNA-seq analysis of the cornea and the epidermis confirms the shared and specific transcription factor expression patterns in the stem cells of these tissues. Finally, we showed that genes associated with corneal opacity can cooperatively be targeted by the shared and limbal stem cell-specific transcription factors. By characterizing molecular signatures, our study uncovers the distinct regulatory circuitry controlling limbal stem cell fates and corneal opacity.

overall_design: siRNA knockdown of PAX6 and FOSL2 in various Limbal stem cells

first: Jos

middle: G

last: Smits

first: Dulce

middle: L

last: Cunha

first: Maryam

last: Amini

first: Marina

last: Bertolin

first: Camille

last: Laberthonnière

first: Jieqiong

last: Qu

first: Nicholas

last: Owen

first: Lorenz

last: Latta

first: Berthold

last: Seitz

first: Lauriane

last: Roux

first: Tanja

last: Stachon

first: Stefano

last: Ferrari

first: Mariya

last: Moosajee

first: Daniel

last: Aberdam

first: Nora

last: Szentmary

first: Simon

last: van Heeringen

first: Huiqing


title: Transcriptional regulation of human dorsal root ganglia cultures by type 2 cytokines

city: Cambridge

first: Madison

last: Mack

state: Massachusetts

address: 350 Water St

department: Immunology & Inflammation Research

institute: Sanofi

summary: Cutaneous afferent neurons express receptors for type 2 cytokines, including interleukins (IL)-4, IL-13, and IL-33. These neuronal cytokine receptors were found to be required in several murine models of itch. The objective of the present study was to determine if type 2 cytokine stimulation sensitizes sensory neurons to future itch stimuli in a fully human ex vivo system. Human dorsal root ganglia were isolated and dissociated to establish ex vivo cultures and subjected to 24 hours of type 2 cytokine exposure with either vehicle, IL-4, IL-13, IL-33, or IL-4 and IL-13 together. After 24 hours, cells were harvested and RNA extracted and sequenced to determine the transcriptomic effects of type 2 cytokine exposure.

overall_design: Cultures were stimulated with 500 nM IL-4, IL-13, IL-33 or the combination of IL-4 and IL-13 for 24 hours. Vehicle alone and a cocktail of inflammatory mediators "CIS" were as negative and positive controls.


title: Multi-omics analysis identifies coordination and hierarchy of transcription factors controlling specific epithelial cell fates in corneal epithelium [scRNA-seq]

first: Jo Huiqing

last: Zhou

overall_design: 2 samples of Limbal stem cells

first: Jos

last: Smits

first: Dulce


title: Effect of methanol fixation on single cell RNA sequencing of the murine dentate gyrus (droplet-based scRNAseq).

city: Jena

first: Anja

last: Urbach

address: Am Klinikum 1

department: Neurology

institute: Universitätsklinikum Jena

summary: We evaluated the effect of methanol (MeOH) fixation on adult murine dentate gyrus (DG) single cell suspensions processed with droplet-based scRNA-seq.

overall_design: Methanol-fixed neuronal and non-neuronal cells from the DG of adult C57BL/6J mice were isolated and proccessed with 10x Genomics Chromium controller, kit 3’ v3.1 to perform library construction.

first: Marta

last: Sánchez-Carbonell

first: Patricia

last: Jiménez Peinado

first: Cathrin

last: Bayer-Kaufmann

first: Jean-Christopher

last: Hennings

first: Yvonne

last: Hofmann

first: Silvio

last: Schmidt

first: Otto

middle: W

last: Witte


title: Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease [CUT&RUN]

summary: The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells, while the non-transparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human limbal stem cells from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors that define specific cell fates, and established their regulatory hierarchy. Single-cell RNA-seq analyses of the cornea and the epidermis confirmed these shared and cell type-specific transcription factors. Notably, the shared and limbal stem cell-specific transcription factors can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the limbal stem cell fate and its association with corneal opacity.

overall_design: FOSL2 CUT&RUN in  Limbal stem cells

last: Lima Cunha

first: Maryam

last: Amini

first: Marina

last: Bertolin

first: Camille

last: Laberthonnière

first: Jieqiong

last: Qu

first: Nicholas

last: Owen

first: Lorenz

last: Latta

first: Berthold

last: Seitz

first: Lauriane

last: Roux

first: Tanja

last: Stachon

first: Stefano

first: Mariya


title: Effect of methanol fixation on single cell RNA sequencing of the murine dentate gyrus.

first: Anja

last: Urbach

summary: We evaluated the effect of methanol (MeOH) fixation on adult murine dentate gyrus (DG) single cell suspensions.

overall_design: Freshly and methanol-fixed neuronal and non-neuronal cells from the DG of adult C57BL/6J mice were isolated by Fluorescence-activated cell sorting (FACS), according to the presence or absence of Sytox AADvanced™ Dead Cell Stain, and analyzed using SOrting and Robot-assisted Transcriptome SEQuencing (SORT-seq), a partially robotized version of the CEL-seq2 protocol.

first: Marta

last: Sánchez-Carbonell

first: Patricia


title: Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease [ChIP-seq]

first: Jo Huiqing

last: Zhou

overall_design: Comparative TF binding analysis of ChIP-seq data for Limbal Stem Cells and Keratinocytes

first: Jos

last: Smits

first: Dulce

last: Cunha

last: Ferrari

last: Moosajee

first: Daniel

last: Aberdam

first: Nora

last: Szentmary

first: Simon

last: van Heeringen

first: Huiqing


title: CBFA2T3::GLIS2 pediatric acute megakaryoblastic leukemia is sensitive to BCL-XL inhibition by navitoclax and DT2216

city: Montreal

first: Sonia

last: Cellot

state: Quebec

address: 3175 Côte-Sainte-Catherine

institute: Centre Hospitalier Universitaire (CHU) Sainte-Justine Research Center

summary: We engineered human models of CBFA2T3::GLIS2  acute megakaryoblastic leukemia and compared gene expression profiles to primary pediatric samples.

overall_design: RNA sequencing of human models of CBFA2T3::GLIS2 and NUP98 rearranged acute megakaryoblastic leukemia. CD34+ cord blood hematopoietic stem/progenitor cells were used as control.

first: Verena

last: Gress

first: Mathieu

last: Roussy

first: Luc

last: Boulianne

first: Mélanie

last: Bilodeau

first: Sophie

last: Cardin

first: Nehme

last: El-Hachem

first: Véronique

last: Lisi

first: Banafsheh

last: Khakipoor

first: Alexandre

last: Rouette

first: Azer

last: Farah

first: Louis

last: Théret

first: Léo

last: Aubert

first: Furat

last: Fatima

first: Éric

last: Audemard

first: Pierre

last: Thibault

first: Éric

last: Bonneil

first: Jalila

last: Chagraoui

first: Louise

last: Laramée

first: Patrick

last: Gendron

first: Loubna

last: Jouan

first: Safa

last: Jammali

first: Bastien

last: Paré

first: Shawn

middle: M

last: Simpson

first: Thai

middle: H

last: Tran

first: Michel

last: Duval

last: Teira

first: Henrique

last: Bittencourt

first: Raoul

last: Santiago

first: Frédéric

last: Barabé

first: Guy

last: Sauvageau

first: Martin

last: Smith

first: Josée

last: Hébert

first: Philippe

middle: P

last: Gruber

first: Vincent-Philippe

last: Lavallée

first: Brian

last: Wilhelm


title: Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease [scRNA-seq]

first: Jo Huiqing

last: Zhou

overall_design: Comparative gene expression profiling analysis of scRNA-seq data for Limbal Stem Cells and Keratinocytes

first: Jos

last: Smits

first: Dulce

last: Cunha

first: Maryam

last: Amini

first: Marina

last: Bertolin

first: Camille

last: Laberthonnière

first: Jieqiong

last: Qu

first: Nicholas

last: Owen

first: Lorenz

last: Latta

first: Berthold

last: Seitz

first: Lauriane

middle: N

last: Roux

first: Tanja

last: Stachon

first: Stefano

last: Ferrari

first: Mariya

last: Moosajee

first: Daniel

last: Aberdam

first: Nora

last: Szentmary

first: Simon

middle: J

last: van Heeringen

first: Huiqing


title: Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease [RNA-seq]

first: Jo Huiqing

last: Zhou

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Limbal Stem Cells and Keratinocytes

first: Jos


title: Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease [ATAC-seq]

overall_design: Comparative genome accesibilty analysis of ATAC-seq data for Limbal Stem Cells and Keratinocytes

last: Smits

first: Dulce

first: Maryam


title: Human fatty liver RT-PCR array on primary human hepatocytes grown with 3t3-J2 stromal cells in a micropatterned co-culture

city: Baltimore

first: Karissa

last: Cottier

state: Maryland

address: 1450 S. Rolling Rd.

institute: BioIVT

summary: Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, progressive disorder and growing public health concern. To address this issue considerable research has been undertaken in pursuit of new NAFLD therapeutics. Development of effective, high-throughput in vitro models is an important aspect of drug discovery. Here, a micropatterned hepatocyte co-culture (MPCC) was used to model liver steatosis. The MPCC model (HEPATOPAC) involves hepatocytes and 3T3-J2 mouse stromal cells patterned onto a standard 96-well plate, increasing throughput and allowing the cultures to be handled and imaged like 2D cultures. These studies employed high content imaging (HCI) analysis to assess lipid content in cultures. HCI analysis of lipid accumulation allows large numbers of samples to be imaged and analyzed in a relatively short period of time compared to manual acquisition and analysis methods. Treatment of MPCC with free fatty acids (FFA), high glucose and fructose (HGF), or a combination o  f both induces hepatic steatosis. MPCC treatment with ACC1/ACC2 inhibitors, as either a preventative or reversal agent showed efficacy against FFA induced hepatic steatosis. Drug induced steatosis was also evaluated. Treatment with valproic acid showed steatosis induction in a lean background, which was significantly potentiated in a fatty liver background. Additionally, these media treatments changed expression of fatty liver related genes. Treatment of MPCC with FFA, HGF, or a combination reversibly altered expression of genes involved in fatty acid metabolism, insulin signaling, and lipid transport. Together, these data demonstrate that MPCC is an easy to use, long-term functional in vitro model of NAFLD having utility for compound screening, drug toxicity evaluation, and assessment of gene expression changes.

overall_design:
MPCC (HEPATOPAC) cultures were created using microfabrication processes to generate an organized co-culture containing cryopreserved primary human hepatocytes (PHH) from BioIVT (Baltimore, MD) and 3T3-J2 murine embryonic fibroblasts. Cryopreserved primary human hepatocytes from  donor lot TWJ were thawed at 37°C, counted, and plated on collagen-patterned 96 or 24 well plates. The cells were allowed to adhere until the following day, when 3T3-J2 fibroblasts were introduced to the culture plates. The resulting culture contained multiple hepatocyte islands having a 500 μm diameter spaced 1200 μm apart, center-to-center; with 3T3-J2 murine fibroblasts filling the area surrounding the hepatocyte islands. MPCC cultures were stabilized in serum containing medium for 7 days. At maturity, approximately 3,100 hepatocytes and 15,000 fibroblasts were present/well in a 96 well plate and about 21,000 hepatocytes and 90,000 fibroblasts were present/well in a 24 well plate. Treatment was initiated on day 7 after initiation of culture manufacture.
Cells were treated with control medium containing 1.0g/L glucose, 1.1 µM insulin and 10% bovine serum or steatosis inducing media containing 0.5 mM free fatty acid (FFA) (Palmitate/Oleate 2:1, Sigma), 10g/L glucose and 1.0g/L fructose (HGF), or a combination of FFA and HGF with media exchanges every other day. Cells were treated with steatosis inducers continuously until day 7 of treatment or switched back to control media on day 4 for 3 days, until day 7 of treatment (reversal samples).
RT-PCR array using Qiagen Human Fatty Liver Array

middle: E

first: Devika

last: Bhalerao

first: Jeannemarie

last: Gaffney

first: Candice

last: Lewis

first: Scott

middle: A

last: Heyward


title: Transcriptomic Analysis of Isolated Monocytes from heathy donors and JIA patinets using Nanostring Technology

status: Public on Sep 21 2023

city: Paris

first: Nassima

last: Bekaddour

address: 45 rue des Saints-Pères

institute: Université Paris Cité

summary: Among immune cells, activated monocytes play a detrimental role in chronic and viral-induced  inflammatory pathologies. The uncontrolled activation of monocytes and the subsequent  excessive production of inflammatory factors damage bone-cartilage joints in Juvenile  Idiopathic Arthritis (JIA), a childhood rheumatoid arthritis (RA) disease. The moderate  beneficial effect of current therapies and clinical trials highlights the need of alternative  strategies targeting monocytes to treat RA disease. Here, we show that targeting CXCR4 with  small amino compound such as the histamine analog clobenpropit (CB) inhibits spontaneous  and induced-production of a set of key inflammatory cytokines by monocytes isolated from  blood and synovial fluids of JIA patients. Moreover, daily intraperitoneal CB treatment of  arthritic mice results in significant decrease in circulating inflammatory cytokine levels,  immune cell infiltrates, joints erosion, and bone resorption leading to reduction of disease  progression. These overall data show that targeting CXCR4 with CB-like molecules may  represent a promising therapeutic option for chronic and viral-induced inflammatory diseases.

overall_design: Total of 6 Samples: Three from Healthy Donors and Three from OligoAJI Patients

first: Nikaia

last: Smith

first: Jean-Philippe

last: Herbeuval


title: Polarized microtubule remodeling transforms the morphology of reactive microglia cells and drives cytokine release

city: South San Francisco

first: Mitchell

last: Rezzonico

state: CA

address: 1 DNA Way

institute: Genentech

summary: Microglial reactivity is a pathological hallmark in many neurodegenerative diseases. During stimulation, microglia undergo complex morphological changes, including loss of their characteristic ramified morphology, which is routinely used to detect and quantify inflammation in the brain. However, the underlying molecular mechanisms and the relation between microglial morphology and their pathophysiological function are unknown. Here, proteomic profiling of lipopolysaccharide (LPS)-reactive microglia identified microtubule remodeling pathways as an early factor that drives the morphological change and subsequently controls cytokine responses.  We found that LPS-reactive microglia reorganize their microtubules to form a stable and centrosomally anchored array to facilitate efficient cytokine trafficking and release. We identified cyclin-dependent kinase 1 (Cdk-1) as a critical upstream regulator of microtubule remodeling and morphological change in-vitro and in-situ. Cdk-1 inhibition also rescued tau and amyloid fibril-induced microglia changes. These results demonstrate a critical role for microtubule dynamics and reorganization in microglial reactivity and modulating cytokine-mediated inflammatory responses.

overall_design: To investigate the effect of LPS on microglia, primary murine microglia were treated with LPS (5 µg/ml) or vehicle control (n = 4 per condition). 24-hours later, total RNA was isolated and subjected to RNA-sequencing.

first: Max

last: Adrian

first: Casper

last: Hoogenraad


title: Multi-species atlas resolves an axolotl limb development and regeneration paradox

status: Public on Sep 20 2023

city: Dresden

first: Tatiana

last: Sandoval-Guzmán

state: Saxony

address: Fetscherstrasse 74

department: Department of Internal Medicine III, Center for Healthy Aging, Medical Faculty Carl Gustav Carus

institute: Technische Universität Dresden

summary: Humans and other tetrapods are considered to require apical-ectodermal-ridge, AER, cells for limb development, and AER-like cells are suggested to be re-formed to initiate limb regeneration. Paradoxically, the presence of AER in the axolotl, the primary regeneration model organism, remains controversial. Here, by leveraging a single-cell transcriptomics-based multi-species atlas, composed of axolotl, human, mouse, chicken, and frog cells, we first established that axolotls contain cells with AER characteristics. Surprisingly, further analyses and spatial transcriptomics revealed that axolotl limbs do not fully re-form AER cells during regeneration. Moreover, the axolotl mesoderm displays part of the AER machinery, revealing a novel program for limb (re)growth. These results clarify the debate about the axolotl AER and the extent to which the limb developmental program is recapitulated during regeneration.

overall_design: Sample collected from a 8.5 cm axolotl amputated in the calcified region of the lower arm after 5 days of regeneration (5 dpa)

first: Rita

last: Aires

first: Jixing

last: Zhong

first: Georgios

last: Tsissios

first: Evangelia

last: Skoufa

first: Kerstin

last: Brandt

first: Can

last: Aztekin


title: Systematic phenotypic characterization and integrated analysis show heterogeneous neomorphic activities of different missense mutant p53 proteins

city: Tempe

first: Jin

last: Park

state: AZ

address: 727 E. Tyler St., Suite A220C

institute: Arizona State University

summary: The phenotypic and transcriptomic data on ten normal breast epithelial cell lines each expressing a distinct missense mutant p53 protein, and their systems-level analysis through ChIP-Seq and RNA-seq defines the molecular basis for phenotypic heterogeneity imparted by the different missense mutant p53 proteins. The focus of the study is to gain mechanistic insight on the neomorphic function of mutant p53 proteins and their manifestation into phenotypic heterogeneity in the context of a TNBC model.

overall_design: Examining the transcriptomics of 10 different p53 missense mutant protein expressing MCF10A cell lines using RNA-seq, and DNA binding and promoter occupancy of 5 p53 mutant MCF10A cell lines using ChIP-Seq. The missense mutant p53 proteins have a V5 tag at the C-term and for ChIP, the mutant p53 proteins were pulled down using the V5 antibody. For the ChIP-seq, the MCF10A cells with endogenous wild type p53 served as the negative control and the cells expressing WT p53 with the V5 tag at C-term as the positive control.

first: Anasuya

last: Pal

first: Laura

last: Gonzalez-Malerva

first: Chenxi

last: Xu

first: Seron

last: Eaton

first: Grant

last: Christensen

first: Joy

last: Blain

first: Nicholas

last: Mellor

first: Jason

last: Steel

first: Chitrak

last: Gupta

first: Ellen

last: Streitwieser

first: Abhishek

last: Singharoy

first: Joshua

last: LaBaer


title: Endogenous renal adiponectin drives gluconeogenesis through enhancing pyruvate and fatty acid utilization

first: Toshiharu

last: Onodera

address: 5323 Harry Hines Boulevard

department: Touchstone diabetes center

institute: UT Southwestern medical center

summary: Adiponectin is an important secretory protein, primarily produced in adipose tissue. However, low but detectable expression of adiponectin can be observed in cell types beyond adipocytes, particularly in kidney tubular cells, but its local renal role is unknown. We assessed the role of renal adiponectin by utilizing male doxycycline inducible kidney tubular cell-specific adiponectin overexpression or knockout mice. Kidney-specific adiponectin overexpression induces a doubling of phosphoenolpyruvate carboxylase expression and enhanced pyruvate-mediated glucose production, tricarboxylic acid cycle intermediates and an upregulation of fatty acid oxidation (FAO). The inhibition of FAO reduces the adiponectin-induced enhancement of glucose production, highlighting the role of FAO in the induction of renal gluconeogenesis. In contrast, kidney-specific adiponectin deleted mice exhibit enhanced glucose tolerance as well as a lower utilization and greater accumulation of lipid species. In summary, renal adiponectin is a potent inducer of gluconeogenesis by driving enhanced FAO in the kidney and underline the important systemic role of renal gluconeogenesis.

overall_design: Comparison of control kidney and adiponectin overexpressed kidney under normal chow diet condition.

first: Philipp

last: Scherer


title: KDM2B regulates hippocampal morphogenesis by transcriptionally silencing Wnt signaling in neural progenitors (RNA-Seq)

first: zhao

last: chen

overall_design: Comparative gene expression profiling analysis of RNA-seq data for P0 mouse cortex of control and Kdm2b-cKO(Emx1-?CxxC), and P0 mouse hippocampi of control and Rnf2-cKO.

first: Bo

last: Zhang

first: Chen

last: Zhao

first: Wenchen

last: Shen

first: Wei

last: Li

first: Yue

last: Zheng

first: Xiangfei

last: Kong

first: Junbao

last: Wang

first: Xudong

last: Wu

first: Ying

last: Liu

first: Yan

last: Zhou


title: Transcriptional linkage analysis with in vivo AAV-Perturb-seq

city: Basel

first: Antonio

middle: Jose

last: Santinha

address: Mattenstrasse 26

department: D-BSSE

institute: ETH Zurich

summary: The ever-growing compendium of genetic variants associated with human pathologies demands novel methods to study genotype-phenotype relationships in complex tissues in high-throughput. Here, we introduce AAV-mediated direct in vivo single-cell CRISPR screens, termed AAV-Perturb-seq, a tunable and broadly applicable method for high-throughput and high-resolution phenotyping of genetic perturbations in vivo. We applied AAV-Perturb-seq using gene editing and transcriptional inhibition to systematically dissect the phenotypic landscape underlying 22q11.2 deletion syndrome genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and novel pathways orchestrating neuronal functions in vivo that explain approximately 40% of the transcriptional changes observed in a 22q11.2 deletion mouse model. Our findings suggest that the 22q11.2 deletion syndrome transcriptional phenotype found in mature neurons may in part be due to the broad dysregulation of a class of disease susceptibility genes important for dysfunctional RNA processing and synaptic function. Our work establishes a flexible and scalable direct in vivo method to facilitate causal understanding of biological and disease mechanisms with potential applications to identify genetic interventions and therapeutic targets for treating disease.

overall_design: Pooled screen data: A single dose of 5.0E+9 AAV particles carrying gRNAs to target 22q11.2 locus genes were injected intravenously into 8 weeks old male LSL-Cas9 mice. Animals were kept for four weeks under standard conditions before tissue extraction and processing. Arrayed confirmation experiments and CRISPRi experiment: Virus carrying gRNAs to target specific genes were individually prepared. Animals were split into cages accordingly to their experimental groups before tail vein injection of 5.0E+9 viral particles carrying unique gRNAs. Animals were kept for four weeks under standard conditions before tissue extraction and processing. For all experiments, animals were intravenously injected with a lethal dose of pentobarbital before transcardial perfusion with 15 mL of ice-cold PBS followed by 15 mL of ice-cold artificial cerebrospinal fluid. The brain was removed, sliced into 1 mm slices, and the region of interest was manually dissected. Nuclei isolation was performed with mechanical and chemical tissue dissociation procedures followed by FANS to isolate infected nuclei (pooled screen, arrayed and CRISPRi experiments) or purify nuclei from debris (LgDel model). Single cell libraries were generated using a modified version of the Chromium Single Cell 5ʹ Reagent Kit v1 (10x Genomics) to capture gene expression and gRNA information from each nucleus. Gene expression and gRNA libraries were sequenced with an Illumina NovaSeq 100 cycle kit.

first: Esther

last: Klingler

first: Maria

last: Kuhn

first: Rick

last: Farouni

first: Sandra

last: Lagler

first: Georgios

last: Kalamakis

first: Ulrike

last: Lischetti

first: Denis

last: Jabaudon

first: Randall

last: Platt


title: Alternative promoters and splicing create multiple functionally distinct isoforms of estrogen receptor alpha in breast cancer and healthy tissues

city: Lund

first: Helena

last: Persson

address: Scheelevagen 2

department: Dept of Clinical Sciences, Oncology

country: Sweden

institute: Lund University

summary: Estrogen receptor alpha (ER) is involved in cell growth and proliferation and functions as a transcription factor, a transcriptional coregulator, and in cytoplasmic signalling. The diverse roles of the ER include effects on e.g. bone, endometrium, ovaries, and mammary epithelium. The ER is a key biomarker in clinical management of breast cancer, where it is used as a prognostic and treatment-predictive factor, and a therapeutical target. Several functionally distinct ER isoforms have been described, but current transcript annotation in public databases is incomplete and inconsistent. We analysed short- and long-read RNA sequencing data from breast tumours, breast cancer cell lines, and normal tissues to create a comprehensive annotation of ER transcripts and combined it with experimental studies of full-length protein and six alternative isoforms to compare transcription factor activity, subcellular localisation, and response to the ER-targeting drug fulvestrant. Antibodies differ in ability to detect alternative isoforms, which raises concerns for the interpretation of ER-status in routine pathology. Future work should investigate the effects of alternative isoforms on patient survival and therapy response. An accurate annotation of ER isoforms will aid in interpretation of clinical data and inform functional studies to improve our understanding of the ER in health and disease.

overall_design: Isoform detection through long-read sequencing (Oxford Nanopore Technologies) of ESR1 RT-PCR amplicon pools from breast cancer cell lines (BT-474, MCF7 and T47D) and 12 commercially available healthy human tissues (adipose, bladder, breast, cervix, kidney, liver, lung, ovary, prostate, skeletal muscle, spleen, testis).

first: Carlos

last: Balcazar Lopez

first: Juliane

last: Albrecht

first: Völundur

last: Hafstað

first: Cornelia

last: Börjesson Freitag

first: Johan

last: Vallon-Christersson

first: Jari

last: Häkkinen


title: Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease

first: Jo Huiqing

last: Zhou

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

overall_design: Refer to individual Series


title: Fibroblast growth factor 18 stimulates the proliferation of hepatic stellate cells, thereby inducing liver fibrosis

city: Tokyo

first: Takao

last: Seki

state: Ota-ku

address: 5-21-16 Omori-Nishi

department: Department of Biochemistry

institute: Toho University School of Medicine

summary:
Purpose: Analyze the transcriptomic differences between hepatic non parenchimal cells (HNPCs) of Non-Tg and Fgf18 Tg mice
Methods: Fresh isolated HNPCs suspensions of Non-Tg and Fgf18 Tg were loaded on the BD RhapsodyTM Single-Cell Analysis System
Results: We revealed that CD31-CD34+ cells comprise Lrat+ hepatic stellate cells (HSCs), Acta2+ myofibroblasts, and Thy1+ portal fibroblasts.

first: Shigeyuki

last: Shichino

first: Hiroyasu

last: Nakano


title: Spinal cord regeneration at single-cell resolution restores walking after paralysis

city: Vancouver

first: Jordan

last: Squair

state: BC

address: 818 West 10th Avenue

institute: University of British Columbia

summary: Although axon regeneration can now be induced experimentally across anatomically complete spinal cord injury (SCI), restoring meaningful function after such injuries has been elusive. This failure contrasts with the spontaneous, naturally occuring repair that restores walking after severe but incomplete SCI. Here, we applied projection-specific and comparative single-nucleus RNA sequencing to uncover the transcriptional phenotype and connectome of neuronal subpopulations involved in natural spinal cord repair. We identified a molecularly defined population of excitatory projection neurons in the thoracic spinal cord that extend axons to the lumbar spinal cord where walking execution centers reside. We show that regrowing axons from these specific neurons across anatomically complete SCI and guiding them to reconnect with their appropriate target region in the lumbar spinal cord restores walking in mice. These results demonstrate that mechanism-based repair strategies that recapitulate the natural topology of molecularly defined neuronal subpopulations can restore neurological functions. Expanding this principle to different classes of neurons across the central nervous system may unlock the framework to achieve complete repair of the injured spinal cord.

overall_design: We used single-nucleus RNA sequencing to profile the low thoracic spinal cord of mice, we devised three experiments to identify  and target the specific neuron populations involved in natural recovery following spinal cord injury. Experiment 1: projection specific sequencing of neurons in the low thoracic spinal cord with projections to walking execution centres in the lumbar spinal cord. Experiment 2: comparative snRNAseq experiment of neurons in the low thoracic spinal cord from uninjured mice and mice that underwent natural spinal cord repair. Experiment 3: projection specific sequencing of regenerating neurons.

first: Marco

last: Milano

first: Matthieu

last: Gautier

first: Alexandra

last: de Coucy

first: Michael

last: Skinnider

first: Nicholas

last: James

first: Newton

last: Cho

first: Anna

last: Lasne

first: Claudia

last: Kathe

first: Thomas

last: Hutson

first: Steven

last: Ceto

first: Laetitia

last: Baud

first: Katia

last: Galan

first: Quentin

last: Barraud

first: Timothy

first: Bernard

last: Schneider

first: Zhigang

last: He

middle: V

last: Sofroniew

first: Gregoire

last: Courtine

first: Mark

last: Anderson


title: effect of CD44 overexpression on luminal type breast cancer cell MCF7

city: shanghai

first: Yaqi

last: Qiu

state: shanghai

address: 上海市徐汇区宜山路600号上海市第六人民医院

institute: shanghaijiaotong university

summary: To further identify the downstream target of CD44-dependent lamellipodia formation during luminal type BrCa progression, we firstly established a stable cell line by transfection of CD44 cDNA into MCF7 cells which expresses low level of CD44. Then we used RNA-sequencing (RNA-seq) to compare mRNA expression profiles between the MCF7vector and MCF7CD44 cells.

overall_design: We firstly established a stable cell line MCF7CD44 , and then we used RNA-sequencing (RNA-seq) to compare mRNA expression profiles between the MCF7vector and MCF7CD44 cells. There are 3 replications for each group.

first: Hui

last: Wang

first: Yan

last: Du

first: Feng

last: Gao