title: Long noncoding RNA EPCART regulates translation through PI3K/AKT/mTOR pathway and PDCD4 in prostate cancer

city: Tampere

first: Tapio

last: Visakorpi

address: Kalevantie 4

department: Faculty of Medicine and Health Technology

country: Finland

institute: Tampere University

summary: While hundreds of cancer-associate long noncoding RNAs (lncRNAs) have been discovered, their functional role in cancer cells is still a mystery for the majority of them. Increasing number of lncRNAs are recognized to function in the cytoplasm, including as modulators of translation. Here, we investigated the detailed molecular identity and functional role of EPCART, a lncRNA we previously discovered to be a potential oncogene in AR- and ERG-regulated prostate cancer (PCa) tumors. First, we confirmed EPCART to be a non-peptide-coding lncRNA with in silico methods and validated the transcript structure of EPCART in vitro. Pathway analysis of differentially expressed protein-coding genes in EPCART knockout cells implied EPCART to inhibit translational machinery of PCa cells. While drastic changes in the global translation were not detected in EPCART knockout cells by polysome profiling, EPCART located largely in the cytoplasm and at the sites of translation. With quantitative whole proteome studies on EPCART knockout cells we discovered PDCD4, an inhibitor of protein translation, to be increased by EPCART reduction. Further studies indicated that the inhibitory effect of EPCART on translation was mediated by reduced activation of AKT and inhibition of mTORC1 pathway. Together, our findings identify EPCART as a translation-associated lncRNA that functions via modulation of AKT/mTORC1/PDCD4 pathway in PCa cells. Furthermore, we provide evidence for prognostic potential of PDCD4 in PCa tumors in connection with EPCART.

overall_design: Read counts of RNA-seq data for two EPCART knockout cell clones (del-4 and del-56) and one wild-type clone (WT) created by CRISPR-Cas9 in LNCaP cells. Three biological replicates were sequenced on each clone.

first: Annika

last: Kohvakka

first: Mina

last: Sattari

first: Janika

last: Nättinen

first: Ulla

last: Aapola

first: Pavlína

last: Gregorová

first: Teuvo

last: Tammela

first: Hannu

last: Uusitalo

first: Peter

last: Sarin

first: Leena

last: Latonen


title: The Drosophila histone methyl-transferase SET1 coordinates multiple signaling pathways in regulating male germline stem cell maintenance and differentiation

status: Public on Jul 31 2024

city: Baltimore

first: Velinda

last: Vidaurre

state: Maryland

address: 3400 N Charles St

country: USA

institute: Johns Hopkins

type: Expression profiling by high throughput sequencing

summary: Many cell types come from tissue-specific adult stem cells that maintain the balance between proliferation and differentiation. Here, we study how the H3K4me3 methyltransferase, Set1, regulates early-stage male germ cell proliferation and differentiation in Drosophila. Early-stage germline-specific knockdown of set1 results in a temporally progressed defects, arising as germ cell loss and developing to overpopulated early-stage germ cells. These germline defects also impact the niche architecture and cyst stem cell lineage in a non-cell-autonomous manner. Additionally, wild-type Set1, but not the catalytically inactive Set1, could rescue the set1 knockdown phenotypes, highlighting the functional importance of the methyl-transferase activity of the Set1 enzyme. Further, RNA-seq experiments reveal key signaling pathway components, such as the JAK-STAT pathway gene stat92E and the BMP pathway gene mad, that are upregulated upon set1 knockdown. Genetic interaction assays support the functional relationships between set1 and JAK-STAT or BMP pathways, as mutations of both the stat92E and mad genes suppress the set1 knockdown phenotypes. These findings enhance our understanding of the balance between proliferation and differentiation in an adult stem cell lineage. The germ cell loss followed by over-proliferation phenotypes when inhibiting a histone methyl-transferase raise concerns about using their inhibitors in cancer therapy.

overall_design: To Investigate the role of Set1 in the drosophila male germline, we knocked down Set1 in the early male germline. We then performed gene expression prolfiling analysis using data obtained from RNA-seq of control and set1 knockdown testes at 4 different time points.

first: Annabelle

last: Song

first: Taibo

last: Li

first: Wai

middle: L

last: Ku


title: Mitochondrial microRNA profiles are altered in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) during hibernation

city: Waterloo

first: Karyn

last: Robichaud

state: ON

address: 200 University Ave West, B1 171B

department: Biology

country: Canada

institute: University of Waterloo

summary: Thirteen-lined ground squirrels (TLGS) are obligate hibernators that cycle between torpor (low metabolic rate and body temperature) and interbout euthermia (IBE; typical euthermic body temperature and metabolism) from late autumn to spring. Many physiological changes occur throughout hibernation, including a reduction in liver mitochondrial metabolism during torpor, which is reversed during arousal to interbout euthermia. Nuclear-encoded microRNA (small post-transcriptional regulator molecules) differ in abundance throughout TLGS hibernation and have been shown to regulate mitochondrial gene expression in mammalian cell culture (where they are referred to as mitomiRs). This study characterized differences in mitomiR profiles from TLGS liver mitochondria isolated during summer, torpor, and IBE, and predicted their mitochondrial targets. Using small RNA sequencing, differentially abundant mitomiRs were identified between hibernation states and, using qPCR analysis we quantified expression of predicted mitochondrial mRNA targets. Most differences in mitomiR abundances were seasonal (i.e. between summer and winter) with only one mitomiR differentially abundant between IBE and torpor. Multiple factor analysis revealed unique clustering of hibernation states, predominantly driven by mitomiR abundances, and nine of these differentially abundant mitomiRs had predicted mitochondrial RNA targets, including subunits of electron transfer system complexes I and IV, 12S rRNA and two tRNAs. Overall, mitomiRs were predicted to suppress expression of their mitochondrial targets and may have some involvement in regulating protein translation in mitochondria. This study found differences in mitomiR abundances between seasons and hibernation states of TLGS and suggests potential mechanisms in regulating the mitochondrial electron transfer system.

overall_design: Mitochondrial fractions from thirteen-lined ground squirrel livers were collected during summer and winter (torpor and interbout euthermia). Total RNA was extracted from the samples (enriched for microRNA), then sequenced to investigate if there were any differences in mitochondrial microRNA abundance between hibernation states.

first: Brynne

last: Duffy

first: James

middle: F

last: Staples

first: Paul

middle: M

last: Craig


title: Genomic binding of NF-Y in mouse and human cells

status: Public on Aug 01 2024

city: Milano

first: Diletta

last: Dolfini

state: MI

address: Via Celoria, 26

department: Bioscience

country: Italy

institute: University of Milan

type: Genome binding/occupancy profiling by high throughput sequencing

summary: NF-Y is a Transcription Factor that regulates transcription through binding to the CCAAT-box. To understand its strategy, we analyzed 16 ChIP-seq datasets from human and mouse cells. Shared loci, mostly located in promoters of expressed genes of cell cycle, metabolism and gene expression pathways, are associated with histone marks of active chromatin and specific modules of TFs. Other peaks are in enhancers and Transposable Elements -TE- of retroviral origin in human and mouse. We evaluated the relationship with USF1, a common synergistic partner in promoters and MLT1 TEs, upon NF-YB inactivation: USF1 binding decreases in promoters, modestly in MLT1, suggesting a pioneering role of NF-Y in formers, not in the latters. These data define a common set of NF-Y functional targets across different mammalian cell types, suggesting a pioneering role in promoters with respect to TEs.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: NF-YB ChIP-seq data analysis on human and mouse cell lines for the identification of shared and specific binding locations.

first: Mirko

last: Ronzio


title: POGZ loss increases TGFβ signaling and potentiates TNBC metastasis

city: Montréal

first: Claudia

last: Kleinman

state: Québec

address: 3999 Côte Ste-Catherine Road

department: Human Genetics

institute: Lady Davis Institute for Medical Research

summary: The pogo transposable element derived zinc finger protein, POGZ, is notably associated with autism-like or intellectual disabilities through its role in gene transcription. Indeed, many proteins involved in neurological development are often dysregulated in cancer. We provide the first experimental evidence that POGZ influences the growth and metastatic spread of triple negative breast cancers (TNBC). Utilizing a well-characterized immunocompetent model of TNBC, we show that POGZ exerts a dual role, both as a tumor promoter and metastasis suppressor. Mechanistically, we show that POGZ loss potentiates TGFβ pathway activation in TNBC, to exert cytostatic effects while simultaneously increasing the mesenchymal and migratory properties of breast tumors. Finally, we demonstrate that whereas POGZ levels are elevated in human breast cancers, the most aggressive TNBC tumors, including those with increased mesenchymal and metastatic properties, dampen POGZ levels, associated with inferior clinical outcomes. Combined these data suggest that POGZ is a critical regulator of the early stages of metastatic cascade

overall_design: POGZ was genetically ablated in murine 4T1 TNBC. Cells lines (3 KO replicates, 3 VC replicates) and syngeneic BalbC derived tumors (3 KO replicates, 3 VC replicates) were subjected to RNAsequencing.

first: John

last: Heath

first: Valerie

last: Sabourin

first: Steven

last: Hebert

last: Findlay

first: Caitlynn

last: Mirabelli

first: Alexandre

last: Orthwein

first: Josie

last: Ursini-Siegel


title: ARID3A coordinates the proliferation-differentiation switch of transit-amplifying cells in the intestine

city: London

first: Vivian

address: 1 Midland Road

country: United Kingdom

institute: The Francis Crick Institute

summary: Intestinal stem cells (ISCs) at the crypt base divide and give rise to progenitor cells that have the capacity to proliferate and differentiate into various mature epithelial cell types in the transit-amplifying (TA) zone. Here, we identified the transcription factor ARID3A as a novel regulator of intestinal epithelial cell proliferation and differentiation at the TA compartment. We show that ARID3A forms an expression gradient from villus tip to the early progenitors at the crypts mediated by TGF-β and WNT signalling. Intestinal epithelial-specific deletion of Arid3a reduces proliferation of TA cells. Bulk and single cell transcriptomic analysis shows increased enterocyte differentiation and reduced secretory cells in the Arid3a cKO intestine. Interestingly, upper-villus gene signatures of both enterocytes and secretory cells are enriched in the mutant intestine. We find that the enhanced enterocyte differentiation in the Arid3a cKO intestine is caused by increased binding of HNF1 and HNF4. Finally, we show that loss of Arid3a impairs irradiation-induced regenerative process by altering the dynamics of proliferation and apoptosis. Our findings imply that ARID3A may play a gatekeeping role in the TA compartment to maintain the “just-right” proliferation-to-differentiation ratio for tissue homeostasis and plasticity

overall_design: Sorted single cell suspension from VillinCreERT2 (control) and VillinCreERT2, Arid3a fl/fl intestines. Two biological replicates per experimental condition were processed. Both Control and cKO animals were injected with tamoxifen at 8-10 weeks old and harvested at 1 month after the first injection.

first: Nikolaos

last: Angelis

first: Anna

last: Baulies

first: Stefan

last: Boeing


title: Role of Arid3a in proliferation and differentiation of intestinal epithelial cells [RNA-seq]

last: Li

summary: To investigate the role of Arid3a in the intestinal epithelium, we generated an inducible conditional knockout model (VillinCreERT2;Arid3afl/fl -Arid3a cKO) and we compared the gene expression analysis to WT animals (ViliinCreERT2). Animals were injected intraperitoneally with tamoxifen to induce gene deletion.

overall_design: Differential expression analysis between WT and Arid3a cKO animals at 1 month after tamoxifen administration.

first: Gavin

last: Kelly


title: Engineered T cell therapy for central nervous system injury [CSF RNAseq + VDJ]

city: Saint Louis

first: Jonathan

last: Kipnis

state: MO

address: 4515 McKinley Ave

department: Pathology and Immunology

institute: Washington University in Saint Louis

summary: Traumatic injuries to the central nervous system (CNS) affect millions of people worldwide yet lack an effective treatment. These injuries contain infiltrating immune cells that can promote tissue repair and could be exploited for therapeutic benefit. Here, using single-cell RNA-sequencing of T cells infiltrating the injured CNS we demonstrate their clonal expansion and antigen specificity towards CNS derived self-peptides. We confirm the beneficial effect of these injury-associated autoimmune CD4+ T cells in murine models of optic nerve and spinal cord injury. Subsequently, using mRNA-based transient T cell receptor (TCR) reconstitution, we demonstrate a therapeutic T cell strategy to alleviate CNS injury. Treatment of CNS-injured mice with this therapy improved locomotion and alleviated histological signs of damage, through regulation of myeloid cells, without detrimental autoimmune side effects. This strategy provides a means of developing custom-designed T cell therapies for CNS injury, and possibly for other neurodegenerative disorders.

overall_design: Human CSF was collected via a lumbar drain daily from SCI patients. 10 mL of CSF was used at each time point for cell isolation. CSF was transferred into a 15 mL centrifuge tube and centrifuged at 500 g for 5 minutes. The supernatant was removed and cell pellet was resuspended with PBS containing 0.04 % BSA and filtered with 40 μm cell strainer. Cell number and viability were determined using Viastain AOPI Staining Solution (CS2-0106, Nexcelom Bioscience) and the CellDrop FL Fluorescence Cell Counter (DeNovix). Cell samples were loaded onto a 10X Genomics Chromium platform for Gel Bead-in Emulsions (GEMs) and Human T cell Chromium Single Cell V(D)J Enrichment Kit and Chromium Single Cell 5’ Library & Gel Bead Kit were used separately to generate a cDNA library targeting the TCR and for transcriptome expression.

first: Wenqing

last: Gao

first: Taitea

last: Dykstra


title: Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

city: Oxford

first: Linzhen

last: Kong

address: University of Oxford Old Road Campus Research Building

department: Nuffield Department of Medicine

institute: Ludwig Institute for Cancer Research

summary: Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of Ψ at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a Ψ-to-C transition. Compared to previous methods, BACS allowed the precise identification of Ψ positions, especially in densely modified Ψ regions and consecutive uridine sequences. BACS successfully detected all known Ψ sites in human rRNA and spliceosomal snRNAs and generated the first quantitative Ψ map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant Ψ114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of Ψ in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of Ψ in future studies.

overall_design: Dataset1: 8 total RNA samples from HeLa. Sample1-8 corresponds to samples containning different fractions of NNψNN spike-in.(0%,20%,50%,75%, respectively).Dataset2(sample 9-12):4 Ribo-depleted library for ncRNAs. Dataset3(sample 13-18): 4 PolyA+ RNA samples from HeLa and 4 for IVT samples. Dataset4(sample 19-32): Ribo-depleted libraries for PUS7,PUS1 and TRUB1 knockout cell lines in Hela. Dataset5(sample 33-50): Ribo-depleted libraries for Epstein–Barr virus(EBV), SARS-CoV-2, Hepatitis C(HCV),Zika, Hepatitis Delta Virus and Sindbis virus samples. Ribo-depleted libraries for Epstein-Barr virus(EBV) using modified BID-seq (Samples 51-54).

first: Haiqi

last: Xu

first: Jingfei

last: Cheng

first: Khatoun

middle: A

last: Moussawi

first: Xiufei

last: Chen

first: Aleema

last: Iqbal

first: Peter

last: Wing

first: James

last: Harris

first: Senko

last: Tsukoda

first: Jane

last: McKeating

first: Azman

last: Embarc

first: Guifeng

last: Wei

first: Alfredo

last: Castello

first: Skirmantas

last: Kriaucionis

first: Xin

last: Lu

first: Chun-Xiao

last: Song


title: Gene expression profile of single endocardial cells in 48 and 72 hpf zebrafish embryos/larvae. In the study "The heart is a niche for hematopoietic stem and progenitor cells in zebrafish."

city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

country: Germany

institute: MPI for heart and lung research

summary: We used single-cell RNA expression to uncover the cellular and molecular heterogeneity of isolated endocardial cells in the heart. From this study, we uncovered the presence of an endocardial-hematopoietic cluster, along with other significant clusters including valve endocardial and interstitial cells, and non-valve cells.

overall_design: Endocardial cells were isolated using FACS from physically extracted Tg(kdrl:nls-mCherry) and Tg(cd41:GFP) hearts according to presence of mCherry-positive cells, and further analyzed using scRNAseq.

first: Dorothee

last: Bornhorst

first: Amulya

middle: V

last: Hejjaji

first: Lena

last: Steuter

first: Nicole

last: Woodhead

first: Paul

last: Maier

first: Alessandra

last: Gentile

first: Alice

last: Alhajkadour

first: Octavia

middle: S

last: Larrain

first: Michael

last: Weber

first: Khrievono

last: Kikhi

first: Jan

last: Huisken

first: Owen

middle: J

last: Tamplin

first: Didier

middle: Y

last: Stainier

first: Felix

last: Gunawan


title: Interactome of the HIV-1 proteome and human host RNA

city: Gent

first: Wim

last: Trypsteen

state: East-Flanders

address: Corneel Heymanslaan 10

department: Internal Medicine

country: Belgium

institute: UGent

summary: The human immunodeficiency virus (HIV-1) is highly dependent on a variety of host factors. Beside proteins, host RNA molecules have been reported to aid HIV-1 replication and latency maintenance. Here we report the use of multiple workflows of native RNA immunoprecipitation and sequencing (nRIPseq) to determine direct host RNA interaction partners of all 18 HIV-1 (poly)proteins. We identified 1,727 HIV-1 protein – human RNA interactions in the Jurkat cell line and 1,558 interactions in SupT1 cells for a subset of proteins.  We discovered different cellular pathways that seem to be used or controlled by HIV-1 on the RNA level: Tat binds for example mRNAs of proteins involved in the super elongation complex (AFF1-4, Cyclin-T1). Correlation of the interaction scores (based on binding abundancy) allowed to identify the highest confidence interactions, for which we performed a small-scale knockdown screen that led to the identification of three HIV-1 protein binding RNA interactors involved in HIV-1 replication (AFF2, H4C9 and RPLP0).

overall_design:
RIPseq was performed in batches of 3 HIV-1 protein IPs and 1 FLAG-tagged GFP background control IP (all in triplicate). For each HIV-1 protein IP also a background control IP was included in triplicate using 10 µg anti-mouse IgG rabbit IgG antibody (ThermoFisher, no. 11895905). For Streptavidin-based RIPseq a similar procedure was performed using 50 µL preconjugated MagStrep “type3” XT beads (iba, no. 2-4090-002) for IP and unconjugated MagStrep “type3” XT beads for preclearance and background control.
This RIPseq series contains 4 data sets, assayA is Jurkat FLAG uninfected, assayB is Jurkat STREP uninfected, assayC is SupT1 FLAG uninfected, assayD is SupT1 FLAG HIV infected

first: Tinus

last: Schynkel

first: Linos

last: Vandekerckhove

first: Pieter

last: Mestdagh


title: Upregulation of HOXA3 by Isoform-Specific Wilms Tumor 1 Drives Chemotherapy Resistance in Acute Myeloid Leukemia

city: Portland

first: Peter

last: Ryabinin

state: OR

address: 3181 SW Sam Jackson Park Rd

institute: Oregon Health and Science University

summary: Upregulation of the Wilms' Tumor 1 (WT1) gene is common in acute myeloid leukemia (AML) and is associated with poor prognosis, including disease relapse, decreased overall survival (OS), and chemotherapy resistance. WT1 produces 12 primary transcripts through different translation initiation sites and alternative splicing, but the specific transcripts and mechanisms responsible for chemoresistance are unknown. We found that overexpression of short WT1 transcripts lacking exon 5 with and without the KTS motif (sWT1+/- and sWT1-/-) led to reduced cell growth. However, only sWT1+/- resulted in decreased CD71 expression, G1 arrest, and cytarabine resistance. Primary AML patient cells with low CD71 expression exhibit relative resistance to cytarabine, suggesting that CD71 may serve as a potential biomarker for cytarabine therapy. RNAseq differential gene expression analysis identified two transcription factors, HOXA3 and GATA2, that are specifically upregulated in sWT1+/- cells. Overexpression of either HOXA3 or GATA2 reproduced the effects of sWT1+/-, including decreased cell growth, G1 arrest, reduced CD71 expression, and cytarabine resistance. Furthermore, sWT1+/-, HOXA3, and GATA2 regulate cell growth and cytarabine sensitivity in a context-dependent manner, likely dependent on HOXA3 expression. HOXA3 expression is significantly correlated with chemotherapy response, OS, and ELN subtypes in NPM1-negative leukemia specimens. Overexpression of HOXA3 leads to drug resistance against a broad spectrum of chemotherapeutic agents. Our results suggest that WT1 modulates drug sensitivity in an isoform-specific manner by promoting HOXA3 expression. Both HOXA3 mRNA and CD71 cell surface expression could serve as potential biomarkers for predicting responses to cytarabine-based treatment in AML.

overall_design: To investigate  the transcriptome alterations associated with various sWT1 transcripts and HOXA3, we conducted an overexpression experiment in AML cell lines. The introduction of sWT1 involved using a constitutive sWT1 vector and a empty vector as a control, while HOXA3 was introduced using a doxycycline-inducible vector. Subsequently, RNAseq differential gene expression analysis was performed.

first: Reid

last: Chen

first: Basil

last: Allen

first: Daniel

last: Bottomly

first: Lindsey

last: Savoy

first: Bonnie

last: Goff

first: Anthony

last: Wang

first: Shannon

last: McWeeney

first: Haijiao

last: Zhang


title: RNA-Seq Time-Course Analysis of NPCs Transcriptome in Response to HSV-1 Infection

city: pittsburgh

first: Leonardo

last: D'aiuto

state: pa

address: 3811 O'Hare st

department: psychiatry

institute: UPMC

summary:
The neurogenic niches within the central nervous system serve as essential reservoirs for neural precursor cells (NPCs), playing a crucial role in neurogenesis. However, these NPCs are particularly vulnerable to infection by the herpes simplex virus type 1 (HSV-1).
In the present study, we investigated the changes in the transcriptome of NPCs in response to Herpes simplex virus 1 (HSV-1) infection. Using bulk RNA-Seq, the transcriptomes of HSV-1-infected NPCs were compared to those of uninfected samples at different time points in the presence or absence of antivirals.

overall_design: To investigate the effects of HSV-1 infection on human NPCs we conducted a time-course RNA-Seq study using NPCs-derived neurospheres. The method for generating NPCs from human iPSC is detailed in the methods section. NPCs were infected with a genetically engineered HSV-1 KOS strain that expresses enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) under the control of immediate early and late gene promoters, respectively. Cells were infected at the multiplicity of infections (MOIs) of 0.001 and 0.0001 in the presence or absence of antivirals antivirals (E)-5-(2-bromovinyl)-2'-deoxyuridine (5BVdU, 30 μM) and interferon-a (IFN-a, 125 U/ml). After one hour the inocula were removed, cells were washed and dissociated, and were transferred into low attachment 6-well plate for the generation of neurospheres. Uninfected and infected neurospheres were harvested at days 3, 5, and 7 post-infection (p.i.). The expression of the fluorescent reporter genes at the different time points confirmed the infection of NPCs (data not shown).

last: D'Aiuto

first: Vishwajit

last: Nimgaonkar


title: Role of Arid3a in proliferation and differentiation of intestinal epithelial cells

first: Vivian

last: Li

overall_design: Differences in chromatin accessibility between WT and Arid3a cKO animals and footprinting analysis at 1 month after tamoxifen administration.

first: Miriam

last: Llorian Sopena

first: Nikolaos

last: Angelis


title: Nuclear PKM2 binds pre-mRNA at folded G-quadruplexes and reveals their gene regulatory role

city: Bethesda

first: Markus

last: Hafner

state: MD

address: 50 South Drive

department: NIAMS

institute: NIH

summary: Nuclear translocation of the key metabolic enzyme Pyruvate Kinase Isoform 2 (PKM2) is widely observed in cancer, but its contribution to gene regulation and pathogenesis remains unclear. Here we find that PKM2 specifically recognized folded RNA G-quadruplex (rG4) structures in vitro with high affinity. In human cells, nuclear PKM2 bound thousands of precursor mRNAs at potential rG4 sites and translocation of PKM2 to the nucleus resulted in increased expression of rG4-containing mRNAs.

sample_organism:
Homo sapiens
Escherichia coli BL21(DE3)

platform_organism:
Homo sapiens
Escherichia coli BL21(DE3)

overall_design: PKM2 PARCLIP , RNA-Seq after nuclear PKM2 depletion or forced nuclear PKM2 expression

first: Dimitrios

middle: G

last: Anastasakis

first: Maria

last: Apostolidi

first: Jesse

last: Rinehart


title: Engineered cytokine/antibody fusion proteins improve delivery of IL-2 to pro-inflammatory cells and promote antitumor activity

status: Public on Jul 30 2024

first: Jyothi

last: Padiadpu

address: 9000 Rockville Pike

department: NHLBI

summary: Progress in cytokine engineering is driving therapeutic translation by overcoming these proteins’ inherent limitations as drugs. The interleukin-2 (IL-2) cytokine harbors great promise as an immune stimulant for cancer treatment. However, the cytokine’s concurrent activation of both pro- inflammatory immune effector cells and anti-inflammatory regulatory T cells, toxicity at high doses, and short serum half-life limit clinical application. One promising approach to improve the selectivity, safety, and longevity of IL-2 is complexation with anti-IL-2 antibodies that bias the cytokine towards activation of immune effector cells. Although this strategy shows therapeutic potential in preclinical cancer models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multi-protein drug and concerns about complex stability. Here, we introduce a versatile approach to designing intramolecularly assembled single- agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokine’s activities towards immune effector cells. We establish the optimal IC construction and further engineer the cytokine/antibody affinity to improve immune biasing function. We demonstrate that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared to natural IL-2, both alone and in combination with immune checkpoint inhibitors. Moreover, therapeutic efficacy is observed without inducing toxicity, as measured by weight loss, pulmonary edema, cytokine secretion, and liver damage markers. This work presents a roadmap for the design and translation of cytokine/antibody fusion proteins.

overall_design: human CD8+ T cells were cultured in complete RPMI 1640 medium and stimulated for 4 or 24 hr with 1 µM IL-2 or 0.5 µM (equivalent stoichiometric quantity) Control IC or F10 IC.

first: Elissa

middle: K

last: Leonard

first: Jakub

last: Tomala

first: Joseph

middle: R

last: Gould

first: Michael

middle: I

last: Leff

first: Jian-Xin

last: Lin

first: Peng

first: Mitchell

last: Porter

first: Eric

last: Johansen

first: Ladaisha

last: Thompson

first: Shanelle

middle: D

last: Cao

first: Shenda

last: Hou

first: Tereza

last: Henclova

first: Maros

last: Huliciak

first: Paul

last: Sargunas

first: Charina

last: Fabilane

first: Ondřej

last: Vaněk

first: Marek

last: Kovar

first: Bohdan

last: Schneider

first: Giorgio

last: Raimondi

first: Warren

first: Jamie

middle: B

last: Spangler


title: Effect of Carbon Nanoparticle Treatment in Rice Adult plants

city: Bhubaneswar

first: Madhusmita

last: Panigrahy

state: Orissa

address: Siksha O Anusandhan University

country: India

institute: Government

summary: Carbon Nanoparticle (CNP, Graphite nano powder,Type 1, APS : 400 nm, Sisco Research Laboratories Pvt. Ltd. India ) was applied to roots of rice (Oryza sativa) plants (SWARNA (MTU7029) seeds were dehulled, surface genotype) in potted condition. The treatment was given in vegetative stage and the effect was noted in reproductive stage by taking several agronomic parameters. Experiment was repeated in 3 different seasons to take a conclusion.

overall_design: Agilent one-color experiment,Organism: Rice ,Agilent Microarray Gene Expression 8x60k Arrays AMADID: 64722

first: Kishore

middle: C

last: Panigrahi


title: Insights into deuterostome evolution from the biphasic transcriptional programmes of hemichordates [RNA-Seq]

city: Sevilla

first: Juan

middle: J.

last: Tena

address: Univ. Pablo Olavide, Ctra. Utrera km1

country: Spain

institute: CABD/CSIC

summary: Evolutionary history of deuterostomes has remained unsolved and is intimately related to how chordates originated. Within the clade of deuterostomes, hemichordates and echinoderms (together called Ambulacraria) are sister groups of chordates; comparative studies among the three are insightful for understanding deuterostome evolution. Indirect-developing hemichordates, such as Ptychodera flava, develop into planktonic larvae, which are morphologically similar to echinoderm larvae, before metamorphosing into the adult body plan that retains an anteroposterior polarity homologous to that of chordates. Therefore, deciphering developmental programmes of indirect-developing hemichordates may provide a key for understanding the evolution of deuterostomes and chordate origins. Here, we characterise transcriptomes and chromatin accessibility across multiple developmental stages of P. flava and uncover biphasic developmental programmes controlled by different sets of transcription factors and their corresponding cis-regulatory elements. Transcriptome age and network analyses reveal that the gastrula transcriptome is relatively ancient and highly connected. By comparing developmental transcriptomes of hemichordate, sea urchin and amphioxus, a high conservation of gene expression during gastrulation is identified and extended to neurula stages of amphioxus, in addition to the highly similar larval transcriptomes among the three species. Moreover, P. flava possesses conserved interactions of transcription factors required for the development of echinoderm endomesoderm and chordate axial mesoderm. These results suggest a deuterostome phylotypic stage during gastrulation (corresponding to amphioxus gastrulation and neurulation) controlled by gene regulatory networks with conserved cis-regulatory interactions, and support the hypothesis that the deuterostome ancestor is an indirect-developer.

overall_design: RNAseq experiments in different developmental stages in Ptychodera flava

first: Alberto

last: Perez-Posada

first: Che-Yi

last: Lin

first: Ching-Yi

first: Yi-Chih

last: Chen

first: José

last: Skarmeta

first: Jr-Kai

last: Yu

first: Yi-Hsien

last: Su


title: TBC1 domain-containing proteins are frequently involved in triple-negative breast cancers in connection with the induction of a glycolytic phenotype

first: Stefano

last: Confalonieri

address: Via Adamello 16

department: Department of Experimental Oncology

institute: IEO, European Institute of Oncology IRCCS, Via Ripamonti 435, 20141, Milan, Italy.

summary: Metabolic plasticity is a hallmark of cancer, and metabolic alterations represent a promising therapeutic target. Since cellular metabolism is controlled by membrane traffic at multiple levels, we investigated the involvement of TBC1 domain-containing proteins (TBC1Ds) in the regulation of cancer metabolism. These proteins are characterized by the presence of a RAB-GAP domain, the TBC1 domain, and typically function as attenuators of RABs, the master switches of membrane traffic. However, a number of TBC1Ds harbor mutations in their catalytic residues, predicting biological functions different from direct regulation of RAB activities. Herein, we report that several genes encoding for TBC1Ds are expressed at higher levels in triple-negative breast cancers (TNBC) vs. other subtypes of breast cancers (BC), and predict prognosis. Orthogonal transcriptomics/metabolomics analysis revealed that the expression of prognostic TBC1Ds correlates with elevated glycolytic metabolism in BC cell lines. In-depth investigations of the three top hits from the previous analyses (TBC1D31, TBC1D22B and TBC1D7) revealed that their elevated expression is causal in determining a glycolytic phenotype in TNBC cell lines. We further showed that the impact of TBC1D7 on glycolytic metabolism of BC cells is independent of its known participation in the TSC1/TSC2 complex and consequent downregulation of mTORC1 activity. Since TBC1D7 behaves as an independent prognostic biomarker in TNBC, it could be used to distinguish good prognosis patients who could be spared aggressive therapy from those with a poor prognosis who might benefit from anti-glycolytic targeted therapies. Together, our results highlight how TBC1Ds connect disease aggressiveness with metabolic alterations in TNBC. Given the high level of heterogeneity among this BC subtype, TBC1Ds could represent important tools in predicting prognosis and guiding therapy decision-making.

overall_design:
High expression of TBC1D7 correlates with worse prognosis in multivariate analysis within the Triple Negative subtype (TNBC) of breast cancer patients. To investigate the underlying molecular mechanism, we selected TNBC cell lines carrying high or low amount of the TBC1D7 protein.
From an initial screening, we identified three lines with these characteristics, MDA-MB-468 (used also in the screening in Fig. 3A) and MDA-MB-231 are high expressors, while Hs578T is a low expressing cell line (Fig. 7A). Silencing of TBC1D7 (Fig. 7A) significantly decreased the intracellular levels of L-lactate in the high expressors (MDA-MB-468 and MDA-MB-231, for a rescue experiment, see Fig. 7C), while it did not affect the low-expressing cell line Hs578T (Fig. 7B). The reduction in L-lactate production was mirrored by a reduction in glucose uptake, which is also a hallmark of glycolytic metabolism (Fig. 7C). Thus, in model TNBC cell lines, high levels of TBC1D7 expression are needed to maintain active glycolysis.
To gain insights into the glycolytic function of TBC1D7 we performed RNAseq analysis from MDA-MB-468 cells in which we downregulated the expression of TBC1D7. This was achieved by transient transfection of a pool of 4 siRNA oligos for TBC1D7 (siTBC1D7) or non-silencing control oligos (siCTRL)

first: Mariadomenica

last: Lupi

first: Daniele

last: Avanzato

first: Flavia

last: Martino

first: Rosa

last: Pennisi

first: Emanuela

last: Pupo

first: Valentina

last: Audrito

last: Freddi

first: Giovanni

last: Bertalot

first: Francesca

last: Montani

first: Bronislava

last: Matoskova

first: Sara

last: Sigismund

first: Pier

middle: P

last: Di Fiore

first: Letizia

last: Lanzetti


title: Insights into deuterostome evolution from the biphasic transcriptional programmes of hemichordates [ATAC-Seq]

first: Juan

last: Tena

overall_design: ATACseq experiments in different developmental stages in Ptychodera flava

first: Alberto

last: Perez-Posada

first: Che-Yi

last: Lin

first: Ching-Yi

first: Yi-Chih

last: Chen

last: Skarmeta


title: Androgen regulation of T cell-intrinsic mechanisms contributes to the sex bias in autoimmunity.

city: Chicago

first: Alexander

last: Chervonsky

state: IL

address: 924 E 57th St

institute: University of Chicago

summary: Major autoimmune diseases such as systemic lupus erythematosus (SLE), multiple sclerosis, rheumatoid arthritis and Graves’ disease display a striking female bias, with a female-to-male incidence ratio ranging up to 9:1 in SLE. Sex hormones contribute to protection of males from autoimmunity, but precise molecular mechanisms of such protection are poorly understood. Here, we find that Androgen Receptor (AR), a nuclear receptor regulating a plethora of genes, is active in T cells during development and regulates directly genes involved in T cell activation or indirectly through regulation of other transcription factors. A gene encoding a phosphatase Ptpn22, a negative regulator of T cell receptor signaling, was found to be dependent of the presence of androgen receptor (AR) in males. Castration or deletion of AR reduced expression of Ptpn22. In a mouse model of Systemic Lupus Erythematosus (SLE), Ptpn22 deletion led to the loss of sexual dimorphism. Moreover, analysis of the regulatory regions of Ptpn22 gene revealed a highly conserved sequence that was necessary for upregulation of the gene’s expression by androgens. Mutation of this sequence in Non-Obese Diabetic (NOD) mice led to enhanced ability of T cells to cause Type 1 diabetes. Thus, PTPN22 is likely to participate in disease pathogenesis making it and other AR-regulated genes fair targets for therapeutic interventions in major autoimmune diseases.

overall_design: Pancreatic lymph nodes from three mice per group were harvested and single cell RNA-Seq was performed.

first: Lee

last: Jean

first: Reiman

last: Derek

first: Khan

last: Aly

first: Chervonsky

last: Alexander


title: Combination anti-PD-1 and anti-CTLA-4 therapy generates waves of clonal responses that include progenitor-exhausted CD8 T cells [dataset 1]

city: Philadelphia

last: Huang

state: PA

address: 421 Curie Boulevard

department: Department of Hematology/Oncology

institute: University of Pennsylvania

summary: Combination checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies has shown promising efficacy in melanoma. However, the underlying mechanism in humans remains unclear. We performed RNA+TCR single-cell analysis across time in 36 stage IV melanoma patients treated with anti-PD-1, anti-CTLA-4, or combination therapy. We developed the algorithm Cyclone to track temporal clonal dynamics and underlying cell states. Checkpoint blockade induced waves of clonal T cell responses that peaked at distinct timepoints. Combination therapy resulted in greater magnitude of clonal responses at 6 and 9 weeks compared to single-agent therapies, including melanoma-specific CD8 T cells and exhausted CD8 T cell (TEX) clones. Focused analyses of TEX identified that anti-CTLA-4 induced robust expansion and proliferation of progenitor TEX, which synergized with anti-PD-1 to reinvigorate TEX during combination therapy. These next generation immune profiling approaches can guide the selection of drugs, schedule, and dosing for novel combination strategies.

overall_design:
We performed single-cell RNA+TCR+ADT sequencing on PBMCs collected from 32 melanoma patients at baseline and every 3 weeks after checkpoint blockade (aPD-1, aCTLA-4, or combination aPD-1+aCTLA-4).

The 'cd8_nn_labeled_FINAL.RDS' is a Seurat object which contains all the non-naive CD8 T cell single-cell data used for the associated manuscript.

first: Kevin

last: Wang

first: Coutifaris

last: Paulina

first: David

last: Brocks

first: Guanning

first: Tarek

last: Azar

first: Sabrina

last: Solis

first: Ajeya

last: Nandi

first: Shaneaka

last: Anderson

first: Nicholas

last: Han

first: Sasikanth

last: Manne

first: Evgeny

last: Kiner

first: Chirag

last: Sachar

first: Minke

last: Lucas

first: Sangeeth

last: George

first: Patrick

last: Yan

first: Melanie

middle: W

last: Kier

first: Amy

last: Laughlin

first: Shawn

last: Kothari

first: Josephine

last: Giles

first: Divij

last: Mathew

first: Reem

last: Ghinnagow

first: Cecile

last: Alanio

first: Ahron

last: Flowers

first: Wei

last: Xu

first: Daniel

last: Tenney

first: Xiaowei

first: Ravi

last: Amaravadi

first: Giorgos

last: Karakousis

first: Lynn

last: Schuchter

first: Marcus

last: Buggert

first: Derek

last: Oldridge

first: Andy

last: Minn

first: Christian

last: Blank

first: Jeffrey

last: Weber

first: Tara

last: Mitchell

first: Michael

last: Farwell

first: Ramin

last: Herati


title: Identification of a new family of peptidoglycan transpeptidases reveals unusualcrosslinking is essential for viability inC. difficile

city: Iowa city

first: Craig

last: Ellermeier

state: Iowa

address: 431 Newton Road, Room 200 E

department: Department of Microbiology and Immunology

institute: University of Iowa

summary: Most bacteria are surrounded by a peptidoglycan cell wall composed of glycan strands held together by short peptide crosslinks. There are two major types of crosslinks, termed 4-3 and 3-3 based on the amino acids involved. 4-3 crosslinks are created by penicillin-binding proteins (PBPs), while 3-3 crosslinks created byL,D-transpeptidases (LDTs). In well studied bacteria 3-3 crosslinks comprise only about 10% of the total and are not essential. However, in the opportunistic intestinal pathogenClostridioides difficile,about 70% of the crosslinks are 3-3. We show here that 3-3 crosslinks and LDTs are essential for viability inC. difficile. We also show thatC. difficilehas five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many Gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficileVanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a new family of peptidoglycan crosslinking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 crosslinking is essential for viability inC. difficile, the first time this has been shown in any bacterial species.

overall_design: We compared the mRNA profile of WT C. difficile with that of our ∆ldt1∆ldt1∆ldt3 mutant to identify any change in gene expression between our mutant and WT

first: Kevin

last: Bollinger

first: David

last: Weiss


title: Transcriptomic heterogeneity of EGFR-mutant non-small cell lung cancer evolution towards small cell lung cancer

city: Seoul

first: Songji

last: Oh

address: 101, Daehak-ro, Jongno-gu

country: South Korea

institute: Cancer Research Institute, Seoul National University

summary: Histological transformation from epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is the major resistance mechanism of EGFR tyrosine kinase inhibitors (TKIs). In our analysis of 59 regions of interest from EGFR-mutant NSCLC or combined SCLC/NSCLC tumors, we compared the transcriptomic profiles before and after transformation.

overall_design: In this study, clinical specimens from 10 patients were selected for spatial and whole transcriptome analysis. To decipher the molecular changes that occur during transformation, we conducted DEG analysis by categorizing the patients into NSCLC and SCLC groups, irrespective of their EGFR-TKI treatment state. This study was approved by the Institutional Review Board of the Seoul National University Hospita and performed in accordance with the Declaration of Helsinki.

first: Jaemoon

last: Koh

first: Tae Min

last: Kim


title: Muscle-resident mesenchymal progenitors sense and repair peripheral nerve injury via the GDNF-BDNF axis

first: Kyusang

last: Yoo

address: 1 Gwanak-ro, Gwanak-gu

department: Biological Sciences Department

institute: Seoul National University

summary: Fibro-adipogenic progenitors (FAPs) are muscle-resident mesenchymal progenitors that can contribute to muscle tissue homeostasis and regeneration, as well as postnatal maturation and lifelong maintenance of the neuromuscular system. Recently, traumatic injury to the peripheral nerve was shown to activate FAPs, suggesting that FAPs can respond to nerve injury. However, questions of how FAPs can sense the anatomically distant peripheral nerve injury and whether FAPs can directly contribute to nerve regeneration remained unanswered. Here, utilizing single-cell transcriptomics and mouse models, we discovered that a subset of FAPs expressing GDNF receptors Ret and Gfra1 can respond to peripheral nerve injury by sensing GDNF secreted by Schwann cells. Upon GDNF sensing, this subset becomes activated and expresses Bdnf. FAP-specific inactivation of Bdnf (Prrx1-Cre; Bdnf-fl/fl) resulted in delayed nerve regeneration owing to defective remyelination, indicating that GDNF-sensing FAPs play an important role in the remyelination process during peripheral nerve regeneration. In aged mice, significantly reduced Bdnf expression in FAPs was observed upon nerve injury, suggesting the clinical relevance of FAP-derived BDNF in the age-related delays in nerve regeneration. Collectively, our study revealed the previously unidentified role of FAPs in peripheral nerve regeneration, and the molecular mechanism behind FAPs’ response to peripheral nerve injury.

overall_design: Single cell RNA-sequencing analysis was performed using FACS-sorted FAPs from limb muscles below knee after either sciatic nerve crush injury or denervation, at days 3, 7, 14, and 28 post injury, using 12-week-old B6 mice. An uninjured control was also sampled for analysis.

first: Young-Yun

last: Kong


title: Temporal maturation of Sertoli cells during the establishment of the cycle of the seminiferous epithelium

city: Pullman

last: Griswold

state: WA

address: Biotechnilogy Life Sciences

department: School of Molecular Biosciences; Center for Reproductive Biology

institute: Washington State University

summary: Sertoli cells, omnipresent, somatic cells within the seminiferous tubules of the mammalian testis are essential to male fertility. Sertoli cells maintain the integrity of the testicular microenvironment, regulate hormone synthesis, and of particular importance, synthesize the active derivative of vitamin A, all trans retinoic acid (atRA), which is required for germ cell differentiation and the commitment of male germ cells to meiosis. Stages VII-IX, when atRA synthesis occurs in the testis, coincides with multiple germ cell development and testicular restructuring events that rely on Sertoli cell gene products to proceed normally. In this study, we have synchronized and captured the mouse testis at four recurrent points of atRA synthesis to observe transcriptomic changes within Sertoli cells as mice age and the Sertoli cells are exposed to increasingly developed germ cell subtypes. This work provides comprehensive, high-resolution characterization of when known, functional Sertoli cell genes are induced across the first wave of spermatogenesis, and outlines in silico predictions of germ cell derived signaling mechanisms targeting Sertoli cells.

overall_design: High throughput expression profiling of synchronized whole testis samples at successive occurences of stages VIII-IX of the cycle of the seminiferous epithelium.

first: Shelby

middle: L

last: Havel


title: Insights into deuterostome evolution from the biphasic transcriptional programmes of hemichordates

first: Juan

last: Tena

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

overall_design: Refer to individual Series


title: Abundant mRNA m1A modification in dinoflagellates: a new layer of gene regulation [Nanopore]

city: Shenzhen

first: Hao

last: Chen

address: 1088 Xueyuan Avenue

country: China

institute: Southern University of Science and Technology (SUSTech)

summary: Dinoflagellates, a class of unicellular eukaryotic phytoplankton, exhibit minimal transcriptional regulation, representing a unique model for exploring gene expression. The biosynthesis, distribution, regulation, and function of mRNA N1-methyladenosine (m1A) remain controversial due to its limited presence in typical eukaryotic mRNA. This study found that m1A, rather than N6-methyladenosine (m6A), is the most prevalent internal mRNA modification in various dinoflagellate species, with an asymmetrically distribution along mature transcripts. In Amphidinium carterae, we identified 6549 m1A sites characterized by a non-tRNA T-loop-like sequence motif within the transcripts of 3196 genes, many involved in regulating carbon and nitrogen metabolism. With enrichment within 3'UTR, dinoflagellate mRNA m1A exhibits negative correlation with translation efficiency. Notably, nitrogen depletion led to a significant decrease in mRNA m1A. Furthermore, our analysis revealed that distinctive patterns of m1A modification influence the expression of metabolism-related genes through translation control. Thus, this study provides a comprehensive map of m1A in dinoflagellate mRNA, highlighting its crucial role as a post-transcriptional regulatory layer and enhancing the understanding of mRNA m1A modification.

overall_design: To determine the polyA length of transcripts, we directly sequenced the full-length mRNA using Direct RNA Sequencing (DRS)strategy with PromethION DNA sequencer (Oxford Nanopore).

first: Chongping

last: Li

first: Ying

first: Yuci

last: Wang

first: Jia

last: Guo

first: Jiawei

last: Xu


title: Human Kidney RNA seq

city: Birmingham

first: Kelly

middle: Anne

last: Hyndman

state: Al

address: 1918 University BLVD

department: Medicine

institute: University of Alabama at Birmingham

summary: Deidentified Total RNA from human kidney samples was purchased from Origene (Rockville, MD).  cDNA libraries were prepared and sequenced. Samples were deemed normal by a pathologist associated with Origene.  Samples were from men and women, and were either kidney cortex or contained some medulla.

overall_design: Deidentified total RNA from human kidney samples that were deemed normal (no pathology) from cortex and medulla samples were obtained from Origene (Rockville, MD).  100 ng of RNA was used to generate cDNA libraries using the Illumina platform and sequenced with the Novaseq500.

first: Anna

last: Montgomery


title: Abundant mRNA m1A modification in dinoflagellates: a new layer of gene regulation


title: Transcription factor PATZ1 promotes adipogenesis by controlling promoter regulatory loci of adipogenic factors [ChIPseq_D-2_D0_D5_D8]

status: Public on Jul 29 2024

city: New York

first: Prashant

last: Rajbhandari

state: New York

address: 1468 Madison Ave, Annenberg 18-10

institute: Icahn School of Medicine at Mount Sinai

summary: White adipose tissue (WAT) plays a central role in lipid storage and systemic energy, lipid, and glucose homeostasis. Understanding the intricacies of adipocyte formation could inform therapies for obesity and metabolic disorders. We have identified the POZ/BTB and AT Hook Containing Zinc Finger 1 (PATZ1) protein as an adipogenic transcription factor through an unbiased high-throughput cDNA screen for transcriptional modulators of adipogenesis. PATZ1 is expressed by both human and mouse adipocyte precursor cells (APCs) and adipocytes, and in cell models, PATZ1 expression promotes adipogenesis through a mechanism dependent on protein-protein interaction and DNA binding. Both adipocyte-specific and APC-specific ablation of PATZ1 in mice leads to decreased fat mass and hypertrophied adipocytes. Genome-wide PATZ1 DNA binding analyses using ChIP-Seq suggest PATZ1 facilitates adipogenesis through interactions with transcription factor machinery at the promoter regions of critical early adipogenic factors and histone modifiers. Purification of the PATZ1 complex showed that General Transcription Factor 2I (GTF2I) associates with PATZ1 in a differentiation-dependent manner. Downregulation of GTF2I levels during adipogenesis markedly augments PATZ1 adipogenic function, suggesting a repressive interaction between GTF2I and PATZ1. These findings identify PATZ1 as a regulator of both adiposity and adipocyte differentiation programs and advance our understanding of the complex transcriptional mechanisms underlying adipose tissue development and homeostasis.

overall_design: PATZ1 ChIP-seq was performed on D0 and D5 differentiated 10T1/2 cells

first: P


title: snRNAseq RAT HEART WT BMD and DMD 12months

city: Creteil

first: Valentina

last: Taglietti

address: 8 rue General Sarrail

country: France

institute: Univ Paris-Est Créteil, INSERM, U955 IMRB

summary: Unrevealing Tmem65-Connexin43 cardiac alterations in a novel preclinical   Becker muscular dystrophy animal model

overall_design: RAT Heart WT, BMD and DMD 12months

first: Kaouthar

last: Kefi

first: Iwona

last: Bronisz-Budzyńska

first: Busra

last: Mirciloglu

first: Alan

last: Jorge

first: Vassiliki

last: Gkouni

first: Carlotta

last: Ferri

first: Christel

last: Gentil

first: France

last: Pietri-Rouxel

first: Peggy

last: Lafuste

first: Laurent

last: Tiret

first: Frederic

last: Relaix

first: Edoardo

last: Malfatti


title: Transcription factor PATZ1 promotes adipogenesis by controlling promoter regulatory loci of adipogenic factors [ChIPreChIPseq_D0_D5]


title: Single-cell/single-nucleus RNA-seq of diffuse hemispheric gliomas, H3G34-mutant.

city: Boston

first: Ilon

last: Liu

state: MA

address: 360 Longwood Avenue

department: Pediatric Oncology

institute: DFCI

summary: As part of a multi-omics and CRISPR/Cas9-screen based study to extensively characterize the molecular basis of diffuse hemispheric gliomas, H3G34-mutant (DHG-H3G34), we applied single-cell/single-nucleus RNA-sequencing to a patient cohort of 9 fresh and frozen untreated DHG-H3G34.

overall_design:
Single-cell/single-nucleus RNA-seq (Smart-seq2) of 9 DHG-H3G34.

>>>Submitter states that raw data have not been provided due to patient privacy concerns<<<

first: Mariella

last: Filbin


title: Resolving Resident Colonic Muscularis Macrophage Diversity and Plasticity During Colitis

first: Rhian

last: Stavely

address: 185 Cambridge Street, CPZN, 6100

department: Pediatric Surgery

institute: Massachusetts General Hospital

summary: Background: Immune cell populations within the intestinal muscularis propria are poorly resolved during colitis. Maintaining homeostasis in this unique niche is of critical importance, highlighted by the poorer prognosis of inflammatory bowel disease associated with inflammation in the muscularis propria. Methods: This study utilizes single-cell RNA sequencing to survey the immune cell populations within the muscularis propria of normal colon and DSS-induced colitis. Results: In naïve conditions, transcriptional duality is observed in MMφ with two major supopulations: conventional resident Cx3cr1+ MMφs and Lyve1+ MMφs. During colitis, significant changes occur within the muscularis propria, with increases in B-cells, T-cells, monocytes, neutrophils, and dendritic cells. Unlike in the mucosa, single cell transcriptomics indicates that resident MMφs are retained during colitis and exhibit plasticity toward an inflammatory profile. Lyve1+ MMφs, which express anti-inflammatory marker CD163, are absent during colitis. In contrast, resident Cx3cr1+ MMφs remain during colitis. Conclusions: Our findings provide a resource for understanding the immune system in the muscularis propria niche during colitis and resolve the heterogeneity and origins of proinflammatory MMφs during colitis by demonstrating the plasticity of the persistent MMφ population.

overall_design: To induce acute colitis B6 mice were provided ad libitum access to drinking water containing 2.5% DSS (36,000 - 50,000 Da, MP Biomedicals, CA) for 7 days before sacrifice. Naïve mice and littermates exposed to DSS to induce colitis of each gender were euthanized and colons collected for isolation of the muscularis propria to analyze leukocytes and enteric glia.

first: Kensuke

last: Ohishi

first: David

last: Dora

first: Christopher

last: Han

first: Richard

middle: A

last: Guyer

first: Takahiro

last: Ohkura

first: Nicole

last: Picard

first: Abigail

last: Leavitt

first: Nandor

last: Nagy

first: Ryo

last: Hotta

first: Allan

middle: M

last: Goldstein


title: Diametral influence of Deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) on the growth of Campylobacter jejuni with consequences on the bacterial transcriptome

city: Vienna

first: Wageha

middle: A.

last: Awad

address: Veterinarplatz 1/23

department: Department for Farm Animals and Veterinary Public Health

country: Austria

institute: University of Veterinary Medicine

summary: Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly found in cereals and grains worldwide. The presence of this fungal secondary-metabolite raises public-health concerns at both the agriculture and food industry level. The toxicity of DON is mainly characterized by its ability to inhibit ribosomal protein biosynthesis. Recently, we have shown that DON has a negative impact on gut integrity, a feature also noticed for Campylobacter (C.) jejuni. We further demonstrated that DON increased the load of C. jejuni in the gut and inner organs. In contrast, feeding the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) to broilers reduced the Campylobacter load in vivo. Consequently, it can be hypothesized that DON and DOM-1 have a direct or indirect effect on the growth profile of C. jejuni. The aim of the present study was to further resolve the nature of this interaction in vitro by co-incubation and RNA-sequencing.  The co-incubation of C. jejuni with DON resulted in significantly higher bacterial growth rates from 30 h of incubation onwards. On the contrary, the co-incubation of C. jejuni with DOM-1 reduced the CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni in birds, altogether confirming in vivo data. Furthermore, the transcriptomic profile of C. jejuni following incubation with either DON or DOM-1 differed. Co-incubation of C. jejuni with DON significantly increased the expression of multiple genes which are critical for Campylobacter growth, particularly members of the Flagella gene family, frr (ribosome-recycling factor), PBP2 futA-like (Fe3+ periplasmic binding family) and PotA (ATP-binding subunit). These organelles are required for pathogenicity-related phenotypes including motility, biofilm formation, host cell interactions, and host colonization, which may explain the high Campylobacter load in the intestine of DON-fed broiler chickens. On the contrary, DOM-1 downregulated the Flagella gene family and upregulated ribosomal proteins.  The results highlight the adaptive mechanisms involved in the transcriptional response of C. jejuni to DON and its metabolite DOM-1, based on the following effects: (a) ribosomal proteins; (b) flagellar proteins; (c) engagement of different metabolic pathways. The results provide insight into the response of an important intestinal microbial pathogen against DON and lead to a better understanding of the luminal or environmental acclimation mechanisms in chickens.

overall_design: Bacteria (Campylobacter jejuni) were harvested at 30h and 48h. For each time point, four pooled biological replicates were used as control for C. jejuni and three pooled biological replicates for C. jejuni + DON, and for C. jejuni + DOM-1, respectively. A toatl of 20 samples were used for RNA-Seq analysis.

first: Bertrand

last: Grenier

first: Daniel

last: Ruhnau

first: Claudia

last: Hess

first: Dian

last: Schatzmayr

first: Michael


title: Transcription factor PATZ1 promotes adipogenesis by controlling promoter regulatory loci of adipogenic factors [RNA-seq]

first: Prashant

last: Rajbhandari

overall_design: RNA-seq was performed on D8 differentiated 10T1/2 cells stably expressing Vector or PATZ1

first: P


title: Transcription factor PATZ1 promotes adipogenesis by controlling promoter regulatory loci of adipogenic factors [ChIPseq_D0_D5]


title: Early Moderate Prenatal Alcohol Exposure and Maternal Diet Impact Offspring DNA Methylation Across Species

city: Perth

first: Nikki

last: Schultz

address: 15 Hospital Ave Nedlands

country: Australia

institute: Telethon Kids Institute

type: Methylation profiling by high throughput sequencing

summary: Alcohol consumption in pregnancy can affect genome regulation in the developing offspring but results have been contradictory. We employed a physiologically relevant murine model of short-term moderate prenatal alcohol exposure (PAE) resembling common patterns of alcohol consumption in pregnancy in humans. Early moderate PAE was sufficient to affect site-specific DNA methylation in new-born pups without altering behavioural outcomes in adult littermates. Whole-genome bisulfite sequencing of neonatal brain and liver revealed stochastic influence on DNA methylation that was mostly tissue-specific, with some perturbations likely originating as early as gastrulation. DNA methylation differences were enriched in non-coding genomic regions with regulatory potential indicative of broad effects of alcohol on genome regulation. Replication studies in human cohorts with fetal alcohol spectrum disorder suggested some effects were metastable at genes linked to disease-relevant traits including facial morphology, intelligence, educational attainment, autism, and schizophrenia. In our murine model, a maternal diet high in folate and choline protected against some of the damaging effects of early moderate PAE on DNA methylation. Our studies demonstrate that early moderate exposure is sufficient to affect fetal genome regulation even in the absence of overt phenotypic changes and highlight a role for preventative maternal dietary interventions.

overall_design:
To investigate the effects of early moderate prenatal alcohol exposure in mice and the effect of a high methyl donor diet, we designed an experiment with four treatment arms: prenatal alcohol exposure-normal chow, prenatal alcohol exposure-high methyl donor diet, water-normal chow and water-high methyl donor diet.    We conducted whole-genome bisulfite sequencing on newborn mouse brain and liver, selecting two males and two females  from each group (n = 16).
We used human datasets generated from Illumina Human Methylation EPIC array to validate observations from the murine model.

first: Mitchell

last: Bestry

first: Alexander

middle: N

last: Larcombe

first: Nina

last: Kresoje

first: Emily

last: Chivers

first: Chloe

last: Bakker

first: James

last: Fitzpatrick

first: Elizabeth

middle: J

last: Elliott

first: Jeffrey

last: Craig

first: Evelyne

last: Muggli

first: Jane

last: Halliday

first: Delyse

last: Hutchinson

first: Sam

last: Buckburry

first: Ryan

last: Lister

first: Martyn

last: Symons

first: David

last: Martino


title: Mechanism of homology search expansion during recombinational DNA repair

city: Lyon

first: Aurele

last: Piazza

address: 46 Allee d'Italie

institute: ENS Lyon

summary: Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination. How it operates in cells remains elusive. Here we developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembly of extensive NPFs. DSB end-tethering promotes coordinated homology search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search orchestrated by chromatin organizers, long-range resection, end-tethering, specialized genetic elements, and that exploits the stiff NPF structure conferred by Rad51 oligomerization.

overall_design: Hi-C and a Hi-C-based experiment enabling detection of ssDNA contacts were performed on S. cerevisiae cultures following site-specific formation of a DNA double-strand break on chr. V, in wild-type cells and various mutants and treatment conditions affecting homologous recombination and/or chromosome structure..

first: Agnès

last: Dumont

first: Nicolas

last: Mendiboure

first: Jérôme

last: Savocco

first: Aurèle


title: Discovery and prediction study of the dominant pharmacological action organ of Aconitum carmichaeli Debeaux using multiple bioinformatic analyses

city: Daejeon

first: Musun

last: Park

address: 1672 Yuseong-daero, Yuseong-gu

institute: Korean Institute of Oriental Medicine

summary: This study aimed to identify the modes of action (MOA) of Aconitum carmichaeli Debeaux water extract (ACDW) in HT29 and SW1783 cell line.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for ACDW (500 ug/mL, 100 ug/mL, and 20 ug/mL, respectively) and vehicle control (2% DMSO).


title: Immune profiling of lymph nodes drained to cold vs hot triple-negative breast tumors.

city: Duarte

first: Peter

last: Lee

state: CA

address: 1500 E. Duarte Rd.

department: Immuno-Oncology

institute: Beckman Research Institute at City of Hope

summary: NanoString pan-cancer immune panel was used to quantified the expression of 730 immune genes of 15 tumor-free TDLNs and paired tumors. FFPE slides were used to quantified the gene expression.

overall_design: We collected and analyzed 7 cold tumor-TDLNCold pairs and 8 hot tumor-TDLNHot pairs. FFPE samples were subjected to NanoString analyses. By using the NanoString Pan-Cancer immune panel, we quantified the expression of 730 immune genes of these 15 tumor-free TDLNs and paired tumors. Stromal TIL percentage of each tumor was scored by a pathologist from H&E-stained slides.  Tumors were graded as cold (<10% stromal TIL), hot (>60% stromal TIL), or intermediate (10-60% stromal TIL).

first: Weihua

last: Guo

first: Minhui

last: Lim

first: Colt

last: Egelston

first: Lei

last: Wang


title: Abundant mRNA m1A modification in dinoflagellates: a new layer of gene regulation [m1A-seq-TGIRT]

first: Hao

last: Chen

overall_design: To gain further insight into the roles of m1A, we sought to characterize m1A sites at single-base resolution using m1A-seq-TGIRT. For m1A-seq-TGIRT, 25 µL of prewashed Protein A/G Dynabeads were incubated with 3 µL of anti-m1A antibody (MBL #D345-3) at 4 °C for two hours. Subsequently, 500 ng of fragmented mRNA was used for immunoprecipitation, with 50 ng of purified mRNA taken as input. After digestion with Proteinase K, the RNA was purified using RNA Extraction Reagent (Shanghai Acmec Biochemical Co., Ltd, China) and then subjected to library construction. Briefly, the purified mRNA was treated with Antarctic Phosphatase (NEB) and T4 PNK (NEB) at 37 °C for 30 minutes, followed by purification using the Oligo Clean & Concentrator kit (Zymo Research). The dephosphorylated RNA was then ligated to the 3'-RNA adaptor (/5'Phosphate/AGAUCGGAAGAGCGUCGUG/ddC) using T4 RNA Ligase I (NEB) at 23°C for two hours, purified by Oligo Clean & Concentrator again, and reverse transcribed using TGIRT-III (Haigne, China) with the RT primer (5'-ACACGACGCTCTTCCGA-3') in RT reaction buffer (50 mM Tris–HCl pH 8.3, 75 mM KCl, and 3 mM MgCl2) at 57 °C for two hours. The reaction was terminated with 25 mM EDTA and RNA was degraded with 150 mM NaOH, heated to 70 °C for 12 minutes, and purified with Oligo Clean & Concentrator kit. The 5'-DNA adaptor (/5'Phosphate/AGATCGGAAGAGCACACGTCTG/ddC) was ligated using T4 RNA ligase I (NEB) at 23 °C overnight. The single-stranded cDNA was then amplified by PCR using specific primers (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' and 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3', NNNNNN denotes the 6-bp barcode sequence), and the product was purified with AMPure XP beads (0.9×). Both input and IP samples were sequenced by GenePlus (Shenzhen, China) on DNBSEQ-T7 platform with paired-end 150 bp mode.

first: Chongping

last: Li

first: Ying

first: Yuci

first: Jia

first: Jiawei

last: Xu


title: RT-PCR analysis of placental tissue with chronichistiocytic intervillositis

city: BORDEAUX

first: Estibaliz

last: LAZARO

address: Avenue Magellan

institute: Bordeaux Hospital University

summary: Chronic histiocytic intervillositis of unknown origin (CHI) is a rare placental disorder associated with adverse pregnancy outcomes, frequent recurrence, and a lack of effective preventive strategies. Recent insights indicate a potential link between CHI-associated inflammatory lesions and the inflammasome pathway, suggesting innovative therapeutic avenues. Here we show a potential role of the inflammasome pathway in CHI through comprehensive transcriptomic analysis of grade 2 or 3 histopathologic CHI samples, paired with placental controls. Additionally, we present case studies of three individuals with recurrent CHI, who have undergone treatment with anakinra and colchicine throughout pregnancy, resulting in improved perinatal outcomes. Notably, all cases are characterised by the birth of healthy, full-term infants, with reduced or absent intervillositis recurrence. Placental assessment unveils heightened activation of the NLRP3-PYCARD inflammasome pathway and IL-1β processing in CHI samples, with downregulation observed in treated pregnancy samples, devoid of intervillositis. Collectively, these findings suggest a potential therapeutic role for targeting the inflammasome pathway in preventing recurrent CHI in pregnant individuals

overall_design: 24 placental samples analysed including 18 intervillositis samples matched in a 3:1 ratio with placental controls (6 samples) based on gestational age, maternal age, and fetal sex

first: A

last: MATTUIZZI

first: E


title: Profiling gene expressions of cancer-associated canonical pathways between cold and hot triple-negative breast tumors.

summary: NanoString nCounter pan-cancer pathway panel was used to quantified the expression of 730 genes related to 13 canncer-assocaited canonical pathways from 15 triple-negative breast tumors. FFPE slides were used to quantified the gene expression.

overall_design: We collected and analyzed 7 cold tumors and 8 hot tumors. FFPE samples were subjected to NanoString analyses. By using the NanoString Pan-Cancer Pathway panel, we quantified the expression of 730 genes related to 13 cancer-associated canonical pathways from these primary tumors. Stromal TIL percentage of each tumor was scored by a pathologist from H&E-stained slides.  Tumors were graded as cold (<10% stromal TIL), hot (>60% stromal TIL), or intermediate (10-60% stromal TIL).


title: Deletion of a state-specific PD-1 enhancer modulates exhausted T cell fate and function [RNA-Seq]

first: Amy

last: Huang

address: 25 Shattuck St

department: Immunology

institute: Harvard Medical School

summary: T cell exhaustion is a state of CD8+ T cell dysfunction elicited by chronic exposure to antigen and inflammation, arises in both cancer and chronic viral infection. The co-inhibitory receptor PD-1 plays a key role in mediating exhaustion, but complete ablation of PD-1 by gene knock-out leads to deeper functional deficits and poor T cell survival. We hypothesized that an intermediate level of PD-1 expression may confer an improved balance of exhausted CD8+ T cell functionality, so we deleted an exhaustion-associated enhancer of PD-1 which indeed resulted in a reduced expression level. We compared EnhDel, WT and PD-1 KO T cells using single-cell RNA-Seq and found that PD-1 KO but not EnhDel cells are strongly biased towards the terminally exhausted subset. EnhDel cells also uniquely enrich for effector-associated genes and gene signatures. However, all three genotypes (EnhDel, WT and PD-1 KO) exhibit a similar chromatin accessibility landscape by ATAC-Seq, controlling for exhausted subset. These data suggest that tuning of PD-1 expression may uniquely permit the maintenance of an “effector” transcriptional profile in exhausted CD8+ T cells.

overall_design: Enhancer-deleted and WT P14+ CD8+ T cells were isolated from donor mice, mixed at a 50:50 ratio, and transferred to congenically-distinct recipient mice (n = 8). Recipients were infected with LCMV Clone 13. Transferred CD8+ T cells were isolated from spleens at day 9. Genotypes (Enhancer-deleted, WT) and cell types (progenitor exhausted SLAMF6+TIM3-, terminally exhausted SLAMF6-TIM3+CX3CR1-) were then isolated by sorting, pooled in pairs to create four samples, and processed for bulk RNA-seq. We then performed differential gene expression analysis using DESeq2 in R to compare the transcriptomic profiles of EnhDel and wild type CD8+ T cells at the D9 time point, controlling for exhausted subset.

first: Sarah

last: Weiss

middle: Y

first: Megan

middle: E

last: Fung

first: Daniela

last: Martinez

first: Alex

first: Thomas

last: LaSalle

first: Brian

last: Miller

first: Christopher

last: Scharer

first: Mudra

last: Hegde

first: Thao

middle: H

last: Nguyen

first: Jared

last: Rowe

first: Jossef

middle: F

last: Osborn

first: Dillon

middle: G

last: Patterson

first: Natalia

last: Sifnugel

first: C

last: Nolan

first: Richard

last: Davidson

first: Marc

last: Schwartz

first: Alexander

last: Bally

first: Dennis

last: Neeld

first: Martin

last: LaFleur

first: Jeremy

last: Boss

first: John

last: Doench

first: W

last: Haining

first: Arlene

last: Sharpe

first: Debattama

last: Sen


title: An Hepatitis B and D Virus Infection Model Using Human Pluripotent Stem Cell-Derived Hepatocyte-Like Cells for Virus Host Interactions and Antiviral Evaluation

city: Heidelberg

first: Viet Loan

last: Dao Thi

address: Im Neuenheimer Feld 344

department: Virology

institute: Heidelberg University

summary: Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co- infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively release infectious progeny virions. We also show that HLCs expressing HBsAg support extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV/HBV co-entry factor, which was rate-limiting HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions.

overall_design: hPSC were differentiated to HLCs and harvested either on Day 17 or on Day 18 of the differentiation protocol to compare their transcriptome profiles.

first: Huanting

last: Chi

first: Viet


title: The nutrient sensor CRTC and Sarcalumenin/Thinman represent a new pathway in cardiac hypertrophy

city: La Jolla

first: Georg

last: Vogler

address: 10901 N Torrey Pines Road

institute: Sanford Burnham Prebys Medical Discovery Institute

summary: CREB-Regulated Transcription Co-activator (CRTC) regulates metabolism in liver where activation by calcineurin regulates gluconeogenic genes. CaN also has roles in pathological cardiac hypertrophy, however cardiac roles for CRTC have not been identified. In Drosophila, CRTC null mutants exhibit severe cardiac restriction, myofibrillar disorganization, cardiac fibrosis, and tachycardia. Cardiac-specific knockdown (KD) of CRTC mimicked the heart defects of CRTC mutants and cardiac-overexpression (OE) of CRTC or calcineurin caused hypertrophy that was reduced in CRTC mutants, suggesting CRTC mediates calcineurin’s effects. RNAseq of CRTC KD or OE hearts revealed contra-regulated genes involved in glucose, fatty acid, and amino acid metabolism. Genes with conserved CREB binding sites included the fly ortholog of Sarcalumenin, a Ca2+-binding protein. Cardiac KD of this gene recapitulated CRTC KD restriction and fibrotic phenotypes. KD in zebrafish also caused restriction, indicating a conserved role in cardiomyocyte maintenance, and suggesting CaN-CRTC-Sarcalumenin signaling represents a novel pathway underlying cardiac hypertrophy.

overall_design:
We aimed at understanding the cardiac role of the CRTC co-factor by comparing gene expression in different experimental conditions.
For pan-tissue effects we prepared total RNA from wildtype and Crtc knock-out flies, while for heart-specific effects we used the heart-specific driver tinCdelta4-Gal4 to drive Crtc-RNAi and Crtc-overexpression (OE).
We used 6 samples for each genotype and performed differential gene expression analysis for the corresponding pairs.

first: Cristiana

last: Dondi

first: Stanley

last: Walls

first: Anjali

last: Gupta

first: Karen

last: Ocorr


title: Single-cell multimodal profiling of monocytes reveals diverse phenotypes and alterations linked to cardiovascular disease risks [CITE-seq]

first: Chenyi

last: Xue

state: NY

institute: Columbia University

summary: Monocytes are a critical innate immune system cell type that serves homeostatic and immunoregulatory functions. They have been identified historically by the cell surface expression of CD14 and CD16. However, recent single-cell studies have revealed that they are much more heterogeneous than previously realized.  We utilized cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing (scRNA-seq) to describe the comprehensive transcriptional and phenotypic landscape of 437,126 monocytes. This high-dimensional multimodal approach identified vast phenotypic diversity and functionally distinct subsets, including IFN-responsive, MHCIIhi, monocyte-platelet aggregates, as well as non-classical, sand several subpopulations of classical monocytes. Using flow cytometry, we validated the existence of MHCII+CD275+ MHCIIhi, CD42b+ monocyte-platelet aggregates, CD16+CD99- non-classical monocytes, and CD99+ classical monocytes. Each subpopulation exhibited unique characteristics, developmental trajectories, transcriptional regulation, and tissue distribution. In addition, alterations associated with cardiovascular disease (CVD) risk factors, including race, smoking, and hyperlipidemia were identified. Moreover, the effect of hyperlipidemia was recapitulated in mouse models of elevated cholesterol. This integrative and cross-species comparative analysis provides a new perspective on comparison of alterations in monocytes in pathological conditions and offers insights into monocyte-driven mechanisms in CVD and the potential for monocyte subpopulation targeted therapies.

overall_design: CITE-seq was performed on blood monocytes from 10 subjects. 5 of the subjects had an additional library with both monocytes and dendritic cells.

last: Bashore


title: Single-cell multimodal profiling of monocytes reveals diverse phenotypes and alterations linked to cardiovascular disease risks

overall_design: Single-cell RNA sequencing (scRNA-seq) was performed on blood monocytes from 118 subjects that were generally healthy.

first: Alexander


title: PHYTOCHROME-INTERACTING FACTOR 7 and RELATIVE OF EARLY FLOWERING 6 act in shade avoidance memory in Arabidopsis

city: Shanghai

first: Qican

last: Cheng

state: Shanghai

address: 2005 Songhu Road, Yangpu District

department: school of life sciences

institute: Fudan university

summary: Shade avoidance helps plants maximize their access to light for growth under crowding. It is unknown, however, whether a priming shade avoidance mechanism exists that allows plants to respond more effectively to successive shade conditions. Here, we show that the shade-intolerant plant Arabidopsis can remember a first experienced shade event and respond more efficiently to the next event on hypocotyl elongation. The transcriptional regulator PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) and the histone H3K27-demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) were identified as being required for this shade avoidance memory. RNA-sequencing analysis revealed that shade induction of shade memory-related genes was impaired in the pif7 and ref6 mutants. Based on the enrichments of H3K27me3, REF6 and PIF7, we found that priming shade treatment induced PIF7 accumulation, which further recruited REF6 to demethylate H3K27me3 on the chromatin of certain memory-related genes, leading to a state poised for their transcription. Upon the second shade treatment, enhanced shade-mediated induction of these genes resulted in stronger hypocotyl growth responses. We conclude that the transcriptional memory mediated by epigenetic modification plays a key role in the ability of primed plants to remember previously experienced shade and acquire enhanced responses to recurring shade conditions.

overall_design: For REF6-HA binding ChIP, proREF6::REF6-HA/ref6-1 (N_WL and N_SH 8 h) and proREF6::REF6-HA/pif7-1 (N_SH 8 h) seedlings were prepared for each genotype of plants grown under white light and shade conditions (N, 4-day-old seedlings + 8 h white light + 40 h white light + 8 h triggering shade), using an anti-HA antibody (Lumiprobe sc-7392 X).


title: TIM-3 on myeloid cells promotes pulmonary inflammation through increased production of galectin-3

city: Goyang-si

first: Ki Sun

last: Kim

state: Gyeonggi-do

address: 323, Ilsan-ro, Ilsandong-gu

institute: National Cancer Center

summary: T cell immunoglobulin and mucin-containing molecule 3 (TIM-3) exhibits unique, cell type- and context-dependent characteristics and functions. Here, we report that TIM-3 on myeloid cells plays essential roles in modulating lung inflammation. We found that myeloid cell-specific TIM-3 knock-in (FSF-TIM3/LysM-Cre+) mice have lower body weight and shorter lifespan than WT mice. Intriguingly, the lungs of FSF-TIM3/LysM-Cre+ mice display excessive inflammation and features of disease-associated pathology. We further revealed that galectin-3 levels are notably elevated in TIM-3-overexpressing lung-derived myeloid cells. Furthermore, both TIM-3 blockade and GB1107, a galectin-3 inhibitor, ameliorated lung inflammation in FSF-TIM3/LysM-Cre+/- mice. Using an LPS-induced lung inflammation model with myeloid cell-specific TIM-3 knock-out mice, we demonstrated the association of TIM-3 with both lung inflammation and galectin-3. Collectively, our findings suggest that myeloid TIM-3 is an important regulator in the lungs and that modulation of TIM-3 and galectin-3 could offer therapeutic benefits for inflammation-associated lung diseases.

overall_design: To gain insight into the mechanisms underlying the ability of myeloid cell-expressed TIM-3 to influence lung inflammation, we performed bulk RNA-sequencing (RNA-seq) analysis of whole lungs from FSF-TIM3/LysM-Cre+/- and FSF-TIM3 WT mice.

first: KiSun

last: KIM

first: Changju

last: Lee

first: EunJung

last: Park


title: A cellular disease model towards gene therapy of TGM1-dependent Lamellar Ichthyosis

city: Padova

first: Oriana

last: Romano

address: Via U. Bassi 58/B

department: Molecular Medicine

institute: University of Padova

summary: Lamellar Ichthyosis (LI) is a chronic disease, mostly caused by mutations in TGM1 gene, marked by impaired skin barrier formation. No definitive therapies are available and current treatments aim at symptomatic relief. LI mouse models often fail to faithfully replicate the clinical and histopathological features of human skin conditions. To develop advanced therapeutic approaches, as combined ex vivo cell and gene therapy, we established a human cellular model of LI by efficient CRISPR-Cas9-mediated gene ablation of the TGM1 gene in human primary clonogenic keratinocytes. Gene edited cells showed complete absence of Transglutaminase-1 (TG1) expression and recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of LI in vitro. Using a SINγ-RV vector expressing transgenic TGM1 under the control of its own promoter, we tested an ex vivo gene therapy approach and validate the model of LI as a platform for pre-clinical evaluation studies. Gene-corrected TGM1-null keratinocytes displayed proper TG1 expression, enzymatic activity and cornified envelopes formation, hence restored proper epidermal architecture. Single cell multiomic analysis demonstrated proviral integrations in holoclone-forming epidermal stem cells, which are crucial to epidermal regeneration. This study serves as a proof-of-concept for assessing the potential of this therapeutic approach in treating TGM1 dependent LI.

overall_design: Primary human keratinocytes from healthy donor were transduced with a self-inactivating retroviral vector carryng the TGM1 coding sequence under the control of its own promoter (endogenous TGM1 promoter). Multiomic analysys were performed on these cells for the simultaneous evaluation of provirus integration and transcriptome profiling within a single cell. For the analisys of keratinocytes population we used a previously defined reference (DOI: 10.1038/s41467-021-22779-9). TGM1-proviral integrations were identified by mapping scATAC-seq data of TGM1-transduced cells to a custom human reference genome that includes the sequence of the SINγ-RV-TGM1 provirus.

first: Laura

last: Sercia

first: Grazia

last: Marini

first: Elena

last: Enzo

first: Mattia

last: Forcato

last: De Rosa

first: Michele

last: De Luca


title: The innate immune regulator MyD88 dampens fibrosis during zebrafish heart regeneration

city: Bad Nauheim

first: Noah

last: Knoppik

address: Ludwigstrasse 43

department: Bioinformatics

institute: Max-Planck-Institute for Heart and Lung Research

summary: The innate immune response is triggered rapidly after injury and its spatiotemporal dynamics are critical for regeneration, but many questions remain about its exact role.  Here we show that MyD88, a key component of the innate immune response, controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration.  We find in cryoinjured myd88-/- ventricles a significant reduction in neutrophil and macrophage numbers as well as the expansion of a collagen-rich endocardial population.  Further analyses reveal compromised PI3K/AKT pathway activation in the myd88-/- endocardium and increased myofibroblasts and scarring.  Notably, endothelial-specific overexpression of myd88 reverses these neutrophil, fibrotic, and scarring phenotypes.  Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a target of the MyD88-signaling pathway, and using loss- and gain-of-function tools show that it controls neutrophil recruitment.  Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis during tissue regeneration.

overall_design: Aim: Investigation of the MyD88 signaling in endocardial cells during zebrafish cardiac regeneration. Methods: Endocardial cells sorted from the ET(krt4:EGFP)sqet33-1A line at 96 hours post cryoinjury (hpci). Conditions: 3 myd88+/+ and 3 myd88-/- samples

first: Pinelopi

last: Goumenaki

first: Stefan

last: Guenther

first: Kikhi

last: Khrievono

first: Mario

last: Looso

first: Rubén

last: Marín-Juez

first: Didier

last: Stainier


title: Nuclear lamin A/C phosphorylation by loss of Androgen Receptor is a global determinant of cancer-associated fibroblast activation

city: Biella

first: Paola

last: Ostano

address: via Malta 3

institute: Fondazione Edo ed Elvo Tempia

summary: Alterations of nuclear structure and function, and associated impact on gene transcription, are a hallmark of cancer cells. Little is known of these alterations in Cancer-Associated Fibroblasts (CAFs), a key component of the tumor stroma. Here we show that loss of androgen receptor (AR), which triggers early steps of CAF activation in human dermal fibroblasts (HDFs), leads to nuclear membrane alterations and increased micronuclei formation, which are unlinked from induction of cellular senescence. Similar alterations occur in fully established CAFs, which are overcome by restored AR function. AR associates with nuclear lamin A/C and loss of AR results in a substantially increased lamin A/C nucleoplasmic redistribution. Mechanistically, AR functions as a bridge between lamin A/C with the protein phosphatase PPP1. In parallel with a decreased lamin-PPP1 association, AR loss results in a marked increase of lamin A/C phosphorylation at Ser 301, which is also a feature of CAFs. Phosphorylated lamin A/C at Ser 301 binds to the transcription promoter regulatory region of several CAF effector genes, which are upregulated due to the loss of AR. More directly, expression of a lamin A/C Ser301 phosphomimetic mutant alone is sufficient to convert normal fibroblasts into tumor-promoting CAFs of the myofibroblast subtype, without an impact on senescence. These findings highlight the pivotal role of the AR-lamin A/C-PPP1 axis and lamin A/C phosphorylation at Ser 301 in driving CAF activation.

overall_design: A human dermal fibroblast strain plus/minus AR silencing was analyzed by CUT&RUN analysis for total and phospho-Ser301 lamin AC and K27aC3.

first: Ghosh

last: Soumitra

first: Dotto

last: Gian Paolo

first: Isma

last: Jovan

first: Mazzeo

last: Luigi

first: Toniolo

last: Annagiada

first: Ostano

last: Paola

first: Guana

last: Francesca

first: Das

last: Monalisa

first: Simon

last: Christian


title: Identification and multimodal characterization of a specialized epithelial cell type associated with Crohn's Disease

city: nashville

first: Qi

last: Liu

state: TN

address: 2525 West End Ave

institute: Vanderbilt univeristy medical center

summary: Crohn’s disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients,  we identified a distinct epithelial cell type in both terminal ileum and ascending colon (termed “LND”) with high expression of LCN2, NOS2, and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, were rare in non-IBD controls but significantly expanded in active CD. These cells actively interacted with immune cells and specifically expressed IBD/CD susceptibility genes, as demonstrated by multimodal data, suggesting a possible role in CD immunopathogenesis. Furthermore, we discovered early and late LND subpopulations with different origins and developmental potential. A higher ratio of late to early LND cells correlated with better response to anti-TNF treatment. These findings suggest a potential pathogenic role for LND cells in both Crohn’s ileitis and colitis.

overall_design:
65 CD and 18 non-IBD control patients in both TI and AC were collected. Tissues were incubated in a chelating buffer (0.5M EDTA, 0.1M DTT in DPBS) for 1.25hrs, and then transferred to cold active protease (5 mg/ml Protease from Bacillus licheniformis, 2.5 mg/mL DNase in PBS) for 25 minutes at 4˚c. Tissues were then pipetted 10-20 times to yield single cells. Cell suspensions were filtered, washed, and inspected for count and quality before loading onto inDrops for microfluidic capture. inDrops scRNA-seq was performed on the Novaseq6000.
Brief descriptions for the various CD types:
Control - non-IBD control, patient without IBD with normal colon histology
Moderate    - CD with chronic epithelial changes and moderately active disease by histology
Normal_CD     - CD with no chronic epithelial changes or active disease by histology
Mild     - CD with chronic epithelial changes and mildly active disease by histology
Quiescent     - CD with chronic epithelial changes but no active disease by histology
Severe  - CD with chronic epithelial changes and severely active disease by histology

first: Keith

middle: T

last: Wilson

first: Lori

last: Coburn

first: Ken

middle: S

last: Lau


city: bad nauheim

first: Stefan

last: Günther

address: ludwigtr. 43

institute: MPI for heart and lung research

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

The innate immune response is triggered rapidly after injury and its spatiotemporal dynamics are critical for regeneration, but many questions remain about its exact role.  Here we show that MyD88, a key component of the innate immune response, controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration.  We find in cryoinjured myd88-/- ventricles a significant reduction in neutrophil and macrophage numbers as well as the expansion of a collagen-rich endocardial population.  Further analyses reveal compromised PI3K/AKT pathway activation in the myd88-/- endocardium and increased myofibroblasts and scarring.  Notably, endothelial-specific overexpression of myd88 reverses these neutrophil, fibrotic, and scarring phenotypes.  Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a target of the MyD88-signaling pathway, and using loss- and gain-of-function tools show that it controls neutrophil recruitment.  Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis during tissue regeneration.

overall_design: Refer to individual Series


title: The innate immune regulator MyD88 dampens fibrosis during zebrafish heart regeneration (dst139)

overall_design: RNA was isolated from myd88+/+ and myd88-/- untouched ventricles and cryoinjured tissues at 1 and 24 hours post cryoinjury (hpci) and analyzed using bulk RNA sequencing

first: Pinelopi

last: Goumenaki

last: Guenther

first: Kikhi

last: Khrievono

first: Mario

last: Looso

first: Rubén

last: Marín-Juez

middle: R


title: The innate immune regulator MyD88 dampens fibrosis during zebrafish heart regeneration (dst169)

summary: The innate immune system is triggered rapidly after injury and its spatiotemporal dynamics are critical for successful regeneration.  MyD88 is a key component of the innate immune response; however, its role during regeneration remains unclear.  Here we show that MyD88 controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration.  Our data reveal a significant reduction in pro-inflammatory neutrophil and macrophage populations, as well as the expansion of a collagen-rich endocardial population in cryoinjured myd88-/- ventricles.  Consistent with these findings, we observed increased myofibroblasts, fibrin abundance, and scarring in myd88-/- ventricles.  Transcriptomic analyses of endocardial cells and immunostaining experiments reveal compromised PI3K/AKT pathway activation in the myd88-/- endocardium.  Moreover, loss of MyD88 impairs cardiomyocyte proliferation and protrusive activity towards the injured area.  Notably, endothelial-specific overexpression of myd88 reverses the neutrophil, fibrotic, and scarring phenotypes in cryoinjured myd88-/- ventricles.  Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a transcriptional target of the MyD88-signaling axis and using loss- and gain-of-function tools show that it controls neutrophil recruitment.  Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis, thereby emphasizing the strong impact of the innate immune response during tissue regeneration.

overall_design: Live zebrafish ventricular cells were isolated at 24 hour post cryoinjury (hpci) from myd88+/+ and myd88-/- ventricles by Fluorescence-activated cell sorting (FACS) and analyzed using scRNAseq.

first: Didier

last: Stainier


title: Upstream regulator of genomic imprinting in rice is a small RNA-associated chromatin remodeler [bisulfite_PCR]

city: Bangalore

first: Shivaprasad

last: Padubidri V.

address: UAS-GKVK Campus

institute: National Centre for Biological Sciences - TIFR

summary: Knock down of CLSY3 in rice caused endosperm development defects. We investigated the molecular effects of loss of CLSY3 in rice  endosperm by profiling sRNA, targeted bisulfite PCR,whole genome bisulfite sequencing and mRNA transcriptomes .These analyses revealed the  roles of CLSY3 in Endosperm

overall_design: We have performed targeted bisulfite PCR

first: Padubidri

middle: V

last: Shivaprasad

first: Avik

middle: K

last: Pal


title: Upstream regulator of genomic imprinting in rice is a small RNA-associated chromatin remodeler [Bisulfite_seq]

overall_design: We have performed whole genome bisulfite sequencing to understand the methylation status of endosperm upon CLSY3 knockdown