title: analysis of the immune and astrocytic cell fraction 12h after cortical injury in Sema4B knockout mice

city: Jerusalem

first: Oded

last: Behar

address: Ein Kerem. P.O. Box 12271, The Hebrew University Faculty ,, building 3, floor 2, room 3213

department: Developmental Biology and Cancer Research

country: Israel

institute: The Hebrew University

summary: After central nervous system injury, a rapid neuroinflammatory response is induced. This response can be both beneficial and detrimental to neuronal survival in the first few days and increase the risk for neurodegeneration if it persists. Semaphorin4B (Sema4B), a transmembrane protein primarily expressed by cortical astrocytes, has been shown to play a role in neuronal cell death following injury. Our study shows that neuroinflammation is attenuated in Sema4B knockout mice and microglia/macrophage activation is reduced after cortical stab wound injury. In vitro, recombinant Sema4B enhances the activation of microglia following injury, suggesting astrocytic Sema4B functions as a ligand. Moreover, injury-induced activation of microglia is attenuated in the presence of Sema4B knockout astrocytes compared to heterozygous astrocytes. In vitro, experiments indicate Plexin-B2 is the Sema4B receptor on microglia. Consistent with this, microglia-specific Plexin-B2 knockout mice, similar to Sema4B knockout mice, also show a reduction in microglial activation after cortical injury. Finally, in Sema4B/Plexin-B2 double heterozygous mice, microglial activation is also reduced after injury, thus supporting the idea that both Sema4B and Plexin-B2 are part of the same signaling pathway. Taken together, we propose a model in which following injury, astrocytic Sema4B enhances the pro-inflammatory response of microglia/macrophages via Plexin-B2, leading to increased neuroinflammation.

overall_design: To test the changes in gene expression systematically and within specific cell types, Sema4B+/- and Sema4B -/- mice inflicted cortical injury. A 3x3mm square around the injured site was dissected out 12h after injury and dissociated into cells. Using immunopanning astrocytes (collecting the cells adhering to the Acsa-2 plate isolate) and immune cells (collecting cells adhering to a secondary-only plate and CD45 plate). The isolated cell populations were used to extract RNA and perform RNAseq.

first: Natania

last: Casden

first: Vitali

last: Belzer


title: RNA-sequence analysis in periferal blood before and after direct antiviral agent treatment  for chronic hepatitis C

status: Public on Feb 27 2024

city: Kanazawa

first: Kazunori

last: Kawaguchi

address: 13-1 Takara-Machi

department: Gastroenterology

country: Japan

institute: Kanazawa University

type: Expression profiling by high throughput sequencing

summary: RNA-sequencing analysis exhibited different patterns of gene expression between SVR and non-SVR groups.

overall_design: We isolated total RNA from peripheral blood mononuclear cells from 25 sustained virological response (SVR) cases before and after ombitasvir/paritaprevir/ritonavir treatment and 4 non-SVR cases.

first: Eishiro

last: Mizukoshi

first: Miyabi

last: Miura

first: Michiko

last: Nishino

first: Tetsuro

last: Shimakami

first: Masao

last: Honda

first: Taro

last: Yamashita


title: Causative mechanisms and clinical impact of immunoglobulin deficiencies in ataxia telangiectasia

status: Public on Feb 29 2024

city: Huddinge

first: Likun

last: Du

address: Halsovagen 7C, Floor 7, NEO, group PHA

department: BioNut

country: Sweden

institute: Karolinska Institutet

summary: To study the details of class switch recombination in patients with AT, we examined the switch junctions in patients’ B-cells as well as matched controls by using linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) methods.

overall_design: Switch recombination fragments in IgH region from B cells were linear ampified, enriched, and sequenced by Miseq. Switch junctions were then analyzed to explore the role of DNA repair during the CSR in AT patients.

first: Sanami

last: Takada

first: Qiang

last: Pan-Hammarstrom

first: Mirjam

last: van der Burg


title: Circadian rhythm-dependent programs control inflammatory responses and drug metabolism in primary human hepatocytes I.

status: Public on Feb 28 2024

city: Cambridge

first: Charles

middle: Arthur

last: Whittaker

state: MA

country: USA

institute: Koch Institute

summary: Various aspects of physiology and cell function present self-sustained ~24h variations termed circadian rhythms, enabling anticipation and adaptation to the 24-h cycle produced by the Earth’s rotation. The circadian clock system consists of a central clock located in the suprachiasmatic nucleus in the hypothalamus, and peripheral clocks present in distinct tissues. In particular, peripheral clocks in the liver have fundamental roles in maintaining liver homeostasis. Here, we established and characterized a human hepatocyte in vitro system in which human primary hepatocytes displayed self-sustained oscillations independently of external cues a distinguishing feature of circadian clocks. We then used this experimental system to study the impact of circadian oscillations in human liver pathophysiology. By generating hepatocyte circadian transcriptomes, we demonstrated that the transcriptional state of cultured human hepatocytes is dynamic across 24h. Analyses of gene expression data identified a set of cycling genes, including a subset of well-established ‘core clock genes’ and oscillating genes related to inflammation, drug metabolism, and energy homeostasis. We documented a circadian-dependent induction of the pro-inflammatory cytokines IL-1b, IL-6, TNF-a CXCLl1 and CXCL10 triggered by LPS or IFN-b in human hepatocytes. We also demonstrated circadian fluctuations of a subset of drug metabolism enzymes, which regulate drug pharmacokinetics. Among these enzymes, we identified CYP3A4, as one displaying circadian fluctuations at activity levels and a time-dependent induction with rifampin. Finally, we showed the impact of applying chronopharmacotherapy by designing a treatment protocol to minimize atorvastatin and acetaminophen induced hepatotoxicity. Collectively, our findings document that circadian time can influence the magnitude of the inflammatory response in the liver and the efficacy, toxicity, and/or the therapeutic index of drugs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Two or three replicates each of primary human hepatocytes cultured with supporting J2-3T3 fibroblasts collected collected at three hour intervals over 48 hours.

first: Niketa

last: Nerurkar

first: Sandra

last: March

middle: A

first: Sangeeta

last: Bhatia


title: Translation-dependent and independent mRNA decay occur through mutually exclusive pathways that are defined by ribosome density during T Cell activation

city: Evry-Courcouronnes

first: Emmanuel

last: Labaronne

address: Genopôle Campus  4 rue Pierre Fontaine - Pépinière du Génopôle

country: France

institute: ADLIN science

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

overall_design: Refer to individual Series


title: Circadian rhythm-dependent programs control inflammatory responses and drug metabolism in primary human hepatocytes II.

overall_design: Two replicates each of primary human hepatocytes cultured with supporting J2-3T3 fibroblasts collected treated with siRNA targeting BmalI (ARNTL) or a non-targeting control for 24 hours and collected at 36, 42,48 or 54 hours


title: Gene expression profile at single level in tumor draining lymph nodes in wild type (DHO001) versus ATG5LEC-KO mice (DHO002)

city: Zwijnaarde

first: VIB

last: Single Cell Core

address: Technologiepark-Zwijnaarde 71

department: VIB Single cell core

country: Belgium

institute: VIB

summary: We used scSEQ analysis to assess differences in the local tumor draining lymph node microenviroment, both on endothelial cells and immune cells

overall_design: 13 lymph nodes from wild type mice and 9 from ATG5LEC-KO mice were isolated and digested, endothelial cells (viable CD31+ Ter1119- CD45-) and immune cells (viable CD45+ Ter119- CD31-) were sorted and pooled for further sequencing analysis

first: Diede

last: Houbaert

first: Patrizia

last: Agostinis

first: Jana

last: Roels

first: Niels

last: Vandamme


title: Catalytic residues of microRNA Argonautes play a modest role in microRNA star strand destabilization in C. elegans

status: Public on Feb 26 2024

city: Bethesda

first: Katherine

last: McJunkin

state: MD

address: 50 South Drive, Building 50 Room 3148

institute: National Institutes of Health

summary: Many Argonaute proteins can cleave RNA (“slicing”) as part of the microRNA-induced silencing complex (miRISC), even though miRNA-mediated target repression is generally independent of target cleavage. Here we use C. elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with normal development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on slicing for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role may contribute to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.

overall_design: To test the role of miRISC-mediated slicing in invertebrates, we used CRISPR/Cas9-mediated genome editing to mutate the DEDH catalytic tetrad to AEDH in the major C. elegans microRNA Argonautes, ALG-1 and ALG-2 (alg-1(AEDH); alg-2(AEDH)). We performed total RNAseq from these mutants or wild type in young adults (4 replicates), L4s (3 replicates), and mixed stage embryos (4 replicates).

first: Kasuen

last: Kotagama

first: Acadia

middle: L

last: Grimme

first: Leah

last: Braviner

first: Bing

last: Yang

first: Rima

middle: M

last: Sakhawala

first: Guoyun

last: Yu

first: Lars

middle: K

last: Benner

first: Leemor

last: Joshua-Tor


title: A New Approach Methods Strategy for Risk-based Prioritization of PFAS [cardiomyocytes]

city: College Station

first: Ivan

last: Rusyn

state: TX

address: 4458 TAMU

department: Department of Veterinary Integrative Biosciences

institute: Texas A&M University

summary: Per- and polyfluoroalkyl substances (PFAS) are a very large (thousands of chemicals) category; these substances have important industrial and consumer product applications. PFAS are highly persistent in the environment, and some are known to pose human health hazard. Regulatory agencies worldwide are considering restrictions and outright bans of PFAS; however, little data exists to make informed decisions. Therefore, a prioritization strategy is urgently needed for evaluation of potential hazards of PFAS. Structure-based grouping could expedite selection of PFAS for testing; still, the hypothesis that structure-effect relationships exist requires confirmation. We tested 26 structurally diverse PFAS from 8 groups in two human cell types from organs that are thought to be targets for PFAS. We used human induced pluripotent stem cell-derived hepatocytes and cardiomyocytes and tested concentration-response effects on both cell function and gene expression. Few phenotypic effects were observed in hepatocytes, but negative chronotropy was observed for 8 of 26 PFAS. Substance- and cell-dependent transcriptomic changes were more pronounced; however, little evidence of group-specific effects was observed. In hepatocytes, we found up-regulation of stress-related and extracellular matrix organization pathways, and down-regulation of fat metabolism. In cardiomyocytes, contractility-related pathways were most affected. Using these data, we derived phenotypic and transcriptomic point of departure estimates and compared them to predicted PFAS exposures. The conservative estimates for bioactivity and exposure were used to derive margin-of-exposure (MOE) for each PFAS. We found that most (23 of 26) PFAS had MOE>1. Overall, our data suggests that chemical structure-based grouping of PFAS may not be an appropriate strategy to predict their biological effects. This means that testing of the individual PFAS would be needed for confident decision-making. Our proposed strategy of using two human cell types and considering both phenotypic and transcriptomic effects, combined with dose-response analysis, may be used for prioritization of PFAS.

overall_design: Comparative gene expression profiling analysis of TempO-Seq data for iPSC-cardiomyocytes for multiple PFAS exposure. Dose-response effects were analyzed.

first: Han-Hsuan

middle: D

last: Tsai

first: Lucie

middle: C

last: Ford

first: Zunwei

last: Chen

first: Sarah

last: Burnett

first: Allison

middle: N

last: Dickey

first: Fred

last: Wright


title: Catalytic residues of microRNA Argonautes play a modest role in microRNA star strand destabilization in C. elegans [miRNA]

type: Non-coding RNA profiling by high throughput sequencing

overall_design: To test the role of miRISC-mediated slicing in invertebrates, we used CRISPR/Cas9-mediated genome editing to mutate the DEDH catalytic tetrad to AEDH in the major C. elegans microRNA Argonautes, ALG-1 and ALG-2 (alg-1(AEDH); alg-2(AEDH)). To determine the impact of mutating Argonautes’ catalytic residues on C. elegans microRNAs, we profiled small RNAs from embryos, L4 stage larvae and young adults. We also profiled small RNAs from embryos that were single mutants, with catalytic residues mutated in either ALG-1 or ALG-2. To test whether Target-directed microRNA degradation contributed to downregulation of select miRNAs in alg-1(AEDH); alg-2(AEDH), we added mutations to this background that theoretically inactivate all TDMD (ebax-1(null)) or TDMD of a mir-243 alone (Y47(mir-243bs_del)) and sequenced small RNAs in young adults.


title: A New Approach Methods Strategy for Risk-based Prioritization of PFAS [hepatocytes]

overall_design: Comparative gene expression profiling analysis of TempO-Seq data for iPSC-hepatocytes for multiple PFAS exposure. Dose-response effects were analyzed.


title: TET1 catalytic activity is required for reprogrammign of imprinting control regions and the patterning of sperm-specific hypomethylated regions [Mouse Methylation285 12v1-0 BeadChip]

city: Philadelphia

first: Rexxi

middle: Diptya

last: Prasasya

state: PA

address: 3400 Civic Center Blvd

department: Cell and Developmental Biology

institute: University of Pennsylvania

type: Methylation profiling by genome tiling array

summary: DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET1 activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls at 5hmC (Tet1-V). This experiment was done to conduct genome wide DNA methylation profiling of catalytically inactive Tet1HxD/HxD sperm, 5hmC stalling Tet1V/V sperm, KO Tet1-/- sperm, and WT sperm. Sperm samples were collected from adult males (>10 week).

overall_design: Bisulfite converted DNA from 36 mouse sperm samples of 4 genotypes  (Tet1-HxD = 8, Tet1-KO = 10, Tet1-V - 10, WT = 8) were hybridized to the Illumina Mouse Methylation285 12v1-0 BeadChip Array

first: R

first: B

last: Caldwell

first: Z

last: Liu

first: S

last: Wu

last: Cincotta

first: J

last: Fowler

first: D

middle: J

last: Laird

last: Kohli

first: M

middle: S

last: Bartolomei


title: CHD7 and SOX2 Act in a Common Gene Regulatory Network During Mammalian Semicircular Canal and Cochlear Development

city: Ann Arbor

first: Donna

last: Martin

address: 1150 Medical Center Dr

institute: University of Michigan

type: Genome binding/occupancy profiling by high throughput sequencing


title: The complex etiology of autism spectrum disorder due to missense mutations of CHD8

city: Fukuoka

first: Taichi

last: Shiraishi

address: 1-1-3 Maidashi Higashiku Fukuoka

department: Medical institute of bioregulation

institute: Kyushu university

summary: CHD8 is an ATP-dependent chromatin-remodeling factor encoded by the most frequently mutated gene in individuals with autism spectrum disorder (ASD). Although many studies have examined the consequences of CHD8 haploinsufficiency in cells and mice, few have focused on missense mutations, the most common type of CHD8 alteration in ASD patients. We here characterized CHD8 missense mutations in ASD patients according to six prediction scores and experimentally examined the effects of such mutations on the biochemical activities of CHD8, neural differentiation of embryonic stem cells, and mouse behavior. Only mutations with high prediction scores gave rise to ASD-like phenotypes in mice, suggesting that not all CHD8 missense mutations detected in ASD patients are directly responsible for the development of ASD. Furthermore, we found that mutations with high scores cause ASD by mechanisms either dependent on or independent of loss of chromatin-remodeling function. Our results thus provide insight into the molecular underpinnings of ASD pathogenesis caused by missense mutations of CHD8.

overall_design: RNA-seq and ChIP-seq

first: T

first: Y

last: Katayama

last: Nishiyama

first: H

last: Shoji

last: Miyakawa

last: Mizoo

first: A

last: Matsumoto

last: Hijikata

last: Shirai

first: K

last: Mayanagi

middle: I

last: Nakayama


title: Risk-based prioritization of PFAS using phenotypic and transcriptomic data from human induced pluripotent stem cell-derived hepatocytes and cardiomyocytes

first: Ivan

last: Rusyn


title: Epigenome analysis of precursor lesions of endometrial endometrioid carcinoma.

city: Koto-ku

first: Osamu

last: Gotoh

state: Tokyo

address: Ariake 3-8-31

institute: Japanese Foundation For Cancer Research

summary: We performed genome-wide DNA methylation profiling of precursor lesions of endometrial endometrioid carcinoma tissues derived from patients in the Cancer Institute Hospital of Japanese Foundation for Cancer Research.

overall_design: DNA methylation status was analyzed using genomic DNA from each tissue on the Illumina Infinium MethylationEPIC BeadChip Arrays (n=91), according to the manufacturer’s instructions.

first: Seiichi

last: Mori

first: Yuko

last: Sugiyama


title: Effects of mTOR inhibition on the phenotypes of spleen naive CD4+ T (TN) cells.

city: Ichikawa

first: Takashi

last: Sekiya

state: Chiba

address: 1-7-1 Kohnodai

institute: National Center for Global Health and Medicine

summary: To analyze the influence of mTOR activity on TN cells, RNA-seq analysis was performed on splenic TN cells from  mice treated or not treated with the mTOR inhibitor Rapamycin.

overall_design: Comparative gene exprssion profiling analysis of RNA-seq data for total TN cells isolated from speen of mice treated or not treated with Rapamycin.

first: Sekiya

last: Takashi


title: Effect of SOCS2 R96C mutation on mouse mammary gland development and function

city: Jouy-en-Josas

first: Fabienne

last: Le Provost

address: INRAE, Domaine de Vilvert, bât. 440

department: GABI

institute: INRAE

summary: The R96C mutation in SOCS2 (suppressor of cytokine signaling 2) that leads to greater milk production but also a greater sensitivity to mastitis was identified in Lacaune sheep by Rupp et al, 2015. This mutation leads to a loss of ligand recognition for SOCS2 and thus interferes with JAK/STAT signaling pathway regulation. A mouse model carrying this mutation (SOCS2 R96C KI) was developed to study its effects on mammary gland development and lactation.

overall_design: The gene expression in the mammary gland of homozygous wild-type and R96C SOCS2 knock-in C57BL/6 mice was analyzed in order to study the effects of the R96C SOCS2 mutation on mammary gland development and function. Mammary gland tissue with the lymph node removed at two stages of development was sequenced: virgin adult mice and mice at 18 days of gestation. Gene expression data was obtained using the Sureprint G3 Mouse gene expression v2 8*60K microarray kit.

first: Elitsa

last: Ivanova

first: Jérôme

last: Lecardonnel

first: Madia

last: Charlier


title: Single-cell RNA-sequencing of stromal vascular cells (SVCs) from the epididymal fat of obese mice.

city: La Jolla

first: Theresa

last: Rohm

state: CA

address: 9500 Gilman Dr

institute: UCSD

summary: The obesity epidemic continues to worsen worldwide, driving metabolic and chronic inflammatory diseases. Thiazolidinediones (TZDs), such as rosiglitazone (Rosi), are PPARy ligands that promote anti-inflammatory changes in adipose tissue macrophages (ATMs) and cause insulin sensitization. We performed single-cell RNA-seuqnecing (scRNA-seq) to analyze the diversity and the phenotype of ATM subpopulations under Rosi and ATM-Exosome treatment.

overall_design: We performed a single-cell transcriptome analysis of stromal vascular cells of the epididymal fat isolated from obese mice treated for the last month with Rosiglitazone or with ATM-Exosomes or PBS Ctrl liposomes. Obese mice were fed with HFD for 10 weeks and were then treated for 4 weeks with Rosi-ATM-Exosomes (Rosi-ATM-Exos, n=3), HFD-ATM-Exos (n=4), or PBS Ctrl liposomes (n=3) (10^9 Exos twice a week) compared to Rosi diet (HFD containing 3 mg/kg/day Rosiglitazone, n=3).

middle: V

first: Jerrold

last: Olefsky


title: TET1 catalytic activity is required for reprogrammign of imprinting control regions and the patterning of sperm-specific hypomethylated regions

first: Rexxi

last: Prasasya


title: Impact of Hydatid Disease Antigens on the Transcriptomic Profile of Mouse Bone Marrow-Derived Mesenchymal Stem Cells

status: Public on Feb 25 2024

city: Urumqi

first: Zengru

last: Xie

state: Xinjiang

address: 137 Liyushan South Road

country: China

institute: The First Affiliated Hospital of Xinjiang Medical University

summary: This study explores the transcriptomic alterations in mouse bone marrow-derived mesenchymal stem cells (BMSCs) upon exposure to antigens from Echinococcus granulosus, the causative agent of hydatid disease. Utilizing high-throughput sequencing, we analyzed the mRNA expression profiles of BMSCs treated with excretory-secretory products (ESPs), hydatid cyst fluid (HCF), and particles from the laminated layer (pLL). Our objective was to identify differentially expressed genes (DEGs) and investigate their roles in immune response modulation and cell behavior, including cell cycle and migration. The findings aim to enhance understanding of the interactions between parasitic antigens and host stem cells, potentially revealing novel therapeutic targets for hydatid disease.

overall_design: 1. **Antigen Preparation:** ESPs, HCF, and pLL antigens are extracted from Echinococcus granulosus cysts obtained from sheep livers. 2. **BMSC Culture and Treatment:** BMSCs are isolated from C57BL/6 mice, cultured, and then treated with 2 μg/ml of each antigen type for 48 hours. 3. **RNA Extraction and Sequencing:** Total RNA is extracted from treated and control BMSCs, followed by library construction for Illumina NovaSeq sequencing. 4. **Data Analysis:** Sequencing data are analyzed to identify DEGs using Deseq2. Further bioinformatics analyses, including GO and KEGG enrichment, are performed to elucidate the biological implications of these DEGs. 5. **Validation:** Selected DEGs are validated through quantitative PCR and Western blot analysis. 6. **Functional Assays:** The effects of antigens on BMSCs are assessed, including cell cycle, apoptosis, and migration assays.


title: Transcriptional analysis of DENV-elicited inflammation following experimental human DENV-3 challenge

city: Syracuse

first: Adam

middle: Tully

last: Waickman

state: New York

address: 766 Irving Avenue

institute: SUNY Upstate Medical University

summary: The objective of this analysis was to determine the transcriptional signature associated with experimental primary DENV-3 infection in human volunteers. Nine flavivirus naive volunteers were challenged with an attenuated DENV-3 strain - CH53489 - and blood collected for RNA extraction and transcriptional analysis on or around study days 0, 6, 8, 10, 14, and 28 post challenge using PAXgene collection tubes. Total RNA was isolated from the collection tubes and subjected to RNAseq analysis to identify genes and gene sets that were differentially expressed across the infection time course.

overall_design: Whole blood was collected on or around days 0, 6, 8, 10, 14, and 28 post DENV-3 (strain CH53489) infection 9 subjects using Paxgene tubes.

first: Stephen

last: Thomas


title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

city: Belmont

first: Joy

last: Otten

state: Massachusetts

address: 115 Mill Street

institute: McLean Hospital

summary: We used RNA sequencing of mouse hippocampal tissue two and four weeks after viral-mediated knockdown of SKA2, in order to examine if transcriptional changes might be associated with a hyperactivated secretory autophagy pathway.

overall_design: Mice received bilateral viral infusions of pAAV9-U6-shRNA[Ska2#1]-PGK-mCherry (SKA2-knockdown-AAV) or pAAV9-U6-shRNA[SCR]-PGK-mCherry (Ctrl-AAV) into the dorsal hippocampus. Mice were sacrificed two and four weeks after viral-mediated knockdown of SKA2. Brains were removed, snap-frozen in isopentane at -40 °C, and stored at -80 °C until further processing. Tissue punches of hippocampus were collected. Total RNA was isolated using the Agilent absolutely RNA miniprep kit. Samples were processed by mRNA enrichment and rRNA removal. The cDNA library was sequenced using the BGISEQ-500 platform by BGI (Hong Kong, China). Sequencing was done with a 100-bp paired-end sequencing and yielded on average 49.3 million raw reads of which on average 48.3 million (98%) is mapped to the M. musculus (GRC38/mm10) genome using STAR and Salmon was used to quantify the expression of the transcripts (Dobin et al., 2013; Patro et al., 2017). The quality control (QC) of the raw reads was carried out to determine whether the sequencing data was suitable for subsequent analysis (Ewels et al., 2016). No reads were discarded. For additional QC and downstream analyses, the bcbioRNASeq R package was used (Steinbaugh et al., 2018).

first: Torsten

last: Klengel

first: Jakob

last: Hartmann


title: Tonic TCR and IL-1beta signaling mediate phenotypic alterations of naive CD4+ T cells


title: Gene expression changes between naïve and IL-1β + TGF-β2 dual cytokine licensed equine mesenchymal stem cells

city: Raleigh

first: Lauren

middle: V.

last: Schnabel

state: NC

address: 1060 William Moore Dr

department: Clinical Sciences

institute: North Carolina State University

summary: The study objectives were to 1) determine mesenchymal stem cell (MSC) surface expression of major histocompatibility complex (MHC) class I and transcriptome-wide gene expression changes following IL-1β + TGF-β2 dual licensing and 2) evaluate if IL-1β + TGF-β2 dual licensed MSCs had greater ability to positively modulate tenocyte function compared to naïve MSCs.

overall_design:
Paired mesenchymal stem cells (MSCs) from equine donors (n=6) were grown for 72 hours in standard media as naïve MSCs or in standard media containing 2 ng/ml IL-1b and 1 ng/ml TGF-b2 as dual licensed MSCs.
Total RNA was extracted and bulk RNA sequencing was performed to determine differentially expressed genes (DEGs).

first: Drew

middle: W

last: Koch


title: Homeodomain-like domain of Plasmodium berghei HDP1 binds to TGCACA motif.

city: tsu

first: masao

last: yuda

state: mie

address: edobashi 2-174

institute: mie university

summary: To investigate the DNA-binding property of homeodomain-like domain of Plasmodium berghei HDP1, DNA immunoprecipitation followed by high-throughput sequencing (DIP-seq) analysis were performed. Recombinant homeodomain-like domain fused with glutathione S-transferase were mixed with the P. berghei genomic DNA fragmented via sonication, and protein-DNA complex was harvested using glutathione-sepharose resin. The obtained DNA fragments were sequenced via the next generation sequencing.

overall_design: DNA fragment encoding the homeodomain of HDP1 was cloned into the expression vector pGEX-6P-1 (Cytiva). Escherichia coli strain DH5α transformed with this plasmid was cultured for 12 h at 37 °C. Next, expression of the GST-fused protein was induced by adding isopropyl β-D-thiogalactopyranoside (final concentration of 200 nM) in the culture and incubating for 9 h at 25 °C. Recombinant homeodomain fused with GST was purified using glutathione-sepharose 4B resin (Cytiva), and eluted with 10 mM glutathione solution. Subsequently, the GST-fused homeodomain was incubated with P. berghei ANKA genomic DNA fragments in binding/washing buffer (10 μM ZnSO4, 2 mM MgCl2, 2 mM Tris-HCl at pH 7.4, 100 mM KCl, and 10% glycerol) for 30 min. The protein/DNA solution was further mixed with glutathione-sepharose resin and incubated for 30 min. After incubation, the resin was washed three times with binding/washing buffer, and bound protein-DNA complexes were eluted with 10 mM glutathione solution. A sequencing library was prepared from the DNA fragments and sequenced using Illumina NextSeq. Before their use for DIP, genomic DNA fragments were also sequenced as an input.

first: Tsubasa

last: Nishi

first: Izumi

last: Kaneko

first: Shiroh

last: Iwanaga

first: Masao

last: Yuda


title: Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput

first: Samuel

last: Myers

state: California

address: 9420 Athena Circle

institute: La Jolla Institute for Immunology

summary: Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe “signaling-to-transcription network” mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.

overall_design: Various phosphosite mutants were introduced into TPR Jurkat cells via in vitro transcription of sgRNA and ABE8e (base editor) complex. To produce purely edited cell populations, single cell clones were isolated from the corresponding bulk edited cell populations by diluting 0.8 cells per well in 96-well plates. Once isolated, these cells were grown until confluence, then individually genotyped through extraction of its gDNA and PCR amplification of the edited genomic region of interest. Once validated, 4-8 single cell clones were then mixed together to avoid clone-specific effects. Transcriptional profiling was completed via single-cell RNA on five selected phosphosite mutants.

first: Patrick

last: Kennedy

first: Suzie

last: Alarcón


title: A palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis

city: Shanghai

first: Zhang

state: Shanghai

address: 100 Haike Rd, Building 13 Pudong New Area

institute: Chinese Academy of Sciences

overall_design: Refer to individual Series


title: The histone acetyltransferase KAT6B is required for haematopoietic stem cell development and function

city: Parkville

first: Alexandra

last: Garnham

state: Victoria

address: 1G Royal Parade

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

summary: The histone lysine acetyltransferase KAT6B (MYST4, MORF, QKF) is the target of recurrent chromosomal translocations causing haematological malignancies with poor prognosis. Using Kat6b germline deletion and overexpression in mice, we determined the role of KAT6B in the haematopoietic system. We found that KAT6B sustained the fetal haematopoietic stem cell pool but did not affect viability or differentiation. KAT6B was essential for normal levels of histone H3 lysine 9 (H3K9) acetylation but not for a previously proposed target, H3K23. Compound heterozygosity of Kat6b and the closely related gene, Kat6a, abolished haematopoietic reconstitution after transplantation. KAT6B and KAT6A cooperatively promoted transcription of genes regulating haematopoiesis, including the Hoxa cluster, Pbx1, Meis1, Gata family, Erg and Flt3. In conclusion, we identified the haematopoietic processes requiring Kat6b and showed that KAT6B and KAT6A synergistically promoted HSC development, function and transcription. Our findings are pertinent to current clinical trials testing KAT6A/B inhibitors as cancer therapeutics.

overall_design: Hematopoietic stem and multipotent progenitor cells - lineage marker negative, SCA1 positive, c-KIT positive (LSK) cells; were isolated by FACS from gestational day 14.5 fetal mouse livers. Mice were either C57Bl6 or FvbxBalbC background. Of those from C57Bl6, there were n=4 mice of each genotype: wild-type (WT), Kat6b-/- null (DEC/DEC), Kat6a-/+ single heterozygous (CFD/+), and Kat6a-/+ Kat6b-/+ double heterozygous (CFD/+ DEC/+). For those from FvBxBalbC, there were n=4 mice of WT and Kat6b overexpressing Transgenic genotypes. Differential expression analyses between genotype groups were performed independently for each mouse background.

first: Nishika

last: Ranathunga

first: Anne

last: Voss

first: Tim

first: Maria

last: Bergamasco


title: A palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis [II]

summary: Gasdermin D (GSDMD) is the executioner of pyroptosis, which is important for host defense against pathogen infection. After activation, caspase-mediated cleavage of GSDMD liberates an N-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, leading to cell death and subsequent release of proinflammatory cytokines. How this process is spatiotemporally controlled to promote pyroptosis in cells has been a fundamental, unaddressed question. Here, we identify GSDMD as a substrate for reversible S-palmitoylation on cysteine 192 (Cys192) in response to lipopolysaccharide (LPS) stimulation. We found that the palmitoyl acyltransferase DHHC7palmitoylates GSDMD to direct its cleavage by caspases in pyroptosis by promoting the interaction of GSDMD and caspases. We further show that after GSDMD cleavage, palmitoylation of GSDMD-NTpromotes its plasma membrane translocation and binding, and then acyl protein thioesterase 2 (APT2) depalmitoylates GSDMD-NT to unmask the Cys192 residue to promote oxidation-mediated oligomerization and pyroptosis. Perturbation of either palmitoylation or depalmitoylation suppresses pyroptosis, extends the survival of mice from LPS-induced lethal septic shock and sensitizes mice to bacterial infection. Thus. our findings reveal a model through which a palmitoylation-depalmitoylationrelay spatially and temporally controls GSDMD activation in pyroptosis.

overall_design: mRNA profiles of primary bone marrow derived macrophages of wt, Dhhc7+/+, Dhhc7-/- , Apt2+/+,  Apt2-/- mice treated with LFn-Rod and 2-bromopalmitate (2-BP).

last: Zhang

first: Yuanxin

last: Yang

first: Daichao

last: Xu


title: Bulk RNA-Seq datasets supporting a common precursor expression program leading to brain pericyte differentiation from neural crest and mesoderm

city: Calgary

first: Jeff

last: Biernaskie

state: Alberta

address: 3330 Hospital Drive NW

department: Comparative Biology and Experimental Medicine

country: Canada

institute: University of Calgary

summary: Brain pericytes are critical for regulating endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. How the developmental acquisition of pericytes to naked endothelium is regulated, is largely unknown, but is relevant to disorders where vessels are poorly stabilized. Although pericytes are derived from neural crest and mesoderm, brain pericytes from both origins are currently indistinguishable; the genetic pathways leading to a convergent pericyte phenotype are unknown. We show here that a precursor population expressing the transcription factor nkx3.1 with origins in both NCC and mesoderm, develops into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1, foxf2a, and cxcl12b -expressing pericyte precursor population is present around the cxcr4 expressing basilar artery, and that these cells later spread throughout the brain. Cxcl12b- Cxcr4 signaling is required for pericyte attachment and differentiation but not for later pericyte development. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number. Thus, we have defined an undescribed population of pericyte precursors and identified genes critical for their differentiation.

overall_design: 30 hpf nkx3.1ca116 mutant and wild type embryos were collected and RNA prepared using Trizol (ThermoFisher, Waltham MA). Libraries were prepared from total mRNA per sample using the Illumina Ultra II directional RNA library prep (SanDiego, CA), and sequenced via the NovaSeq SP 100 cycle v 1.5 sequencing run with 33 million reads per sample. 4 replicates were sequenced per condition.

first: Suchit

last: Ahuja

first: Cynthia

last: Adjekukor

first: Qing

last: Li

first: Katrinka

last: Kocha

first: Nicole

last: Rosin

first: Elodie

last: Labit

first: Sarthak

last: Sinha

first: Ankita

last: Narang

first: Quan

last: Long

first: Peng

last: Huang

first: Sarah

last: Childs


title: A palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis [I]

first: Zhang

overall_design: mRNA profiles of primary bone marrow derived macrophages, liver, spleen of wt, Dhhc7+/+, Dhhc7-/- ,Apt2+/+,  Apt2-/- mice treated with lipopolysaccharide (LPS) or 2-bromopalmitate (2-BP).


title: Bone Marrow Stromal Cell Induced Chromatin Remodeling and Associated Transcriptional Changes in Multiple Myeloma

city: Boston

first: Mehmet

middle: K.

last: Samur

address: 440 Brookline Avenue

department: Department of Medical Oncology

institute: Dana-Farber Cancer Institute

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


title: Bone Marrow Stromal Cell Induced Chromatin Remodeling and Associated Transcriptional Changes in Multiple Myeloma [RNA-Seq]

summary: Differential gene expression analysis of three multiple myeloma cell lines without stromal co-culture (control) with stromal co-culture (condition) after 72 hours of exposure.

overall_design: Differential gene expression analysis by RNA-seq (whole transcriptome) for INA6, MM1S, and RPMI multiple myeloma cell lines.

first: Raphael

middle: E

last: Szalat

first: Nikhil

last: Munshi


title: A common precursor expression program leading to brain pericyte differentiation from neural crest and mesoderm

summary:
Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

This SuperSeries is composed of the SubSeries listed below.


title: scRNAseq datasets supporting  a common precursor expression program leading to brain pericyte differentiation from neural crest and mesoderm

overall_design: Embryos expressing the nkx3.1NTR-mcherry transgene were dissociated from wildtype 30 hpf zebrafish embryos and mCherry-positive FACs sorted cells were subjected to single cell RNA sequencing. Two replicate libraries were generated from FACS-sorted nkx3.1 expressing cells . To examine molecular changes within specific cell compartments, we isolated single-cells from both biopsies and processed according to 10X Genomics Chromium Single Cell 3’ Reagent Guidelines v3 Chemistry as per the manufacturer’s protocol. In brief, single cells were sorted based on forward versus side scatter gating into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10x GemCodeTM Technology. Next-Generation Sequencing was performed using the Illumina NovaSeq S2 Flow cells. All raw FASTQs were aligned to the human reference genome generated using cellranger mkref pipeline. The resulting gene barcode matrix was processed using Seurat R toolkit.


title: Dynamic regulation of miRNA accumulation during C. elegans larval development [miRNA-Seq]

status: Public on Feb 24 2024

city: Basel

first: Dimos

last: Gaidatzis

address: Maulbeerstrasse 66

department: Computational Biology

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

summary: Here, we profile miRNA expression at high temporal resolution (1 hr interval)  during C. elegans postembryonic development, which identifies several miRNAs with highly dynamic concentration changes

overall_design: Synchronized L1's of N2 and HW3527 (ebax-1(tm2321) IV ) mutant were plated on 10 cm plates (2000 worms/plate) and kept at 25°C for the time-course. Every hour samples were collected from 25h to 42h, extracted RNA, treated with DNAse and subjected to small-RNA sequencing

first: Smita

last: Nahar

first: Helge

last: Grosshans


title: Brown Adipose Tissue

city: Bronx

first: Jeffrey

last: Pessin

state: NY

address: 1301 Morris Park Avenue

department: Medicine and Molecular Pharmacology

institute: Albert Einstein College of Medicine


title: Cooperative role of Lymphotoxin β Receptor and Tumor Necrosis Factor Receptor p55 in murine liver regeneration (PHx Control vs Enbrel)

city: Bonn

first: Joachim

last: Schultze

state: NRW

address: Carl-Troll-Strasse 31

department: Genomics and Immunoregulation

country: Germany

institute: LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)

summary: The liver exhibits a unique capacity for regeneration in response to injury. Lymphotoxin β Receptor (LTβR), a core member of the Tumor Necrosis Factor (TNF)/TNF Receptor (TNFR) superfamily is known to play an important role in this process. However, LTβR functions in the pathophysiological alterations and molecular mechanisms of liver regeneration are so far ill-characterized. Interestingly, LTβR /  mice suffered from increased and prolonged liver tissue damage after 70 % hepatectomy (PHx), a finding accompanied by elevated alkaline phosphatase levels and deregulated bile acid (BA) homeostasis. Pronounced differences in the expression patterns of genes relevant for BA synthesis and recirculation were observed. Transcriptome analysis revealed a marked disparity in gene expression programs in LTβR /  vs. WT liver tissue, where gene ontology (GO) terms related to transcription, gene expression and metabolic pathways were over-represented in the latter. In addition, murinoglobulin 2 (Mug2), a gene product to date not implicated in liver regeneration, was identified as one of the most differentially regulated genes after PHx in WT compared to LTβR /  and TNFRp55-/- livers. LTβR and TNFRp55 share downstream signaling elements. TNFRp55 is known to also play an important role in liver regeneration after PHx. Therefore, LTβR /  mice were treated with Etanercept to create mice functionally deficient in both signaling pathways. Strikingly, the combined blockade of TNFR and LTβR signaling leads to complete failure of liver regeneration resulting in death within 24 to 48 hours after PHx. LTβR is essential for efficient liver regeneration and cooperates with TNFRp55 in this process. Differences in survival kinetics strongly suggest distinct functions for these two cytokine receptors in liver regeneration. Failure of TNFR and LTβR signaling renders liver regeneration impossible.

overall_design: 24 hrs before surgery, LTRβ-/- animals were treated with Etanercept. 12 h after PHx, mice were sacrificed and liver tissue obtained and snap-frozen in liquid nitrogen. Next, RNA was isolated from whole liver tissue after micing of liver tissues and lysis of cells in Trizol (Invitrogen). After purification, RNA was stored at -80°C before further use.

first: U

last: Sorg

first: M

last: Beyer

first: J


title: Single-Nuclei Transcriptomic Profiling of Brown Adipose Tissue with Age

summary: In our investigation of age-related changes in brown adipose tissue, we have identified two distinct populations of brown adipocytes characterized by high and low expression of uncoupling protein 1 (UCP1). We observed that with aging, the proportion of UCP1-low adipocytes increases, and these cells notably express genes associated with pyroptosis, an inflammatory form of programmed cell death.

overall_design: we employed a single-nuclei RNA sequencing (snRNA-seq) approach. Nuclei were isolated from the brown adipose tissue of wild-type (WT) mice at young (2 months), middle-aged (12 months), and old (24 months).

first: Gabrielle

last: Benitez

first: Li

last: Liu

first: Kosaku

last: Shinoda

first: Daorong

last: Feng


title: Dynamic regulation of miRNA accumulation during C. elegans larval development [smallRNA-Seq 5]

summary: Here, we profile miRNA expression at high temporal resolution (1 hr interval) from 5h to 48h at 25°C, during C. elegans postembryonic development, which identifies several miRNAs with highly dynamic concentration changes

overall_design: Synchronized L1's of N2 were plated on 10 cm plates (2000 worms/plate) and kept at 25°C for the time-course. Every hour samples were collected from 5h to 48h, extracted RNA, treated with DNAse and subjected to small-RNA sequencing

first: Gert-Jan

last: Hendriks

first: Yannick

middle: P

last: Hauser


title: Assessing the Impact of STX4 Knockdown on the Transcriptome of Brown Adipose Tissue

summary: In this study, we investigate the effects of Syntax 4 (STX4) knockdown in brown adipocytes and its role in age-related changes in brown adipose tissue (BAT). Our focus was to understand the mechanisms preceding the significant loss of BAT mass observed with aging. By comparing transcriptomic profiles before the onset of this massive tissue loss, we identified a notable increase in markers indicative of pyroptotic cell death. These findings suggest a potential link between STX4 activity, cellular death pathways, and age-related alterations in BAT, providing insights into the molecular underpinnings of BAT aging and loss.

overall_design: We isolated total RNA from the BAT of four male mice with Ucp1 promoter-driven STX4 knockdown (Ucp1STX4KO) and four male wild-type littermates, serving as controls. Following RNA extraction, these samples were submitted for high-throughput RNA sequencing to analyze differential gene expression and related transcriptomic changes.

first: Tszki

last: Wei


title: Dynamic regulation of miRNA accumulation during C. elegans larval development [smallRNA-Seq 4]

overall_design: Synchronized L1's of N2 and HW2643 (mir-235(xe181[seed_mutant])  were plated on 10 cm plates (2000 worms/plate) and kept at 25°C for the time-course. Every hour samples were collected from 18h to 30h, extracted RNA, treated with DNAse and subjected to small-RNA sequencing


title: Spatiotemporally resolved ex vivo colorectal cancer development in engineered mini-colons (RNA-Seq Gpx2 KD)

city: Salamanca

first: L. Francisco

last: Lorenzo-Martín

state: Castilla y León

address: Campus Miguel de Unamuno

country: Spain

institute: University of Salamanca (USAL) – Centro de Investigación del Cáncer (CIC)

summary: Here we developed topobiologically complex mini-colons able to undergo tumorigenesis ex vivo by integrating microfabrication, optogenetic, and tissue engineering approaches. With this system, tumorigenic transformation can be spatiotemporally controlled by directing oncogenic activation through blue-light exposure, and emerging colon tumors can be tracked in real-time with single-cell resolution for several weeks without breaking the culture. These induced mini-colons display rich intra- and inter-tumoral diversity and recapitulate key pathophysiological hallmarks displayed by colorectal tumors in vivo. By fine-tuning cell-intrinsic and  extrinsic parameters, mini-colons can be leveraged to unveil tumorigenic determinants, including dietary patterns, microbiota-derived metabolites, and pharmacological therapies.

overall_design: Gene expression profiling analysis of bulk RNA-seq data from control and Gpx2 knockdown colon organoids, both before and after blue light-mediated AKP oncogenic recombination.

first: L

middle: F

first: Tania

last: Hübscher

first: Nicolas

last: Broguiere

first: Amber

last: Bowler

first: Jakob

last: Langer

first: Lucie

last: Tillard

first: Mikhail

last: Nikolaev

first: Freddy

last: Radtke

first: Matthias

last: Lutolf


title: Dynamic regulation of miRNA accumulation during C. elegans larval development [smallRNA-Seq 2]

first: Dimos

last: Gaidatzis

overall_design: Synchronized L1's of N2 and HW3527 (ebax-1(tm2321) IV ) mutant were plated on 10 cm plates (2000 worms/plate) and kept at 25°C for the time-course. Every hour samples were collected from 18h to 31h, extracted RNA, treated with DNAse and subjected to small-RNA sequencing

first: Smita

last: Nahar

first: Helge

last: Grosshans


title: Dynamic regulation of miRNA accumulation during C. elegans larval development [smallRNA-Seq 1]

summary: Here, we profile miRNA expression at high temporal resolution (1 hr interval) from 18h to 33h at 25°C, during C. briggsae postembryonic development, which identifies several miRNAs with highly dynamic concentration changes

overall_design: Synchronized L1's of WT C. briggsae were plated on 10 cm plates (2000 worms/plate) and kept at 25°C for the time-course. Every hour samples were collected from 18h to 33h


title: Cooperative role of Lymphotoxin Beta Receptor and Tumor Necrosis Factor Receptor p55 in murine liver regeneration (PHx WT vs KO)

first: Joachim

last: Schultze

overall_design: 12 h after PHx, mice were sacrificed and liver tissue obtained and snap-frozen in liquid nitrogen. Next, RNA was isolated from whole liver tissue after micing of liver tissues and lysis of cells in Trizol (Invitrogen). After purification, RNA was stored at -80°C before further use.

first: U

last: Sorg

first: M

last: Beyer

first: J

middle: L


title: Spatiotemporally resolved ex vivo colorectal cancer development in engineered mini-colons (scRNA-Seq)

first: L. Francisco

overall_design: Single-cell RNA-seq of tumor bearing mini-colons labeled with CellTag


title: Dynamic regulation of miRNA accumulation during C. elegans larval development [RNA-Seq]

overall_design: Synchronized L1's of N2 and HW3527 (ebax-1(tm2321) IV) mutant were plated on 10 cm plates (2000 worms/plate) and kept at 25°C for the time-course. Every hour samples were collected from 18h to 31h, extracted RNA, treated with DNAse and subjected to RNA sequencing


title: Spatiotemporally resolved ex vivo colorectal cancer development in engineered mini-colons (RNA-Seq AKP lines)

overall_design: Gene expression profiling analysis of bulk RNA-seq data from colon organoid lines derived from: healthy colon (control), OptoCre-induced AKP organoids (organoid AKP), OptoCre-induced AKP mini-colons (mini-colon AKP), in vivo colon tumors (in vivo AKP).

first: L


title: Dynamic regulation of miRNA accumulation during C. elegans larval development [smallRNA-Seq 3]

overall_design: Synchronized L1's of HW1993 (xeSi312[eft-3p::luc::gfp::unc-54 3'UTR, unc-119(+)] IV)  and HW2706 (lin-28(xe192) I; xeSi312 [Peft-3::luc::gfp::unc-54 3’UTR, unc-119(+)] IV) were cultured in liquid culture (S-Basal supplemented with OP50, OD600=2.7, 1000 animals/mL) in a temperature controlled incubator at 25°C, shaking at 180 rpm.  Every hour samples were collected from 0 h to 23h, RNA was extracted and treated with DNAse, then subjected to sequencing reactions.

first: Jana

last: Brunner


title: Vesicular miRNAs as predictive biomarkers for benzo(a)pyrene's exposure

city: RENNES

first: Lydie

last: SPARFEL

address: 2 avenue du Pr Léon Bernard

department: IRSET U1085

institute: Inserm

summary: Benzo(a)pyrene is a widespread environmental pollutant associated with the development of toxic effects on human health. This work explores the potential of microRNAs extracted from extracellular vesicles from circulating blood as biomarkers of exposure to this pollutant.

overall_design: 12 independent EVs from PBMC cultures each grown in untreated (DMSO) and 2 µM B[a]P-treated conditions for 48 h, were combined in 3 equal pools (with 4 separate blood donors per pool) for each condition. A volume of 5 µl was used to isolate the small RNA fraction and processed using Qiaseq miRNA library prep kit.

first: Jérémy

last: Amossé

first: Rima

last: Souki

first: Maguy

last: El Hajjar

first: Marie

last: Marques

first: Valentine

last: Genêt

first: Alexis

last: Février

first: Morgane

last: Le Gall

first: Benjamin

last: Saintpierre

first: Franck

last: Letourneur

first: Eric

last: Le Ferrec

first: Dominique

last: Lagadic-Gossmann

first: Christine

last: Demeilliers

last: Sparfel


title: GENETIC INDEPENDENCE BETWEEN TRAITS SEPARATED BY METAMORPHOSIS IS WIDESPREAD BUT VARIES WITH BIOLOGICAL FUNCTION

city: Nouzilly

first: Julie

last: Collet

address: Centre INRAE

institute: INRAE

summary: Why is metamorphosis so pervasive? Does it facilitate the independent (micro)evolution of quantitative traits in distinct life-stages, like it does for discrete characters such as limbs and organs? We tested this hypothesis by measuring the expression of 6400 genes in 41 Drosophila melanogaster inbred lines at larval and adult stages. Only 32% of the genes showed significant genetic correlations between larval and adult expression. By contrast, 44% of the traits showed some level of independence between stages. Gene ontology terms enrichment of the functions most and least constrained among stages revealed that traits related to immunity emerged as largely correlated between larvae and adults. Direct comparisons with other datasets showed that inter-stage constraints were lower than inter-sexual or cross-environment genetic constraints. These results show that metamorphosis enables a large part of the transcriptome to evolve independently at different life-stages and identify biological functions under high and low genetic constraints.

overall_design:
DGRP lines and sample production
We used the Drosophila Genetic Reference Panel [DGRP, 18], a set of inbred lines derived from an outbred population of Drosophila melanogaster. We chose 50 lines that showed no inversion and scored across the range of starvation resistance measured in a previous study [18]. Our final analyses focussed on 41 DGRP lines after removing samples that could not be produced or failed to meet the quality control analyses criteria.
All lines were reared on cornmeal-molasses-agar medium at 25°C and 12-hour light and dark cycles. Grand-parental generation consisted of two replicate vials per line of two virgin males and two virgin females placed together for 2 days and further tipped in in another vial to lay for another two days. Egg density was manually regulated to approximate 50 eggs per vial. In the following generation, 12 virgin males and 12 virgin females aged two to six days were placed together for 24 hours to ensure mating occurred. Up to three males and three females (36 out of the 328 vials contained fewer adults as there were not enough flies available) of these 12 were subsequently placed together in a vial for 48 hours. Egg density was approximately kept to 50 per vial. We independently used the larvae and adults that emerged from those vials for RNA sampling.
Not all of lines were synchronous; in order to avoid creating among-line variance resulting from difference in development times, we did not collect all flies on the same day. During larva collections, once the first ‘wanderer’ (fully grown 3rd instar larva that stop eating and start searching for a pupation site) was observed in the vial, up to 10 larvae per vial were collected from the medium (i.e. not wanderers) and flash frozen with liquid nitrogen. When possible, a further 10 larvae were collected as a backup supply (and used in 12 of the 82 samples). The remainder of the flies were discarded (i.e. adults were never collected from a larva vial). Freezing occurred randomly during the day for the different samples, between 10am and 12am. The collected larvae were not sexed and we expect our samples to be a random mix of males and females. During the collection of adult flies, we scanned vials every day. When the first adults were observed in a vial, we removed them and collected all emerged adults within the following 48 hours. The collected adult flies were placed in fresh vials for 4 days to finish maturing and flash frozen in the same manner as were the larvae.

middle: M

first: Sabine

last: Nidelet

first: Simon

last: Fellous


title: Mutational signature-based identification of DNA repair deficient gastroesophageal adenocarcinomas for therapeutic targeting

status: Public on Feb 23 2024

first: David

last: Chen

state: MA

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana Farber Cancer Institute

summary: Homologous recombination (HR) and nucleotide excision repair (NER) are the two most frequently disabled DNA repair pathways in cancer. HR-deficient breast, ovarian, pancreatic and prostate cancers respond well to platinum chemotherapy and PARP inhibitors. However, the frequency of HR deficiency in gastric and esophageal adenocarcinoma (GEA) still lacks diagnostic and functional validation. Using whole exome and genome sequencing data, we found that a significant subset of GEA, but very few colorectal adenocarcinomas, show evidence of HR deficiency by mutational signature analysis (HRD score). High HRD gastric cancer cell lines demonstrated functional HR deficiency by RAD51 foci assay and increased sensitivity to platinum chemotherapy and PARP inhibitors. Of clinical relevance, analysis of three different GEA patient cohorts demonstrated that platinum treated HR deficient cancers had better outcomes. A gastric cancer cell line with strong sensitivity to cisplatin showed HR proficiency but exhibited NER deficiency by two photoproduct repair assays. Single-cell RNA-sequencing revealed that, in addition to inducing apoptosis, cisplatin treatment triggered ferroptosis in a NER-deficient gastric cancer, validated by intracellular GSH assay. Overall, our study provides preclinical evidence that a subset of GEAs harbor genomic features of HR and NER deficiency and may therefore benefit from platinum chemotherapy and PARP inhibitors.

overall_design: NUGC3 cells were treated with the IC50 concentration of Cisplatin, Oxaliplatin, 5-FU and DMSO for 2 days. Single-cell RNA-sequencing was used to analyze the mechanism of increased sensitivity of NER-deficient gastric cancer to cisplatin.


title: Premature skewing of T cell receptor clonality and delayed memory expansion in HIV-exposed infants

city: Stanford

first: Aaron

middle: James

last: Wilk

address: 300 Pasteur Dr Bldg S131, Grant

institute: Stanford University

summary: While preventing vertical HIV transmission has been very successful, HIV-exposed uninfected infants (iHEU) experience an elevated risk to infections compared to HIV-unexposed and uninfected infants (iHUU). Here we present a longitudinal multimodal analysis of infant immune ontogeny that highlights the impact of HIV/ARV exposure. Using mass cytometry, we show alterations in T cell memory differentiation between iHEU and iHUU being significant from week 15 of life. The altered memory T cell differentiation in iHEU was preceded by lower TCR Vβ clonotypic diversity and linked to TCR clonal depletion within the naïve T cell compartment. Compared to iHUU, iHEU had elevated CD56loCD16loPerforin+CD38+CD45RA+FcεRIγ+ NK cells at 1 month postpartum and whose abundance pre-vaccination were predictive of vaccine-induced pertussis and rotavirus antibody responses post 3 months of life. Collectively, HIV/ARV exposure disrupted the trajectory of innate and adaptive immunity from birth which may underlie relative vulnerability to infections in iHEU.

overall_design: Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples from HIV-exposed uninfected and HIV-unexposed infants. T cells were sorted into CD4 naïve, CD8 naïve, CD4 memory, and CD8 memory subsets, and RNA isolated for bulk TCR sequencing.

first: Sonwabile

last: Dzanibe

first: Catherine

middle: A

last: Blish

first: Clive

last: Gray


title: scRNA-Seq from stroma vascular fraction of abdominal and gluteofemoral subcutaneous white adipose tissue

city: orlando

first: Adeline

last: Divoux

state: FL

address: 301 E. Princeton St

department: Translational Research Institute

institute: Adventhealth

summary: SVF was isolated from ABD and GF-adipose tissue biopsies by collagenase digestion and centrifugation, from 5 healthy women (38yo ±4.4; 31.8kg/m-2 ±3.12; WHR 0.86 ±0.05). After isolation, cells were counted with a Countess II automated cell counter (Thermofisher Scientific). Single cell suspension (56K/mL) was distributed into eight wells of a 384-well source plate (Takara Bio USA, San Jose, CA) and dispensed onto a iCELL8 350v Chip (Takara Bio USA) using an iCELL8 MultiSample NanoDispenser (Takara Bio USA). We distributed the ABD and GF-SVF of each subject on the same chip. The method used was described in [6]. In brief, mRNA from single cells were isolated, converted to full-length cDNA prior to unbiased amplification of 3’ and 5’ ends using SMART-Seq® ICELL8® Application Kit (Takara Bio, USA). Multiplexed sequencing libraries were generated from cDNA using the Illumina Nextera XT protocol and 150bp paired-end sequencing was performed on an Novogene HiSeqX instrument.

overall_design: SVF was isolated from ABD and GF-adipose tissue biopsies tissue was digested with collagenase (type 1, 1 mg/ml in HBSS) for 45min at 37ºC with shaking (100 rpm). The mixture was centrifuged at 500g for 5 minutes and floating adipocytes were discarded. Cell pellets were treated with erythrocyte lysis buffer (Invitrogen) for 5 min at room temperature. After discarding the supernatant, cells were repelleted with centrifugation, resupend in PBS/EDTA and the mixture was passed through 100 and 40 micron mesh filters. After centrifugation, the cells were resuspended in PBS/10% FBS/1%EDTA and counted with a Countess II automated cell counter (Thermofisher Scientific). SVF were isolated from 5 healthy women (38yo ±4.4; 31.8kg/m-2 ±3.12; WHR 0.86 ±0.05).  Single cell suspension (56K/mL) was distributed into eight wells of a 384-well source plate (Takara Bio USA, San Jose, CA) and dispensed onto a iCELL8 350v Chip (Takara Bio USA) using an iCELL8 MultiSample NanoDispenser (Takara Bio USA). We distributed the ABD and GF-SVF of each subject on the same chip.

first: S

middle: R

last: SMITH

first: T

last: OSBORNE


title: Bone Marrow Stromal Cell Induced Chromatin Remodeling and Associated Transcriptional Changes in Multiple Myeloma [ATAC-Seq]

first: Mehmet

last: Samur

summary: Differential chromatin accessibility analysis of three multiple myeloma cell lines without stromal co-culture (control) with stromal co-culture (condition) after 72 hours of exposure.

overall_design: Differential chromatin accessibility by ATAC-seq (whole transcriptome) for INA6, MM1S, and RPMI multiple myeloma cell lines.

first: Raphael

last: Szalat

middle: K

first: Nikhil

middle: C

last: Munshi


title: TNYA0042: TN-401 dose-dependent efficacy study at about 10 weeks in Pkp2-cKO ARVC mouse model

city: South San Francisco

first: Farshad

last: Farshidfar

address: 171 Oyster Point Blvd Ste 500

institute: Tenaya Therapeutics Inc.

summary: Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Currently, there are no approved treatments that address the underlying genetic cause of this disease, representing a significant unmet need.  Mutations in Plakophilin-2 (PKP2), encoding a desmosomal protein, account for approximately 40% of ARVC cases and result in reduced gene expression. Methods: Our goal is to examine the feasibility and the efficacy of adeno-associated virus 9 (AAV9)-mediated restoration of PKP2 expression in a cardiac specific knock-out mouse model of Pkp2. Results: We show that a single dose of AAV9:PKP2 gene delivery prevents disease development before the onset of cardiomyopathy and attenuates disease progression after overt cardiomyopathy.  Restoration of PKP2 expression leads to a significant extension of lifespan by restoring cellular structures of desmosomes and gap junctions, preventing or halting decline in left ventricular ejection fraction, preventing or reversing dilation of the right ventricle, ameliorating ventricular arrhythmia event frequency and severity, and preventing adverse fibrotic remodeling.  RNA sequencing analyses show that restoration of PKP2 expression leads to highly coordinated and durable correction of PKP2-associated transcriptional networks beyond desmosomes, revealing a broad spectrum of biological perturbances behind ARVC disease etiology. Conclusions: We identify fundamental mechanisms of PKP2-associated ARVC beyond disruption of desmosome function.  The observed PKP2 dose-function relationship indicates that cardiac-selective AAV9:PKP2 gene therapy may be a promising therapeutic approach to treat ARVC patients with PKP2 mutations.

overall_design: RNA sequencing on 4 groups of C57BL6 mice with 1. wild-type background: WT_HBSS (n=8), 2. cKO_HBSS (n=8), 3. cKO_TN-401_3E13 vg/kg (n=6), 4. cKO_TN-401_6E13 vg/kg (n=6). Right ventricle (RV) and left ventricle (LV) tissues from each animal were analyzed simultaneously.

first: Iris

last: Wu

first: Aliya

last: Zeng

first: Amara

last: Greer-Short

first: Alex

middle: J

last: Aycinena

first: Tefera

last: Anley

first: Reva

last: Shenwai

first: Melissa

last: Van Pell

first: Emma

last: Xu

first: Chris

last: Reid

first: Neshel

last: Rodriguez

first: Beatriz

last: Lim

first: Tae

last: Chung

first: Cristina

last: Dee-Hoskins

first: Carolina

last: G Gutierrez

first: Jessie

last: Madariaga

first: Kevin

last: Robinson

first: Yolanda

last: Hatter

first: Renee

last: Butler

first: Stephanie

last: Steltzer

first: Jaclyn

last: Ho

first: James

last: R Priest

first: Xiaomei

last: Song

first: Kristina

last: Green

first: Kathryn

middle: N

last: Ivey

first: Timothy

last: Hoey

first: Jin

last: Yang

first: Zhihong


title: β-cell Jagged1 is sufficient but not necessary for islet Notch activity and insulin secretory defects.

city: New York

first: Utpal

last: Pajvani

address: 1150 St. Nicholas Avenue

institute: Columbia University Medical Center

summary:
Objective
Notch signaling is re-activated in  β cells from obese mice, and is causal to β cell dysfunction. Notch activity is determined in part by expression of transmembrane ligand availability in a neighboring cell. We hypothesized that β cell expression of Jagged1 determines the maladaptive Notch response and resultant β cell dysfunction in obese mice.
Methods
We assessed expression of Notch pathway components in diet-induced obese (DIO) or leptin receptor-deficient (db/db) mice, and performed single cell RNA sequencing (scRNAseq) in islets from patients with and without type 2 diabetes (T2D). We generated and performed glucose tolerance testing in inducible, β cell-specific Jagged1 gain-of- and loss-of-function mice. We also tested effects of monoclonal neutralizing antibodies to Jagged1 in glucose-stimulated insulin secretion (GSIS) assays in isolated islets.
Results
Jag1 was the only Notch ligand that tracked with increased Notch activity in DIO and db/db mice. Consistently, JAG1 tracked with Notch activity in metabolically inflexible β cells enriched in patients with T2D. Neutralizing antibodies to block Jagged1 in islets isolated from DIO and db/db mice potentiated GSIS ex vivo. To demonstrate if β cell Jagged1 is sufficient to cause glucose tolerance in vivo, we generated inducible β cell-specific Jag1 transgenic mice (β-Jag1TG), which showed impaired glucose intolerance due to reduced GSIS. However, β cell-specific Jagged1 loss-of-function (β-Jag1KO) did not protect against HFD-induced insulin secretory defects or glucose intolerance.
Conclusions
Jagged1 is increased in islets from obese mice and in patients with T2D, and neutralizing Jagged1 antibodies lead to improved GSIS, suggesting that inhibition of Jagged1-Notch signaling may have therapeutic benefit. However, genetic loss-of-function experiments suggest that β cells are not a likely source of the Jagged1 signal.

overall_design: RNA-seq of pancreatic islets from β cell Jag1-gain of function model

first: Nina

last: Suda


title: TNYA0016: Time-dependent transcriptional effects of PKP2 mRNA knockdown in wild-type human iPSC-CMs

first: Farshad

last: Farshidfar

overall_design: RNA sequencing on 2 groups of iPSC-CMs with 1. wild-type background: siNeg (n=4 at day 2, 4, 6, and 8, respectively), 2. siPKP2 (n=16 at day 2, 4, 6, and 8, respectively).

first: Iris

last: Wu

first: Aliya

last: Zeng

first: Amara

last: Greer-Short

first: Alex

last: Aycinena

first: Tefera

last: Anley

first: Reva

last: Shenwai

first: Melissa

last: Van Pell

first: Emma

last: Xu

first: Chris

last: Reid

first: Neshel

last: Rodriguez

first: Beatriz

last: Lim

first: Tae

last: Chung

first: Cristina

last: N Ivey


title: Investigation of the role of Il33 in the chronicity of the periodontitis

city: Tokyo

first: ANHAO

last: LIU

state: Tokyo

address: 1-5-45 Yushima, Bunkyo-ku

department: Department of Periodontology

institute: Tokyo Medical and Dental University

summary:
To investigate the function of different tissues in the pathogenesis, we established a modified mouse ligature-induced periodontitis model, supporting the separation of the maxillae into gingiva, peri-root, and bone tissue.
After setting the right molars as the non-ligature control side and the left molars as the ligature side, we performed gene expression profiling and gene enrichment analysis using data obtained from RNA-seq of sixteen individuals after 11 days of induction (32 samples in total). Four individuals were WT mice, and another four were Il33-deficient mice. Sample of peri-root tissues and bone tissues from these mice were compared separately to explore the role of Il33 in the chronicity of periodontitis, especially its function in periodontal ligament and bone tissue.

overall_design: Comparative gene expression profiling and gene enrichment analysis of RNA-seq data for separated maxillae samples(peri-root and bone tissue; control vs. ligature; WT vs KO).

first: Anhao

last: Liu

first: Mikihito

last: Hayashi

first: Yujin

last: Ohsugi

first: Sayaka

last: Katagiri

first: Takanori

last: Iwata

first: Tomoki

last: Nakashima


title: SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination (snRNA-Seq)

city: Saarbrücken

first: Fabian

middle: Michael

last: Kern

state: Saarland

address: Campus E2 1

department: Center for Bioinformatics

institute: Saarland University

summary: Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)—a transcription factor known to regulate expression of actin and actin regulators in other cell types—as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the fundamental role of SRF in oligodendrocyte lineage cells. Here we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in OPCs and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies a novel pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.

overall_design: To investigate the role of Srf in myelination mediated by cells from the Oligodendrocyte lineage, brain tissue and cell cultures from control and knockout mice and rats were repeatedly characterized by different sequencing types (RNA-seq, snRNA-seq, ChIP-seq).

first: Tal

last: Iram

first: Miguel

last: Garcia

first: Jeremy

last: Amand

first: Achint

last: Kaur

first: Micaiah

last: Atkins

first: Manasi

last: Iyer

first: Mable

last: Lam

first: Nicholas

last: Ambiel

first: Danielle

last: Jorgens

first: Andreas

last: Keller

first: Tony

last: Wyss-Coray

first: J

middle: B

last: Zuchero


title: PKP2 Gene Therapy Improves Heart Function and Reduces Mortality in a Pkp2-deficient Mouse Model of Arrhythmogenic Right Ventricular Cardiomyopathy

first: Farshad

last: Farshidfar

state: CA

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

overall_design: Refer to individual Series


title: Tregs in autoimmune polyendocrine syndrome type I

city: Tartu

first: Pärt

last: Peterson

address: Ravila 19-A1038

department: Department of Biomedicine

country: Estonia

institute: Institute of Biomedicine and Translational Medicine

summary: Autoimmune polyendocrine syndrome type 1 (APS-1) relies on failures both in central and peripheral tolerance. A decreased number of regulatory T cells (Tregs) is repeatedly reported, and Tregs based therapy could therefore be considered. We have used a single cell transcriptomic approach to characterize Tregs sorted from blood.

overall_design:
sequencing of B and T cells from Autoimmune polyendocrine syndrome type 1 (APS1)
****Submitter declares that the raw data will not be provided due to patient privacy concerns***

first: Liina

last: Tserel

first: Igor

last: Filippov


title: Group 3 medulloblastomatranscriptional networks collapse under domain-specific EP300/CBP inhibition.

city: Memphis

first: Brian

last: Abraham

state: TN

address: 262 Danny Thomas Blvd

institute: St. Jude Children's Research Hospital

summary: EP300 and CBP are paralogous, multidomain histone acetyltransferases (HATs) that regulate gene expression by binding to and acetylating diverse proteins. With widespread effects on gene regulation, these enzymes are attractive targets for therapeutic development. Discovery efforts for chemical inhibitors typically target protein domains that are amenable to probe binding. In some cases, probes have been identified for more than one domain within a single protein, including high potency inhibitors of both the catalytic HAT domain and bromodomain (BRD) that mediate the enzymatic and protein binding (“reader”) activities of EP300/CBP. In cancer cell systems, combination inhibition of both domains is more toxic than single inhibitor alone, indicating that inhibition of individual domain functions may elicit differential effects. Thus, we hypothesized that determining the relative effects of domain-specific inhibition of EP300/CBP on tumor cell growth may elucidate exceptional-responding tumor types to one inhibitor or the other. This would permit a broader therapeutic index by enabling reduced dosing of domain-targeted inhibitors, while maintaining enhanced inhibition of cancer cell growth. Here, using high-throughput cell line-based screening, we demonstrate that targeting the HAT or BRD domains of EP300/CBP using the chemical probes A485 and CCS1477 has differential effects in select tumor types. Group 3 medulloblastoma (G3MB) cells were especially sensitive to EP300/CBP BRD inhibition, as compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, the effects of bromodomain and HAT-domain specific inhibition are distinct, with bromodomain inhibition causing rapid early disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB cells.

overall_design: Total RNA-seq gene expression profiling in medulloblastoma cells treated with histone acetyltransferase inhibitors.

first: Noha

last: Shendy

first: Melissa

last: Bikowitz

first: Logan

middle: H

last: Sigua

first: Yang

last: Zhang

first: Audrey

last: Mercier

first: Yousef

last: Khashana

first: Stephanie

last: Nance

first: Qi

last: Liu

first: Ian

last: Delahunty

first: Sarah

last: Robinson

first: Vanshita

last: Goel

first: Matthew

middle: G

last: Rees

last: Ronan

first: Tingjian

last: Wang

first: Mustafa

last: Kocak

first: Jennifer

last: Roth

first: Burgess

last: Freeman

first: Brent

last: Orr

first: Martine

last: Roussel

first: Erinst

last: Schonbrunn

first: Jun

last: Qi

first: Adam

middle: D

last: Durbin


title: High-resolution analysis of the pathogenesis of periodontitis

first: ANHAO

last: LIU

summary:
To investigate the function of different tissues in the pathogenesis, we established a novel mouse ligature-induced periodontitis model, supporting the separation of the maxillae into gingiva, peri-root, and bone tissue.
After setting the right molars as the non-ligature control side and the left molars as the ligature side, we performed gene expression profiling and gene enrichment analysis using data obtained from RNA-seq of four individuals after 5-days-induction (24 samples in total).

overall_design: Comparative gene expression profiling and gene enrichment analysis of RNA-seq data for separated maxillae samples (gingiva, peri-root and bone tissue; control vs. ligature).

first: Anhao

first: Mikihito

last: Hayashi

first: Yujin

last: Ohsugi

first: Sayaka

last: Katagiri

first: Takanori

last: Iwata

first: Tomoki

last: Nakashima


title: functional targets of cilia dependent cyst activation (CDCA) and a potential therapeutic targets for treating polycystic kidney disease

city: New Haven

first: Stefan

last: Somlo

state: CT

address: 333 Cedar Street

institute: Yale University

summary: We applied unbiased cell type specific transcriptional profiling by translating ribosome affinity purification (TRAP) to mouse kidneys with polycystin-1 and cilia inactivation at a stage prior kidney tubule cyst formation. We identified differentially expressed actively translating mRNA that correlated with the cilia dependent cyst activation (CDCA) pattern common to male and female mice. This differential translatome offers opportunities for mechanistic discovery in polycystin and cilia related kidney phenotypes.

overall_design: We performed TRAP RNASeq at a stage when cyst formation had not yet occurred following Pkd1 inactivation. To achieve these objectives, we used mice with the following genotypes: Pkd1fl/+; R26Rpl10a; Pax8rtTA; TetOCre (“noncystic”), Pkd1fl/fl; R26Rpl10a; Pax8rtTA; TetOCre (Pkd1KO), Pkd1fl/fl; Kif3afl/fl; R26Rpl10a; Pax8rtTA; TetOCre (Pkd1KO+ciliaKO). All experimental mice were hemizygous for the R26Rpl10a, Pax8rtTA and TetOCre alleles. The R26Rpl10a allele expresses the L10a-EGFP ribosomal protein fusion23 following expression of Cre recombinase in response to doxycycline, allowing selective pulldown of ribosomes only from nephron epithelial cells in which Pax8rtTA is expressed. All mice received oral doxycycline from postnatal day 28 to 42 (P28–P42) to induce TetOCre. Fresh kidney tissue was harvested for ribosomal pulldown at P49 (7 weeks).

first: Chao

last: Zhang

first: Michael

last: Rehman

first: Xin

last: Tian


title: The CoREST Repressor Complex Mediates Phenotype Switching and Therapy Resistance in Melanoma [ChIP-seq]

status: Public on Feb 22 2024

first: Robert

last: Fisher

address: 609 Albany St

department: Dermatology

institute: Boston University

summary: Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through non-mutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated to date. Here, we evaluate the CoREST repressor complex and the recently developed bivalent inhibitor, corin, within the context of melanoma phenotype plasticity and therapeutic resistance. We find that CoREST is a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells leads to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin reveals specific effects on histone marks connected to EMT-associated transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor (BRAFi)-resistant melanomas with corin promotes resensitization to BRAFi therapy. DUSP1 is consistently downregulated in BRAFi-resistant melanomas which is reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity can be recapitulated by the p38 MAPK inhibitor, BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial to patients with BRAFi- resistant melanoma.

overall_design: 451Lu-R or 1205Lu-R melanoma cells were treated with 2.5 uM corin or DMSO for 24 h and grown to 80% confluency in 150 cm² plates. ChIP was conducted as previously described.  The following antibodies were used for IP: H3K27ac (ab4729; lot GR3442884-1), H3K9ac (ab32129; lot GR3359686), or H3K4me2 (ab32356; lot GR3422075-2), with input samples as controls. Two biological replicates were performed for each cell line. Library prep and Illumina sequencing of ChIP DNA was performed by Azenta (Indianapolis, IN) with 40 million 2x150 bp paired-end reads for each sample.

first: Muzhou

last: Wu

first: Ailish

last: Hanly

first: Frederick

last: Gibson

first: Samantha

last: Rogers

first: Kihyun

last: Park

first: Angelina

last: Zuger

first: Kevin

last: Kuang

first: Jay

last: Kalin

first: Sarah

last: Nocco

first: Matthew

last: Cole

first: Amy

last: Xiao

first: Filisia

last: Agus

last: Labadorf

first: Samuel

last: Beck

first: Marianne

last: Collard

first: Philip

first: Rhoda

last: Alani


title: Spatiotemporally resolved ex vivo colorectal cancer development in engineered mini-colons

first: L. Francisco

last: Lorenzo-Martín


title: TNYA0044: AAV9:mPkp2 preventive vs therapeutic modes of treatment at 51 weeks in Pkp2-cKO ARVC mouse model

first: Farshad

last: Farshidfar

overall_design: RNA sequencing on 5 groups of C57BL6 mice with 1. wild-type background: WT_HBSS (n=9), 2. cKO_HBSS (n=4), 3. cKO_AAV9:mPkp2_3E13 vg/kg (n=8), 4. cKO_AAV9:mPkp2_1E14 vg/kg (n=5), 5. cKO_AAV9:mPkp2_1E14 vg/kg_2.5wks (n=6). Right ventricle (RV) and left ventricle (LV) tissues from each animal were analyzed simultaneously.

first: Iris

last: Wu

first: Aliya

last: Zeng

first: Amara

last: Greer-Short

first: Alex

last: Aycinena

first: Tefera

middle: E

last: Anley

first: Reva

last: Shenwai

first: Melissa

last: Van Pell

first: Emma

last: Xu

first: Chris

last: Reid

first: Neshel

last: Rodriguez

first: Beatriz

last: Lim

first: Tae

middle: W

last: Chung

first: Cristina

last: Dee-Hoskins

first: Carolina

last: G Gutierrez

first: Jessie

last: Madariaga

last: Robinson

first: Yolanda

last: Hatter

first: Renee

last: Butler

first: Stephanie

last: Steltzer

first: Jaclyn

last: Ho

first: James

last: R Priest

first: Xiaomei

last: Song

first: Kristina

last: Green

first: Kathryn

last: N Ivey

first: Timothy

last: Hoey

first: Jin

last: Yang

first: Zhihong


title: Time-resolved profiling of RNA binding proteins throughout the mRNA life cycle

status: Public on Feb 21 2024

first: Yeon

last: Choi

address: 1470 Madison Ave

institute: Iicahn school of medicine at mount sinai

summary: mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNP remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA-binding proteins (RBPs) across ten distinct time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed numerous RBPs with unexpected dynamics: the TRanscription-EXport complex (TREX) recruited posttranscriptionally after NXF1 binding, challenging the current view of transcription-coupled mRNA export mechanism, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with GO annotations. Our data can be explored at chronology.rna.snu.ac.kr.

overall_design: Pulse-chased mRNA analysis using 10 min 4sU labeling and MTS-biotin pulldown

first: Buyeon

last: Um

first: Yongwoo

first: Jeesoo

last: Kim

first: Jong-Seo

first: V


title: Quantifying forms and functions of intestinal bile acid pools in mice

city: Lebanon

first: Mark

last: Sundrud

state: NH

address: Dartmouth Hitchcock Medical Center One Medical Center Drive

department: Microbiology and Immunology

institute: Dartmouth Hitchcock Medical center

summary: Bile acids (BAs) are gastrointestinal metabolites that serve dual functions in lipid absorption and cell signaling. BAs circulate actively between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with intestinal cells in vivo remain ill-defined. Through multi-site sampling of nearly 100 BA species in individual wild type mice, as well as mice lacking the ileal BA transporter, Asbt/Slc10a2, we calculate the ileal BA pool in fasting C57BL/6J mice to be ~0.3 mmoles/g. Asbt-mediated transport accounts for ~80% of this pool and amplifies size, whereas passive absorption explains the remaining ~20%, and generates diversity. Accordingly, ileal BA pools in mice lacking Asbt are ~5-fold smaller than in wild type controls, enriched in secondary BA species normally found in the colon, and elicit unique transcriptional responses in cultured ileal explants. This work quantitatively defines ileal BA pools in mice and reveals how BA dysmetabolism can impinge directly on intestinal physiology.

overall_design:
To investigate gene-regulatory functions of endogenous bile acids, ileal fragments from male 4-hours-starved C57BL/6J mice were treated with indicated additives for 8 hours.
We then analyzed differential expressed genes using 3' mRNA-seq.

first: Koichi

last: Sudo

first: Shannon

last: Soucy

first: Akshaya

last: Balasubramanian

first: Henry

last: Dionne

middle: S


title: In Vivo Genome-Wide CRISPR Screening Identifies CITED2 as a Driver of  Prostate Cancer Bone metastasis

first: Juan

last: Arriaga

address: 1425 Madison Ave

department: Oncological Sciences, Urology

institute: Icahn School of Medicine at Mount Sinai

summary: Most cancer deaths are due to metastatic dissemination to distant organs. Bone is the most frequently affected organ in metastatic prostate cancer and a major cause of prostate cancer deaths. Yet, our partial understanding of the molecular factors that drive bone metastasis has been a limiting factor for developing preventative and therapeutic strategies to improve patient survival and well-being. Although recent studies have uncovered molecular alterations that occur in prostate cancer metastasis, their functional relevance for bone metastasis is not well understood. Using genome-wide CRISPR activation and inhibition screens we have identified multiple drivers and suppressors of prostate cancer metastasis. Through functional validation, including innovative organ-on-a-chip invasion platform for studying bone tropism, our study identifies the transcriptional modulator, CITED2, as a novel driver of prostate cancer bone metastasis and uncovers multiple new potential molecular targets for bone metastatic disease.

overall_design:
A CRISPRa screen was performed in non-metastatic human prostate cancer cells to identify drivers of metastasis. This identified CITED2 as a main target
CITED2 was then overexpressed using CRISPRa in isogenic 22Rv1 cells and using cDNA overexpression in LNCaP cells.
RNAseq was performed from control and CITED2 overexpressed cell lines, in duplicate for 22Rv1 and triplicate for LNCaP


title: The neuroimmune CGRP-RAMP1 axis tunes cutaneous adaptive immunity to the microbiota.

city: Bethesda

first: Verena

last: Link

address: 4 Memorial Drive

department: NIAID

institute: NIH

summary: The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of sensory neurons in controlling the adaptive immune system, and more specifically immunity to the microbiota, however, remain elusive. Here, we identified a novel mechanism for direct neuroimmune communication between commensal-specific T lymphocytes and somatosensory neurons mediated by the neuropeptide calcitonin gene-related peptide (CGRP) in the skin. Intravital imaging revealed that commensal-specific T cells are in close proximity to cutaneous nerve fibers in vivo. Correspondingly, we observed upregulation of the receptor for the neuropeptide CGRP, RAMP1, in CD8+ T lymphocytes induced by skin commensal colonization. Neuroimmune CGRP-RAMP1 signaling axis functions in commensal-specific T cells to constrain Type 17 responses and moderate the activation status of microbiota-reactive lymphocytes at homeostasis. As such, modulation of neuroimmune CGRP-RAMP1 signaling in commensal-specific T cells shapes the overall activation status of the skin epithelium, thereby impacting the outcome of responses to insults such as wounding. The ability of somatosensory neurons to control adaptive immunity to the microbiota via the CGRP-RAMP1 axis underscores the various layers of regulation and multisystem coordination required for optimal microbiota-reactive T cell functions under steady state and pathology.

overall_design: bulk RNA-seq: CD4+ T cells from wild-type animals following S. epidermidis colonization were sorted into Th1 and Th17, processed and analyzed for RNA-seq as described in Linehan et al., 2018. Keratinocytes from the same Lck-Cre Ramp1 fl/fl experiment described above were sorted and processed for RNA-seq as described in Lima-Junior et al., 2021.

first: Warakorn

last: Kulalert

first: Yasmine

last: Belkiad


title: The neuroimmune CGRP-RAMP1 axis tunes  cutaneous adaptive immunity to the microbiota

overall_design: single cell RNA-seq: Skin CD8+ lymphocytes labeled with TotalSeqC hashtag antibodies (BioLegend) from S. epidermidis-associated mice (six Lck-Cre- Ramp1 fl/fl animals and six Lck Cre+ Ramp1 fl/fl animals) were sorted as DAPI- CD90.2+ TCR+ CD4- CD8+ TCR- CD49f- NK1.1- B220- CD11b- CD11c- MHCII-, using a Sony MA900 cell sorter. All samples were pooled together, and approximately 15,000 sorted cells were loaded onto the Chromium Single Cell Controller (10X Genomics) to encapsulate cells into droplets. The library was prepared using a Chromium Single Cell 5’ Reagent Kits v2 (10X Genomics) following manufacturer's instructions, and then sequenced on an Illumina Nextseq500 (Next Seq 500/550 High Output Kit v2, Illumina). ; bulk RNA-seq: CD4+ T cells from wild-type animals following S. epidermidis colonization were sorted into Th1 and Th17, processed and analyzed for RNA-seq as described in Linehan et al., 2018.  Keratinocytes from the same Lck-Cre Ramp1 fl/fl experiment described above were sorted and processed for RNA-seq as described in Lima-Junior et al., 2021.


title: Two distinct waves of transcriptome and translatome remodelling drive germline stem cell differentiation

city: Cambridge

first: Tamsin

middle: Joy

last: Samuels

address: Downing Street

department: Department of Genetics

country: United Kingdom

institute: University of Cambridge

summary: Development and tissue maintenance require a supply of new cells, which are generated by division of stem cells and then specialised to perform their function. The differentiation process requires the tight control of gene expression, but this has been challenging to study in a systematic manner in adult stem cells, which are sparsely distributed in tissues. We have overcome these limitations by establishing a robust protocol to synchronise Drosophila female germline stem cell (GSC) differentiation in vivo, allowing us to perform transcriptome and translatome analyses at high temporal resolution during differentiation. As well as observing dynamic changes in mRNA level, promoter usage and exon inclusion, our data reveal that changing translation efficiency is the predominant regulatory mechanism during GSC differentiation. Indeed, changes in mRNA level are frequently buffered by changes in translation efficiency, including the translational repression of transcripts that will only be translated later during oocyte maturation or embryo development. Contrary to the expected linear accumulation of changes from stem cell to differentiated cell, our data reveal that differentiation occurs through two distinct waves of changes in both the transcriptome and translatome level.

overall_design: Time course of RNAseq and Riboseq experiments in synchronised differentiating Drosophila germline stem cells in vivo.

first: Jinghua

last: Gui

first: Daniel

last: Gebert

first: Felipe

last: Teixeira


title: Similarity and dissimilarity in alteration of gene expression profile associated with inhalational anesthesia between sevoflurane and desflurane

city: Hidaka

first: Akihiko

last: Okuda

state: Saitama

address: 1397-1 Yamane

department: Division of Biomedical Sciences, Research Center for Genomic Medicine

institute: Saitama Medical University

summary: Although sevoflurane is one of the most commonly used inhalational anesthetic agent, the popularity of desflurane is increasing to a similar level. The main beneficial property of desflurane is the relatively fast emergence of the patient from the anesthetic state after halting its supply compared with anesthesia using other anesthetic agents. However, there has been no comprehensive comparison of the effects of these two anesthetic agents on alterations in liver gene expression profiles in animals, including humans, to assess the kinds of biological abnormalities/changes and their levels in the host. Thus, we compared alterations in the global gene expression profiles in the livers of rats subjected to inhalational anesthesia by sevoflurane or desflurane by a next-generation sequencing method.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for livers from rats subjected to inhalational anesthesia with either sevoflurane or desflurane and their controls that were treated with sevoflurane or desflurane only briefly until the loss of their consciousness.

first: Takehiro

last: Nogi

first: Kousuke

last: Uranishi

first: Masataka

last: Hirasaki


title: SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination (RNA-Seq)

first: Fabian

last: Kern

overall_design: To investigate the effect of missing SRF on myelation, we established mesenchymal stem cells in which SRF has been knocked down using Olig2-Cre.

first: Tal

last: Iram

first: Miguel

last: Garcia

first: Jeremy

last: Amand

first: Achint

last: Kaur

first: Micaiah

last: Atkins

first: Manasi

last: Iyer

first: Mable

last: Lam

first: Nicholas

last: Ambiel

first: Danielle

middle: M

last: Jorgens

first: Andreas

last: Keller

first: Tony

last: Wyss-Coray

first: J

last: Zuchero