title: STAT1 employs myeloid cell-extrinsic mechanisms to regulate the neutrophil response and provide protection against invasive Klebsiella pneumoniae lung infection

status: Public on Jan 10 2024

city: Pittsburgh

first: Zeyu

last: Xiong

state: Pennsylvania

address: 3459 5th Ave, MUH W631

department: Medicine

institute: University of Pittsburgh

summary: Klebsiella pneumoniae (KP) are extracellular Gram-negative bacteria that cause infections in lower respiratory and urinary tract, and bloodstream. STAT1 is a master transcription factor that acts to maintain T-cell quiescence under homeostatic conditions. Although STAT1 helps defend against systemic spread of acute KP intrapulmonary infection, whether STAT1 regulation of T-cell homeostasis impacts pulmonary host defense during acute bacterial infection and injury is less clear. Using a clinical KP respiratory isolate and a pneumonia mouse model, we found STAT1-deficiency led to an early neutrophil-dominant transcriptional profile and neutrophil-recruitment in the lung preceding widespread bacterial dissemination and lung injury development. Yet, myeloid cell STAT1 was dispensable for control of KP proliferation and dissemination, as myeloid cell-specific STAT1-deficient (LysMCre/WT;Stat1fl/fl) mice showed similar bacterial burden in lung, liver, and kidney as WT littermates. Surprisingly, IL-17 producing CD4+ T-cells infiltrated Stat1-/- mice lungs early during KP infection. Increase in Th17-cells in the lung was not due to preexisting immunity against KP and were consistent with circulating rather than tissue-resident CD4+ T-cells. However, blocking global IL17 signaling with anti-IL17-RC administration led to increased proliferation and dissemination of KP, suggesting IL17 provided by other innate immune cells is essential in defense against KP. Contrastingly, depletion of CD4+ T-cells reduced Stat1-/- mice lung bacterial burden indicating early CD4+ T-cell activation in the setting of global STAT1-deficiency is pathogenic. Altogether, our findings suggest STAT1 employs myeloid cell-extrinsic mechanisms to regulate neutrophil responses and provide protection against invasive KP by restricting non-specific CD4+ T-cell activation and immunopathology in the lung.

overall_design: Lungs were harvested from WT and Stat1-/- mice at 24h after intratracheal infection of Klebsiella pneumoniae. RNA was purified from lung tissue using an RNeasy Plus Universal Mini kit (catalog 73404) according to the manufacturer’s instructions (Qiagen), purified RNA was sequenced by Novogene. Libraries using the Illumina platform and paired-end reads were generated.

first: Shekina

middle: M

last: Gonzalez-Ferrer

middle: J

last: Lee


title: The yeast AMP-activated protein kinase Snf1p phosphorylates the inositol polyphosphate kinase Kcs1p

city: ANN ARBOR

first: Anuj

last: Kumar

state: MI

address: 1105 N. University Avenue, BSB Room 3210

department: MCDB

country: USA

institute: University of Michigan

type: Expression profiling by high throughput sequencing

summary: The yeast Snf1p/AMP-activated kinase (AMPK) maintains energy homeostasis, controlling metabolic processes and glucose derepression in response to nutrient levels and environmental cues. Under conditions of nitrogen or glucose limitation, Snf1p regulates pseudohyphal growth, a morphological transition characterized by the formation of extended multicellular filaments. During pseudohyphal growth, Snf1p is required for wild-type levels of inositol polyphosphate (InsP), soluble phosphorylated species of the six-carbon cyclitol inositol that function as conserved metabolic second messengers. InsP levels are established through the activity of a family of inositol kinases, including the inositol polyphosphate kinase Kcs1p, which principally generates pyrophosphorylated forms of InsP7 and InsP8. Here, we report that Snf1p regulates Kcs1p, affecting Kcs1p phosphorylation and inositol kinase activity. A snf1 kinase-defective mutant exhibits decreased Kcs1p phosphorylation, and Kcs1p is phosphorylated in vivo at Ser residues 537 and 646 during pseudohyphal growth. By in vitro analysis, Snf1p directly phosphorylates Kcs1p, predominantly at amino acids 537 and 646. A yeast strain carrying kcs1 encoding Ser-to-Ala point mutations at these residues (kcs1-S537A,S646A) shows elevated levels of pyrophosphorylated InsP7, comparable to InsP7 levels observed upon deletion of SNF1. The kcs1-S537A,S646A mutant exhibits decreased pseudohyphal growth, invasive growth, and cell elongation. Transcriptional profiling indicates extensive perturbation of metabolic pathways in kcs1-S537A,S646A. Growth of kcs1-S537A,S646A is affected on medium containing glycerol and antimycin A, consistent with decreased Snf1p signaling. This work identifies Snf1p phosphorylation of Kcs1p, collectively highlighting the interconnectedness of AMPK activity and InsP signaling in coordinating nutrient availability, energy homoeostasis, and cell growth.

overall_design: We inoculated three single colonies each of a control strain (SSY128: kcs1 delta/delta with pRS416-KCS1) and kcs1 phosphodefective strain (SSY146: kcs1delta/delta with pRS416-kcs1-S537A,S646A) in 5 ml of SC -Ura media at 30 degrees C with shaking (250 rpm). Cultures were grown to saturation and were subsequently diluted in 50 ml of low-nitrogen media to an absorbance (optical density at 600 nm) of 0.05. Cultures were grown at 30 degrees C with shaking for roughly 18-20 hours to an optical density of approximately 0.5 at 600 nm. We subsequently harvested cells from each culture and stored the cell pellets at -80 degrees C. We isolated RNA from the frozen pellets using the RiboPure Yeast Kit (Thermo Fisher Scientific). RNA samples were treated with DNase, and RNA concentrations were determined using the NanoDrop microvolume spectrophotometer.

first: Sham

last: Sunder

first: Joshua

middle: S

last: Bauman


title: MATR3 pathogenic variants differentially impair its cryptic splicing repression function

city: Houston,

first: Hari Krishna

last: Yalamanchili

state: TX

address: 1250 Moursund St #1125,

institute: Baylor College of Medicine

summary: To investigate the role of MATR3 in splicing regulation, we performed RNA sequencing on HeLa cells treated with either control siRNA or MATR3 siRNA and then conducted alternative and cryptic splicing analysis

overall_design: Alternative and cryptic splicing analysis following RNA sequencing from HeLa cells upon MATR3 knockdown

first: Hari

middle: K

first: Jeehye

last: Park

first: Xiao-Xiao

last: Chen

first: Mashiat

last: Khan

first: Michelle

last: Diaz


title: Reprogramming the Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD1 Therapy (bulk RNA-seq)

status: Public on Jan 15 2024

city: Boston

first: Dan

last: Duda

state: MA

address: 55 Fruit St

department: Radiation Oncology

institute: Massachusetts General Hospital

summary: Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Adding blockade of the PD1 pathway to gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers but with low response rates. Here, we studied the effects of anti-CTLA-4 when combined with anti-PD1 and gemcitabine/cisplatin in orthotopic murine models of ICC. This combination therapy led to substantial survival benefits and reduction of morbidity in two aggressive ICC models that were resistant to immunotherapy alone. Gemcitabine/cisplatin treatment increased tumor-infiltrating lymphocytes and normalized the ICC vessels, and when combined with dual CTLA-4/PD1 blockade, increased the number of activated CD8+Cxcr3+IFNγ+ T cells. CD8+T cells were necessary for the therapeutic benefit because the efficacy was compromised when CD8+ T cells were depleted. Expression of Cxcr3 on CD8+ T cells is necessary and sufficient since CD8+ T cells from Cxcr3+/+ but not Cxcr3–/– mice rescued efficacy in T cell‒deficient mice. Finally, rational scheduling of anti-CTLA-4 “priming” with chemotherapy followed by anti-PD1 therapy achieved equivalent efficacy with reduced overall drug exposure. These data suggest that this combination approach should be clinically tested to overcome resistance to current therapies in ICC patients.

overall_design: Using tumor tissues collected in a time-matched manner, we employed bulk RNA-Seq to evaluate the transcriptional changes in murine 425-ICC tissues after 8 days of treatment with GC alone, dual ICB alone, and GC/dual ICB versus IgG control.

first: Jiang

first: Xin

last: Liu

first: Pin-Ji

last: Lei

middle: G


title: Sonic hedgehog-heat shock protein 90β axis promotes the development of nonalcoholic steatohepatitis (miRNA-Seq)

city: Nanjing

first: Weitao

last: Zhang

address: longmian road

country: China

institute: China Pharmaceutical University

type: Non-coding RNA profiling by high throughput sequencing

summary: The incidence rate of nonalcoholic steatohepatitis (NASH) is on the rise with no effective clinical treatment available to date. Understanding the key molecular mechanism underlying the process of NASH can provide theoretical support for the development of safe and effective therapeutic drugs. Sonic hedgehog (SHH) and heat shock protein 90β (HSP90β) have been implicated in NASH but their molecular mechanisms of action remain elusive. HSP90β was found to be a key SHH downstream molecule that promoted NASH processes In hepatocytes, SHH reduced HSP90β ubiquitylation through deubiquitylase USP31, thus preventing HSP90β degradation and promoting hepatic lipid synthesis. As an important component protein in the formation of exosomes, HSP90β is high in high fructose, high fat, and high cholesterol (HFFC) diet-induced NASH mouse model, leading to secretion of exosomes enriched with miR-28-5p. miR-28-5p directly targets and decreases Rap1b levels, which in turn promote NF-κB transcriptional activity in macrophages and stimulate the expression of inflammatory factors. Genetic deletion, pharmacological inhibition of the SHH-HSP90β axis, or delivery of miR-28-5p to macrophages in the liver, impaired NASH symptomatic development. Importantly, analysis of NASH patient sera revealed a markedly higher abundance of miR-28-5p compared to healthy donors. Taken together, the SHH-HSP90β-miR-28-5p axis offers promising therapeutic targets against NASH, and serum miR-28-5p may serve as a novel NASH diagnostic biomarker.

overall_design: Gene expression analysis using data from RNA-seq for exosomes in NASH or normal mice livers

first: Xiaojun

last: Xu


title: Sonic hedgehog-heat shock protein 90β axis promotes the development of nonalcoholic steatohepatitis (RNA-Seq)


title: The impact of eosinophils on the in vitro differentiation of osteoclasts

status: Public on Jan 09 2024

city: Erlangen

first: Darja

last: Andreev

address: Glückstraße 6

department: Medicine 3

country: Germany

institute: Friedrich-Alexander-University Erlangen-Nürnberg and Universitätsklinikum Erlangen

summary: To investigate the role of eosinophils on osteoclast differentiation from bone marrow-derived monocytes (BMMs), BMMs were cultivated in the presence or absence of eosinophils and eosinophil supernatant as compared with unstimulated control.

overall_design: The mRNA expressional changes in osteoclasts upon stimulation with eosinophils or eosinophil supernatant were anlaysed by bulk RNA sequencing

first: Aline

last: Bozec


title: TGF-beta blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo [scRNA-seq]

status: Public on Jan 08 2024

city: Chicago

first: Jinhee

last: Kim

state: IL

address: 676 North St. Clair Street Arkes Suite 2330

department: Infectious Disease

institute: Northwestern University

summary: HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of the anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirmed the latency reversal properties of in vivo TGF-beta blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on suppressive ART were treated with 4 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption(ATI). One macaque did not rebound, while the remaining rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with the 64Cu-DOTA-F(ab')2-p7D3-probe. Half to 1 Log decrease in cell-associated (CA-)SIV DNA was detected in lymph nodes, gut and PBMC, while intact pro-virus in PBMC decreased by 3-fold. No systemic increase in inflammatory cytokines was observed. High-dimensions cytometry, bulk and single-cell RNAseq revealed a shift toward an effector phenotype in T and NK cells.

overall_design:
To clarify the impact of galunisertib at the single cell level and in lymphoid tissues, we also performed scRNAseq analysis of cells isolated from LNs before (right axillar) and after (right or left inguinal) the first cycle.
8 Macaques, 2 time points Before and After Galunisertib.

first: Deepanwita

last: Bose

first: Mariluz

last: Aranga

first: Muhammad

last: Haque

first: Christine

last: Fennessey

first: Rachel

last: Caddell

first: Yanique

last: Thomas

first: Douglas

last: Ferrell

first: Syed

last: Ali

first: Emanuelle

last: Grody

first: Yogesh

last: Goyal

first: Claudia

last: Cicala

first: James

last: Arthos

first: Brandon

last: Keele

first: Monica

last: Vaccari

first: Ramon

last: Lorenzo-Redondo

first: Thomas

last: Hope

first: Francois

last: Villinger

first: Elena

last: Martinelli


title: TGF-beta blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo

first: Jinhee

last: Kim

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

overall_design: Refer to individual Series


title: Osr1-mediated mesothelial transition of liver mesenchymal cells exacerbates fibrotic liver damage

status: Public on Jan 12 2024

city: Beijing

first: Zebin

last: Mao

state: Beijing

address: No. 38 Xueyuan Road, Haidian District, Beijing

department: Department of Biochemistry and Molecular Biology

institute: Peking University

summary: In chronic liver diseases, hepatic stellate cells (HSCs) are induced to form the myofibroblasts responsible for scar formation, leading to liver fibrosis and cirrhosis. Here, single-cell RNA sequencing with in vivo lineage tracing in non-alcoholic steatohepatitis (NASH) model mice reveals a subpopulation of HSCs transitioning back to a state resembling their developmental precursors, mesothelial cells (MCs), after liver injury. These damage-associated intermediates between HSCs and MCs (DIHMs) can be traced with a dual recombinase system by labeling Krt19-expressing cells within pre-labeled Pdgfrb-positive HSCs, and DIHMs highly express inflammation- and fibrosis-associated genes. Cre and Dre-inducible depletion of DIHMs by administering diphtheria toxin reduces liver fibrosis and alleviates liver damage in NASH model mice. Importantly, knockdown of Osr1, a zinc finger transcription factor of the OSR gene family, can block DIHM induction in vitro. Conditional knockout Osr1 in Pdgfrb-expressing mesenchymal cells in NASH model mice can reduce liver fibrosis in vivo. Our study collectively uncovers an injury-induced developmental reversion process wherein HSCs undergo what we term a mesenchymal-to-mesothelial transition (MMesoT), which can be targeted to develop interventions to treat chronic liver diseases.

overall_design: To characterize the molecular identities and cell dynamics of myofibroblasts or activated HSC underlying the development, progression, and resolution of non-alcoholic steatohepatitis (NASH), we induced a mouse model of NASH using a methionine- and choline-deficient diet combined with a 45% high-fat diet and supplemented with 0.1% L-methionine in drinking water (MCDHF) for eight weeks. In addition, Col1a2-CreERT2; R26RtdTomato reporter mice were used to enrich Col1a2 positive myofibroblasts as an individual sample.

first: Xinxin

last: Nian

first: Pengyan

last: Lin

first: Yunfei

last: Bai

first: Donglin

last: Yu

first: Xinyan

last: Yang

first: Bin

last: Zhou

first: Jie

last: Gao


title: Protection against overfeeding-induced weight gain in mice is preserved in obesity but does not require FGF21 or MC4R

city: Copenhagen

first: Lars

middle: Roed

last: Ingerslev

address: Blegdamsvej 3B

department: NNF Center for Basic Metabolic Research

country: Denmark

institute: Copenhagen University

summary: Experimental overfeeding triggers homeostatic compensatory mechanisms that counteract weight gain. Here, we utilized intragastric overfeeding in mice to investigate the physiological and molecular responses to forced weight gain. Both lean and diet-induced obese (DIO) mice exhibited a potent and prolonged lowering of voluntary food intake following overfeeding-induced weight gain. Although overfeeding resulted in a marked increase in circulating fibroblast growth factor 21 (FGF21), experiments with FGF21 knockout (KO) mice demonstrated that FGF21 is dispensable for the homeostatic defense against experimental weight gain. Targeted proteomics unveiled novel circulating factors linked to overfeeding, including the protease legumain (LGMN). Notably, administration of recombinant LGMN lowered body weight and food intake in DIO mice. The protection against weight gain was also associated with reduced vascularization in the hypothalamus and sustained reductions in transcript levels of the orexigenic neuropeptides, Npy and AgRP, suggesting a role of hypothalamic signaling in the homeostatic recovery from overfeeding. Overfeeding of melanocortin 4 receptor (MC4R) KO mice showed that these mice can suppress voluntary food intake and counteract the enforced weight gain, although their rate of weight recovery is impaired. Collectively, these findings demonstrate that the defense against overfeeding-induced weight gain remains intact in obesity and involves mechanisms independent of both FGF21 and MC4R.

overall_design: 18-week-old chow-fed male C57BL6/J mice were fed by liquid infusion. On day 14, overfeeding was terminated and switched to sterile water infusion. On day 14 control and overfed mice were sacrificed to collect brain. On day 17 (d14+3), control and overfed mice were sacrificed, and the same tissue were collected.

first: Camilla

last: Lund

first: Pablo

last: Ranea-Robles

first: Sarah

last: Falk

first: Dylan

last: Belmont-Rausch

first: Grethe

last: Skovbjerg

first: Victoria

last: Vibe-Petersen

first: Nathalie

last: Krauth

first: Jacob

last: Lercke Skytte

first: Vasiliki

last: Vana

first: Urmas

last: Roostalu

first: Tune

middle: H

last: Pers

first: Jens

first: Christoffer

last: Clemmensen


title: Leukemic stem cells hijack lineage inappropriate signalling pathways to promote growth [RNA-seq]

status: Public on Jan 06 2024

city: Nottingham

first: Sophie

last: Kellaway

department: School of Medicine

country: United Kingdom

institute: University of Nottingham

summary: Acute Myeloid Leukemia (AML) is caused by different mutations which affect growth and differentiation of myeloid cells. Depending on the mutation sub-type, cells adopt different gene regulatory networks maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent cells (leukemic stem cells or LSCs) which escape chemotherapy and can emerge from quiescence to trigger a relapse. The signalling processes that allow such cells to re-grow remain unknown. Here, we examined the well characterised t(8;21) AML sub-type as model to address this question. We show that t(8;21) LSCs aberrantly activate the ectopic VEGF and IL-5 signalling pathways. We uncovered a regulatory circuit consisting of the driver oncoprotein RUNX1-ETO and the AP-1 / GATA2 transcription factor axis allowing LSCs to re-enter the cell cycle.

overall_design: Total RNA-sequencing

first: Peter

last: Keane

first: Salam

last: Assi

first: Constanze

last: Bonifer


title: MyD88 deficiency in podocytes does not change the outcome in experimental glomerulonephritis.

status: Public on Jan 11 2024

city: Cologne

first: Giuliano

last: Crispatzu

address: Joseph-Stelzmann-Straße 26

department: CECAD

institute: University of Cologne (UoC), Germany

summary: Podocytes, together with glomerular endothelial cells, form the kidney filtration barrier. Loss of podocyte function leads to proteinuria and end-stage renal disease. Podocytes are damaged by various diseases. How they respond to damage-associated molecular patterns is however incompletely understood. MyD88 is the central adaptor protein downstream of Toll-like receptor (TLR) / interleukin-1 (IL-1) signaling and key to the initiation of inflammatory responses.

overall_design: Here, we challenged WT and podocyte-specific MyD88 knockout mice (MyD88pko) with experimental glomerulonephritis (GN), nephrotoxic nephritis (NTN). Proteinuria, blood urea nitrogen (BUN) and kidney histology were used as read-outs. Podocyte ultrastructure was imaged by STED and infiltrating cells were quantified by FACS. Transcriptomic changes in podocytes of MyD88pko and WT littermate controls were analyzed under healthy and diseased conditions by bulk RNA-seq.

first: Thomas

last: Schömig

first: Paul

last: Brinkkötter

first: Sebastian

last: Brähler


title: Comparative transcriptomics coupled to developmental grading via transgenic Zebrafish reporter strains identifies conserved features in neutrophil maturation

city: Vienna

first: Florian

last: Halbritter

address: Zimmermannplatz 10

country: Austria

institute: St. Anna Children's Cancer Research Institute (CCRI)

summary: Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defence. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-β, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

overall_design: Kidney marrow from 2 adult, six-month old, male zebrafish was isolated, labelled using lipid-tagged following MULTI-seq protocol, for scRNA-seq using 10x Genomics.

first: Stefanie

last: Kirchberger

first: Mohamed

middle: R

last: Shoeb

first: Daria

last: Lazic

first: Andrea

last: Wenninger-Weinzierl

first: Kristin

last: Fischer

first: Lisa

middle: E

last: Shaw

first: Filomena

last: Nogueira

first: Fikret

last: Rifatbegovic

first: Eva

last: Bozsaky

first: Ruth

last: Ladenstein

first: Bernd

last: Bodenmiller

last: Lion

first: David

last: Traver

first: Matthias

last: Farlik

first: Christian

last: Schöfer

first: Sabine

last: Taschner-Mandl

first: Martin

last: Distel


title: single-cell RNAseq on Klebsiella infected mouse lung

city: pittsburgh

first: Janet

last: Lee

state: PA

address: 3459 fifth ave, MUH NW632

institute: University of pittsburgh

summary: Lung cells have traditionally been considered homogeneous. Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed the diverse responses of lung cells to Klebsiella infection. Furthermore, we compared the differential gene expression between wild-type mice and STAT1-knockout mice.

overall_design: Wild type and KO mice with and without infection were sacrificed 24h after bacterial inoculation and lung cells were harvested for scRNAseq analhysis.


title: Leukemic stem cells hijack lineage inappropriate signalling pathways to promote growth [ATAC-seq]

first: Sophie

last: Kellaway

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: ATAC-sequencing

first: Peter

last: Keane

first: Salam

last: Assi

first: Constanze

last: Bonifer


title: Single-cell multi-omics decodes regulatory programs during development of mouse secondary palate [bulk RNA-seq]

city: HOUSTON

first: Zhongming

last: Zhao

state: Texas

address: 7000 Fannin St

department: School of Biomedical Informatics

institute: UTHealth

summary: Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. However, currently there is no comprehensive multiomics map of the developing secondary palate. Here, we perform single-cell multiome sequencing and profile chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study chart epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research.

overall_design: The anterior (n=3) and posterior (n=3) 1/3 palatal shelves of embryonic day (E) 14.0 C57BL/6J mice were microdissected, isolated, and then subjected to bulk RNA-seq.

first: Fangfang

last: Yan

first: Akiko

last: Suzuki

first: Chihiro

last: Iwaya

first: Guangsheng

last: Pei

first: Xian

last: Chen

first: Hiroki

last: Yoshioka

first: Meifang

last: Yu

first: Lukas

last: Simon

first: Junichi

last: Iwata


title: Mitochondrial complex I activity in microglia sustains neuroinflammation

city: Cambridge

first: Irina

last: Mohorianu

address: Puddicombe Way

department: Wellcome-MRC Cambridge Stem Cell Institute

institute: University of Cambridge

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Clonal barcoding of endogenous adult hematopoietic stem cells reveals a spectrum of lineage contributions

status: Public on Jan 05 2024

city: New York City

first: Boris

last: Reizis

state: New York

address: 550 1st Ave

department: Pathology

institute: NYU Grossman School of Medicine

summary: The hierarchical model of hematopoiesis posits that self-renewing, multipotent hematopoietic stem cells (HSCs) give rise to all blood cell lineages. While this model accounts for hematopoiesis in transplant settings, its applicability to steady-state hematopoiesis remains to be clarified. Here we used inducible clonal DNA barcoding of endogenous adult HSCs to trace their contribution to major hematopoietic cell lineages in unmanipulated animals. Whereas the majority of barcodes were unique to a single lineage, we also observed frequent barcode sharing between multiple lineages, specifically between lymphocytes and myeloid cells. These results suggest a spectrum of multipotent and unipotent HSCs that collectively drive continuous hematopoiesis in the adult, and highlight a close relationship of myeloid and lymphoid development.

overall_design: For transposon integration site cloning, bone marrow cells and thymocytes were isolated from the Pdzk1ip1CreERR26FlipJump mice at months 6 and 10 post tamoxifen induction. Granulocytes, monocytes, MPP, MkP, EryP, ImmB, DN and DP were sorted from isolated bone marrow cells and thymocytes.

first: Jue

last: Feng

first: Eduardo

last: Esteva


title: Spatial Visium FFPE Samples

city: St. Louis

first: Benjamin

last: Humphreys

state: Missouri

address: 4523 Clayton Ave,

department: Nephrology

institute: Washington University School of Medicine in St. Louis

summary: Human Formalin fixed paraffin embedded Samples collected from Donated kidney samples, processed for cortex specific sections

overall_design: Kidney samples were cut into 6-20 mm sections, fixed in formalin for 16-24hrs, embedded in paraffin, and serial sections cut at 5um. Sections were manually placed on 10x Visum v.10 slides and processed per 10x Visium FFPE Protocols to bind and amplify transcriptome probe panels and generate libraries from mRNA. Data from Healthy cortex was compared to Chronic Kidney disease (CKD) or Acute Kidney disease (AKI)

first: Eryn

last: Dixon

first: Nicole

last: Malvin

first: Parker

last: Wilson


title: Transcriptomic Profiling of Immune Cells in Murine Polymicrobial Sepsis

city: Manhasset

first: Ping

last: Wang

address: 350 Community Dr

department: Center for Immunology and Inflammation

institute: The Feinstein Institutes for Medical Research

summary: Introduction: Various immune cell types play critical roles in sepsis with numerous distinct subsets exhibiting unique phenotypes even within the same cell population. In this study, we have unveiled the transcriptomic landscape of immune cells in sepsis through single-cell RNA sequencing (scRNA-seq) analysis. We induced sepsis in mice by cecal ligation and puncture. 20 h after the surgery, the spleen and peritoneal lavage were collected. Single-cell suspensions were processed using a 10× Genomics pipeline and sequenced on an Illumina platform. Count matrices were generated using the CellRanger pipeline, which maps reads to the mouse reference transcriptome, GRCm38/mm10. Subsequent scRNA-seq analysis was performed using the R package Seurat. Results: After quality control, we subjected the entire data set to unsupervised classification.Four major clusters were identified as neutrophils, macrophages, B cells, and T cells according to their putative markers. Based on the differentially-expressed genes, we identified activated pathways in sepsis for each cell type. In neutrophils, pathways related to inflammatory signaling, such as NF‐κB and responses to pathogen-associated molecular patterns (PAMPs), cytokines and hypoxia were activated. In macrophages, activated pathways were the ones related to cell aging, inflammatory signaling and responses to PAMPs. In B cells, pathways related to endoplasmic reticulum stress were activated. In T cells, activated pathways were the ones related to inflammatory signaling, responses to PAMPs, and acute lung injury. Next, we further classified each cell type into subsets. Neutrophils consisted of four clusters. Some subsets were activated in inflammatory signaling or cell metabolism, while others possessed immunoregulatory or aging properties. Macrophages consisted of four clusters, including the ones with enhanced aging, lymphocyte activation, extracellular matrix organization, or cytokine activity. B cells consisted of four clusters, including the ones possessing the phenotype of cell maturation or aging. T cells consisted of six clusters, whose phenotypes include molecular translocation or cell activation. Conclusions: Transcriptomic analysis by scRNA-seq has unveiled a comprehensive spectrum of immune cell responses and distinct subsets in the context of sepsis. These findings are poised to enhance our understanding of sepsis pathophysiology, offering avenues for targeting novel molecules, cells, and pathways to combat infectious disease.

overall_design: Cells from the peritoneal cavity and spleen of sham mice and mice that underwent cecal ligation and puncture (CLP) and then processed through the 10× Genomics single-cell RNA sequencing platform (i.e., barcoding and cDNA library preparation) for sequencing.

first: Atsushi

last: Murao

first: Alok

last: Jha

first: Monowar

last: Aziz


title: Interferon-stimulated neutrophils identified in preclinical models may serve as a potential biomarker for immunotherapy response in human

city: Haifa

first: Timothy

middle: Jon

last: Cooper

address: 1 Efron St.

country: Israel

institute: Technion – Israel Institute of Technology

summary: Despite the remarkable success of immunotherapy, only ~20-30% of cancer patients display an extended durable response. Pre-existing biomarkers for immunotherapy outcome are significantly limited in their predictive power. While mice are the most widely used and cost-effective model to study human disease, translating preclinical biomarkers into clinical practice faces significant obstacles – in part due to the lack of diverse or appropriate models. Here, to improve translatability and identify novel biomarkers, we showcase an approach encompassing several  pre-clinical models - each capturing one possible mechanistic aspect of immunotherapy response, such as tumor- and host-dependency, in order to reflect the variability seen in human cancers. Using this approach, we identify interferon-stimulated, Ly6Ehi neutrophils as a pre-treatment, blood-borne biomarker for anti-PD1 response in mice. We subsequently validate this result in cohorts of immunotherapy-treated non-small cell lung cancer (NSCLC) and melanoma patients, where the abundance of Ly6Ehi neutrophils predicts anti-PD1 response and further validated these results using available datasets for other cancers. Moreover, we demonstrate that these cells sensitize otherwise resistant tumors to anti-PD1 therapy, in part by directly activating cytotoxic T cells and contributing to tumor cell killing, while operate upstream of T cells in the immunotherapy response. Collectively, our study identifies a new and functionally active biomarker to predict immunotherapy outcome with potential clinical relevance.

overall_design: GR1+ myeloid cells were isolated from non-responding or responding 4T1, breast carcinoma murine tumors (5 mice pooled per sample) using magnetic beads and analyzed using scRNAseq.

first: Yuval

last: Shaked


title: Real Time quantitative PCR analysis of human T cells overexpressing AMPKg2

city: Pittsburgh

first: Erica

last: Braverman

address: 4401 Penn Ave

institute: University of Pittsburgh

summary: Human T cells were transduced to overexpress AMPKg2 or an EV control. CD4+ T cells were assessed for expression of TH subset related genes

overall_design: qPCR expression profiling. CD4+ T cells from 2 random human donors were transduced and expanded before RNA was isolated for expression analysis

first: Craig

last: Byersdorfer


title: SARS-CoV-2 infection evokes transcriptional changes in Calu-3 cells and cultured primary mouse trigeminal ganglia cells

city: Berkeley

first: Diana

last: Bautista

state: California

address: University Avenue

institute: University of California, Berkeley

summary: SARS-CoV-2, the virus responsible for COVID-19, infects both human airway epithelial cells and trigeminal ganglia. We assessed the consequences of SARS-CoV-2 infection on gene expression in Calu-3 cells and in primary Mus musculus trigeminal ganglia cells (mmTG). Here, we provide datasets that include raw reads and mapped reads for the following: 1) mock infected mmTG 48 hrs post infection; 2) SARS-CoV-2 infected mmTG 48 hrs post infection; 3) mock infected Calu-3 cells 24 hrs post infection; 4) SARS-CoV-2 infected Calu-3 cells 24 hrs post infection; 5) mock infected Calu-3 cells 48 hrs post infection; 6) SARS-CoV-2 infected Calu-3 cells 48 hrs post infection.

overall_design:
All mice were housed in standard conditions in accordance with standards approved by the Animal Care and Use Committee of the University of California Berkeley (14 hour light: 10 hour dark cycle, 21 °C). C57BL/6N mice were obtained from Charles River. Mouse trigeminal ganglia were cultured as previously described (Hill et al. 2018 J. Neurosci, PMID: 30082422; Wilson et al. 2011, Nature Neuro, PMID: 21460831) and plated in a 96-well plate. Eight trigeminal ganglia from 4 animals were combined and plated across 8 wells. TG cultures were infected with 500,000 SARS-CoV-2 (WA1) virions in medium (10% heat-inactivated FBS, sodium pyruvate, HEPES, L-glutamine, penicillin/streptomycin) or medium (mock infection) for 48 hours.
Human adenocarcinoma lung epithelial (Calu-3) cells (ATCC, HTB-55) were maintained with RPMI (Fisher Scientific) supplemented with fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin. Approximately 500,000 Calu-3 cells were incubated with SARS-CoV-2 (WA1) virions in medium (10% heat-inactivated FBS, sodium pyruvate, HEPES, L-glutamine, penicillin/streptomycin) diluted in phenol-red free RPMI to an MOI of 1 for 24 or 48 hours. After removing supernatant from infected cells, RNA was isolated with DNA/RNA Shield (Zymo Research) according to the manufacturer’s instructions.

first: Zhongyan

last: Gong

first: Sonali

last: Mali


title: TGF-beta blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo [RNA-seq]

first: Jinhee

last: Kim

overall_design:
To understand early and later effect of galunisertib in the context of SIV infection, we performed bulk RNAseq of PBMCs isolated from before cycle 1 (24hrs) and 1hrs after the first dose of galunisertib or after the last dose of cycle 1
We also performed bulk RNAseq of rectal biopsy specimens collected before cycle 1 (24hrs) and after the last dose of cycle 1.
7 Macaques, 3 time points for PBMCs, 2 time points for Rectal Biopsies

first: Deepanwita

last: Bose

first: Mariluz

last: Aranga

first: Muhammad

last: Haque

first: Christine

last: Fennessey

first: Rachel

last: Caddell

first: Yanique

last: Thomas

first: Douglas

last: Ferrell

first: Syed

last: Ali

first: Emanuelle

last: Grody

first: Yogesh

last: Goyal

first: Claudia

last: Cicala

first: James

last: Arthos

first: Brandon

last: Keele

first: Monica

last: Vaccari

first: Ramon

last: Lorenzo-Redondo

first: Thomas

last: Hope

first: Francois

last: Villinger

first: Elena

last: Martinelli


title: Human iPSC-derived closed-loop cardiac tissue for drug assessment

status: Public on Jan 07 2024

city: Suita

first: Junjun

last: Li

address: 2-2 Yamadaoka

country: Japan

institute: Osaka University

summary: Human iPSC-derived cardiomyocytes (hiPSC-CMs) exhibit functional immaturity, potentially impacting their suitability for assessing drug proarrhythmic potential. We previously devised a Traveling Wave (TW) system to promote maturation in 3D cardiac tissue. To align with current drug assessment paradigms (CiPA and JiCSA), necessitating a 2D monolayer cardiac tissue, we integrated the TW system with a multi-electrode array. This gave rise to a hiPSC-derived closed-loop cardiac tissue (iCT), enabling spontaneous TW initiation and swift pacing of cardiomyocytes from various cell lines. The TW-paced cardiomyocytes demonstrated heightened sarcomeric and functional maturation, exhibiting enhanced response to isoproterenol. Moreover, these cells showcased diminished sensitivity to verapamil and maintained low arrhythmia rates with ranolazine—two drugs associated with a low risk of torsades de pointes (TdP). Notably, the TW group displayed increased arrhythmia rates with high and intermediate risk TdP drugs (quinidine and pimozide), underscoring the potential utility of this system in drug assessment applications..

overall_design: To investigate how traveling wave regulate gene expression in hiPSC-derived closed-loop monolayer cardiac tissue


title: Leukemic stem cells hijack lineage inappropriate signalling pathways to promote growth [scRNA-seq]

first: Sophie

last: Kellaway

overall_design: Single-cell RNA sequencing of sorted leukemic stem cell and blast populations from t(8;21) AML patients pooled to form a single library

first: Peter

last: Keane

first: Salam

last: Assi

first: Constanze

last: Bonifer


title: Leukemic stem cells activate lineage inappropriate signalling pathways to promote growth

overall_design: Refer to individual Series


title: Biomarker Validation on the ‘Analysis of geNe Expression and bioMarkers fOr poiNt-of-care dEcision support in Sepsis’ (ANEMONES) study

city: Salisbury

first: Karen

middle: Elizabeth

last: Kempsell

state: Wiltshire

address: Porton Down

department: Science Group

institute: UK Health Security Agency

summary: Early diagnosis of sepsis and discrimination from SIRS is crucial for clinicians to provide appropriate care, management and treatment to critically ill patients. Here, we describe identification of transcriptional mRNA biomarkers able to identify severe systemic inflammation and differentiate Sepsis from SIRS, in adult patients within a multi-center clinical study. All patients were recruited in Intensive Care Units (ICUs) from multiple UK hospitals including 59 patients with abdominal sepsis, 84 patients with pulmonary sepsis, 42 SIRS patients with Out-of-Hospital Cardiac Arrest (OOHCA), at four time points including 30 healthy control donors. Multiple clinical parameters were measured, including SOFA score etc., with many differences observed between SIRS and sepsis groups. Differential gene expression analyses were performed using PBL mRNA microarray hybridization and data analyzed using a combination of parametric and non-parametric statistical tools. Nineteen select high-performance, differentially-expressed mRNA biomarkers were identified between control and combined SIRS/Sepsis groups (FC>20.0, p<0.05), termed ‘indicators of inflammation’ (IoI), including CD177, FAM20A and OLAH. Combinations of these were trialed. Best-performing minimal panels e.g. FAM20A/OLAH showed good accuracy for determination of severe, systemic inflammation (ROC/AUC>0.99). Twenty select entities were differentially-expressed between sepsis and SIRS (FC>2.0, p-value<0.05), termed ‘SIRS or Sepsis’ (SoS) biomarkers. Panels of biomarkers able to differentiate sepsis from SIRS were also identified and performance assessed using AUCROC. The best performing panel was CMTM5/CETP/PLA2G7/MIA/MPP3 using our dataset (AUCROC=0.9758). The IoI and SoS signatures were evaluated on other independent gene expression datasets, with some reduced performance observed, which maybe in part due to study/platform technical variation.

overall_design: Patients  were recruited from four UK hospitals (Royal Glamorgan Hospital, Prince Charles Hospital, Bristol Royal Infirmary and University Hospitals Birmingham) between 2013 and 2015. Healthy volunteers were recruited at UKHSA (Porton, UK) following written informed consent (n=30). Blood samples were collected from sepsis and SIRS patients at day 1, day 2 and day 5 of admittance to an intensive care unit (ICU) and on discharge from ICU. Some time-points were not collected due to patient death, patients leaving ICU or events beyond our control.  Healthy control blood samples were collected from volunteers once and were recorded as day 1 only.

first: Harriet

last: Garlant


title: Gene regulation in t(6;9) AML resembles that of FLT3-ITD/NPM1 AML with an altered HOX/MEIS axis [DNase-hypersensitivity]

status: Public on Jan 04 2024

summary: Acute Myeloid Leukemia (AML) represents a heterogeneous group of hematological malignancies. The t(6;9)(p23;q34) translocation, giving rise to the DEK::NUP214 fusion protein, is a rare mutation which produces a highly aggressive AML associated with extremely poor prognosis. Here, we utilise genome-wide chromatin accessibility to elucidate how a normal gene regulatory networks is disrupted by DEK::NUP214. We find that DEK::NUP214 AML forms a specific GRN related to that of mutant NPM1 AML, but also displays an elevated leukemic stem cell signature, suggesting both similar and unique therapeutic vulnerabilities.

overall_design: Here, we utilise genome-wide chromatin accessibility  by DNase-1-seq to elucidate how a normal gene regulatory networks is disrupted by DEK::NUP214. We find that DEK::NUP214 AML forms a specific GRN related to that of mutant NPM1 AML, but also displays an elevated leukemic stem cell signature, suggesting both similar and unique therapeutic vulnerabilities.


title: Gene regulation in t(6;9) AML resembles that of FLT3-ITD/NPM1 AML with an altered HOX/MEIS axis [RNA-seq]


title: Leukemic stem cells hijack lineage inappropriate signalling pathways to promote growth [ChIP-seq]

overall_design: ChIP-sequencing


title: Leukemic stem cells hijack lineage inappropriate signalling pathways to promote growth [DNase1-seq]

overall_design: DNaseI-sequencing


title: Clinical-grade human skin-derived ABCB5+ mesenchymal stromal cells exert modulatory functions on mRNA expression in a cisplatin-induced kidney injury murine model

city: Mannheim

first: Carsten

last: Sticht

address: Theodor-Kutzer-Ufer

department: ZMF

institute: University Heidelberg

summary:
Acute kidney injury (AKI) is characterized by a rapid reduction in renal function and glomerular filtration rate (GFR). The broadly used anti-cancer chemotherapeutic agent cisplatin often induces AKI as an adverse drug side effect. Therapies targeted at the reversal of AKI and its potential progression to chronic kidney disease or end-stage renal disease are currently insufficiently effective. Mesenchymal stromal cells (MSC) possess diverse immunomodulatory properties that confer upon them significant therapeutic potential for the treatment of diverse inflammatory disorders. Human dermal MSCs expressing ATP-Binding Cassette member B5 (ABCB5) have shown therapeutic efficacy in clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. In preclinical studies, ABCB5+ MSCs have also been shown to reverse metabolic reprogramming in polycystic kidney cells, suggesting a capacity for this cell subset to also improve organ function in kidney diseases. Here, we aimed to explore the therapeutic capacity of ABCB5+ MSCs to improve renal function in a preclinical rat model of cisplatin-induced AKI.
First, ABCB5+ MSCs suppressed cisplatin-induced apoptosis of human conditionally-immortalized proximal tubular epithelial cells in vitro. Second, ABCB5+ MSCs inhibited the proliferation of either human or rat peripheral blood mononuclear cells. Third, ABCB5+ MSCs decreased TNF-α secretion after LPS stimulation in both human and rat macrophages. Fourth, clinical-grade ABCB5+ MSCs grafted intravenously and intraperitoneally to a cisplatin-induced AKI murine model exerted modulatory effects on mRNA expression patterns towards an anti-inflammatory and pro-regenerative state despite an apparent lack of amelioration of renal damage at physiologic, metabolic, and histologic levels.
Our results demonstrate anti-inflammatory and pro-regenerative effects of clinical grade ABCB5+ MSCs in vitro and in vivo and suggest potential therapeutic utility of this cell population for treatment or prevention of cisplatin chemotherapy-induced tissue toxicity.

overall_design: SD rats were treated with 7mg/kgBW cispaltin or Saline Solution (Ctrl). ABCB5+ cells were administered on day 3 either intravenously (iv) or intraperitoneally (ip) and the rats were sacrificed on day 15.

first: Daniele

last: Christina

first: Gretz

last: Nobert


title: Functional drug testing of pediatric acute lymphocytic leukemia cells by single-cell transcriptome sequencing for mapping cellular drug response

city: Uppsala

first: Jessica

last: Nordlund

address: Box 1432, BMC

department: Department of Medical Sciences

country: Sweden

institute: Uppsala University

summary: Functional precision medicine (FPM) aims to match the right patients to the right drugs by using specific features of the individual’s cancer cells. Recently, FPM has been propelled by technologies that enable high throughput ex vivo drug profiling to tailor treatments for individual patients. Here, we present a proof of concept study for an integrated experimental system that incorporates ex vivo treatment response with a single-cell gene expression output that enables barcoding of several drug conditions in one single-cell sequencing experiment. We perform functional annotation of drug resistance using the glucocorticoid-resistant E/R+ REH cells as a cellular model and evaluate three different approaches for single-cell transcriptome sequencing (scRNA-seq). Using this integrated system, we show that all scRNA-seq methods accurately reflected gene expression changes in the system, with high cell recovery and accurate tagging of the different drug conditions. Furthermore, we identified a substantial single-cell transcriptional response to fludarabine, a drug of particular interest for treatment of high-risk ALL.

overall_design: We perform functional annotation of drug resistance using the glucocorticoid-resistant E/R+ REH cells as a cellular model and evaluate three different approaches for single-cell transcriptome sequencing (scRNA-seq). Five conditions were chosen based on dose response curves measured by FMCA. Single-cell sequencing of the treated cells was carried out using three different approaches, Parse Evercode WTK, MULTI-seq and 10x Chromium Fixed RNA Profiling. We investigated whether specific drug conditions influenced the gene expression profiles of the single cells recovered.

first: Henrik

last: Gezelius

first: Anna

middle: P

last: Enblad

first: Anders

last: Lundmark

first: Martin

last: Åberg

first: Kristin

last: Blom

first: Jakob

last: Rudfeldt

first: Arja

last: Harila

first: Amanda

last: Raine

first: Claes

last: Andersson


title: Endometrial transcriptome profiling of patients with recurrent implantation failure (RIF) during hormone replacement therapy (HRT) cycles

status: Public on Jan 03 2024

city: Hangzhou

first: Jiucheng

last: Chen

state: Zhejiang

address: No.866 Yuhangtang Road

institute: Zhejiang University

summary: The molecular mechanisms underlying window of implantation (WOI) displacement in patients with recurrent implantation failure (RIF) remain unclear. This study aims to explore the transcriptomic signatures of endometrium with normal and displaced WOIs during HRT cycles and to identify the causes of endometrial receptivity (ER) abnormalities and WOI displacement in RIF patients. 40 RIF patients were recruited and underwent personalized embryo transfer (pET) guided by the predicted results of endometrial receptivity diagnosis (ERD) model. Transcriptome analysis of endometrium from patients with clinical pregnancies after pET was performed to identify differentially expressed genes (DEGs) associated with WOI displacement. The ERD results indicated that 67.5% of RIF patients (27/40) were non-receptive in the conventional WOI (P+5) of the HRT cycle. The clinical pregnancy rate in RIF patients improved to 65% (26/40) after ERD-guided pET, indicating the effectiveness of transcriptome-based WOI prediction. Among the 26 patients with clinical pregnancy, the gene expression profiles of P+5 endometrium from advanced (n=6), normal (n=10) and delayed (n=10) WOI groups were significantly different from each other.  10 DEGs (CES4A, LRRC1, SLC25A48, TM4SF4, DPP4, CXCR1, CXCR2, OSM, LCN2 and TNFRSF10C ) identified among P+5 endometrium of 3 groups were involved in immunomodulation, transmembrane transport and tissue regeneration, which could accurately classify the endometrium with different WOIs.

overall_design: The diagnostic value of the eight gene expression signatures was evaluated by receiver operator characteristic analysis. This model was validated by an external clinical patient cohort. All samples were surplus tissue from endometrial biopsies obtained from patients for diagnostic purposes. The timing of the endometrial biopsy was 5 days after ovulation (evaluated by ultrasonography), which is equivalent to LH + 7 in a natural cycle.

first: Wen-bi

last: Zhang

first: Jue

last: Li

first: Jiu-cheng

first: Bing-jie

last: Lu

first: Xiao-xi

last: Sun


title: Self-renewing human naïve pluripotent stem cells dedifferentiate in 3D culture and form blastoids spontaneously

city: Guangzhou

first: Jinyi

last: Wu

state: Guangdong

address: No. 9 XingDaoHuanBei Road, Guangzhou International Bio Island

institute: Guangzhou Laboratory

summary: Human naïve pluripotent stem cells (hnPSCs) can generate integrated models of blastocysts termed blastoids upon switch to inductive medium. However, the underlying mechanisms remain obscure. Here we report that self-renewing hnPSCs spontaneously and efficiently give rise to blastoids upon three dimensional (3D) suspension  culture. The spontaneous blastoids mimic early stage human blastocysts in terms of structure, size, and transcriptome characteristics and are capable of progressing to post-implantation stages. This property is conferred by the glycogen synthase kinase-3 (GSK3) signalling inhibitor IM-12 present in 5iLAF self-renewing medium. IM-12 upregulates oxidative phosphorylation-associated genes that underly the capacity of hnPSCs to generate blastoids spontaneously. Starting from day one of self-organization, hnPSCs at the boundary of all 3D aggregates dedifferentiate into E5 embryo-like intermediates. Intermediates co-express SOX2/OCT4 and GATA6 and by day 3 specify trophoblast fate, which coincides with cavity and blastoid formation. In summary, spontaneous blastoid formation results from 3D culture triggering dedifferentiation of hnPSCs into earlier embryo-like intermediates which are then competent to segregate blastocyst fates.

overall_design: We convert human primed pluripotent stem cells to a naïve state by GSK3 inhibitor CHIR99021 to IM-12 switch (CI-hnPSCs). We find that CI-hnPSCs spontaneously and efficiently generate blastocyst-like structures (blastoids) in the 5iLAF self-renewing medium. Removing BRAF and MEK inhibitors from 5iLAF, using 3iLAF from day 3 of blastoid formation results in hypoblast specification.

first: Mingyue

last: Guo

first: Chuanxin

first: Xinggu

last: Wang

first: An

last: Gong

first: Wei

last: Guan

first: Rowan

last: Karvas

first: Kexin

first: Mingwei

last: Min

first: Yixuan

first: Thorold

middle: W

last: Theunissen

first: Shaorong

last: Gao

first: Jose

middle: C

last: Silva


title: ChIP-seq for PIF4-HA plants under different temperatures.

city: Shanghai

first: Wenhao

last: Xie

state: Shanghai

address: No. 2005, Songhu Road

department: School of Life Science

institute: Fudan University

summary: To investigate the effect of the PIF4, we analysed PIF4 binding genes at 28℃ compared to 22℃.

overall_design: ChIP-seq for PIF4-HA plants at 22℃ and 28℃ using HA antibody.

first: Aiwu

last: Dong

first: Chengzhang

first: Jiachen


title: Unraveling the Intricate Network of LncRNAs in Corneal Epithelial Wound Healing: Insights into the Regulatory Role of linc17500.

city: Wenzhou

first: Qiongjie

last: CAO

address: 270 Xueyuan Road

institute: Wenzhou Medical University

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: The precise role of long non-coding RNAs (lncRNAs) as key epigenetic regulators in mediating corneal epithelial wound healing remains elusive. Here, we aim to elucidate the functional contribution of lncRNAs in regulating CEWH. We used Microarray to characterize lncRNA expression profiling during mouse CEWH. The full thickness of the corneal epithelium of C57BL/6 mice, was removed using a 0.5-mm corneal rust ring remover. Subsequently, the entire corneal epithelium was collected from both the injured and fellow eyes for RNA isolation after approximately 48 hours. Microarray analysis revealed dysregulation of numerous lncRNA candidates during CEWH. Out of the 41,655 non-coding RNAs detected by the microarray, 639 were upregulated and 1,006 were downregulated during the process of CEWH.

overall_design: Gene and lncRNA expression profiling in the injured and fellow uninjured corneal epitheliums was measured at 48 hours after removing the full thickness of the corneal epithelium.

last: Cao


title: TDP-43 stable deficiency leads to transcriptional dysregulation and transposable elements activation via global  R-loops and 5hmC landscape alteration [RNA-Seq II]

city: Atlanta

first: Bing

last: Yao

address: 615 Michael Street

department: Department of Human Genetics

institute: Emory University

summary: TAR DNA-binding protein 43 (TDP-43) is an RNA/DNA-binding protein involved in many aspects of RNA metabolism, but its molecular roles in regulating gene and tTransposable eElements (TEs) transcription transcription is not extensively explored. TDP-43 loss-of-function via due to progressive cytoplasmic aggregations serves as a pathological hallmark and potential causality for several neurodegenerative diseases. While rRecent evidence suggest TDP-43 acute knockdown of TDP-43 affects the formation of R-loops formation, a nuclear DNA:RNA hybrid structures implicated in transcription regulation, . However, how its stable and chronic functional perturbation of TDP-43, which more closely resembles age-related neurodegeneration, impact global transcriptome via R-loop dysregulation remains unclear. Here we show demonstrate that stable and prolonged TDP-43 loss-of-function results in slowed impaired cell proliferation and impaired DNA damage response. At the molecular level, TDP-43 stable knockdown impacts key gene expression through concomitantly altering intragenic R-loops dynamics and its crosstalk with and thea DNA covalent modification 5-hydroxymethylcytosine (5hmC) in cis, as well as long- range R-loops-mediated enhancer-promoter interactions in trans. Furthermore, we find TDP-43 stable knockdown induces massive disease-related TEstransposable elements (TEs) activation via influencing R-loop and 5hmC homeostasis in many of these loci. Our results highlight previous underdeveloped transcriptional roles of TDP-43 via R-loops regulation in coding genes, distal regulatory elements and TEs, and suggest that TDP-43 proteinopathies transcriptionally contributes to the etiology of neurodegenerative disorders.

overall_design: TDP-43 knockdown by shRNA, RNA-seq

first: Yingzi

last: Hou

first: Yangping

last: Li


title: MicroRNA profile of extracellular vesicles released by Müller glial cells

city: London

first: William

last: Lamb

state: England

address: UCL Institute of Ophthalmology, 11-43 Bath Street, 11-43 Bath Street

institute: UCL Institute Ophthalmology

summary: As any other radial glia in the central nervous system, Müller glia derive from the same neuroepithelial precursors, perform similar functions and exhibit neurogenic properties as radial glia in the brain. Müller glial cells retain progenitor-like characteristics in the adult human eye, and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Recently, it has been demonstrated that intracellular communication is possible via the secretion of nano-sized membrane-bound extracellular vesicles (EV), which contain bioactive molecules like microRNA (miRNA) and proteins that induce phenotypic changes when internalised by recipient cells. We conducted high-throughput sequencing to profile the microRNA signature of EV populations secreted by Müller glia in culture, and used bioinformatic tools to evaluate their potential role in the neuroprotective signalling attributed to these cells. Sequencing of miRNA within Müller EV suggested enrichment with species associated with stem cells such as miR-21 and miR-16, as well as with miRNA previously found to play a role in diverse Müller cell functions in the retina: miR-9, miR-125b and the let-7 family. A total of 51 miRNA were found to be differentially enriched in EV compared to whole cells from which EV originated. Bioinformatic analyses also indicated preferential enrichment of species demonstrated to regulate genes involved in cell proliferation and survival, including PTEN the master inhibitor of the PI3K/AKT pathway.  The results suggest that the release by Müller cells of miRNA-enriched EV abundant in species that regulate anti-apoptotic signalling networks, is likely to represent a significant proportion of the neuroprotective effect observed after transplantation of these cells into animal models of retinal ganglion cell (RGC)-depletion. Future work will seek to evaluate the modulation of putative genes, as well as the activation of these pathways in in vitro and in vivo models following the internalisation of Müller-EV by target retinal neurons.

overall_design: We conducted Illumina micro-RNA sequencing analysis on samples of RNA recovered from whole MIO-M1 cells as well as on samples recovered from their secreted EV.

middle: D

first: Karen

last: Eastlake

first: Joshua

last: Luis

first: Najam

middle: A

last: Sharif

first: Peng

middle: T

last: Khaw

first: Gloria

last: Limb


title: Identification of a Novel, MSC-Induced Macrophage Subtype via Single-Cell Sequencing: Implications for Intervertebral Disc Degeneration Therapy

city: Minneapolis

first: Elizabeth

last: Bradley

state: Minnesota

address: 2001 6th St SE

department: Department of Orthopedic surgery

institute: University of Minnesota

summary: Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that MSCs promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. MSCs encounter drastic declines in oxygen tension when injected into the avascular IVD; thus, understanding how oxygen tension affects interactions between MSCs and macrophages may offer clues as to how MSCs act therapeutically. We investigated how conditioned medium (CM) derived from MSCs cultured in hypoxia altered macrophage subsets. We cultured human, bone marrow-derived STRO3+ MSCs in hypoxia or normoxia, and collected CM. We then cultured human bone marrow-derived macrophages with IFN-y, followed by either hypoxic or normoxic STRO3+ MSC CM. We also cultured macrophages in hypoxic MSC CM in the presence of IL-4 and collected cells for scRNA-seq analyses. Bioinformatic analyses confirmed multiple macrophage subpopulations. We first performed comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM. Integration of these data sets showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. Gene Ontogeny (GO) analyses revealed enrichment in IL-10, Ccl5/Ccr5, G alpha (i) Signaling, and Rho GTPases within this unique cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-β signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-β neutralization partially limited the effects of MSC CM. Our study identifies a unique macrophage subset induced by MSCs within hypoxic conditions, and supports that MSCs alter macrophage phenotypes through TGF-β-dependent mechanisms


title: BCL7A and BCL7B potentiate SWI/SNF-complex-mediated chromatin accessibility to regulate gene expression and vegetative phase transition in plants

first: Yawen

last: Lei

address: No. 135, Xingang Xi Road

institute: SUN YAT-SEN UNIVERSITY

summary: Switch defective/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes are multi-subunit machineries that establish and maintain chromatin accessibility and gene expression by regulating chromatin structure. However, how the remodeling activities of SWI/SNF complexes are regulated in eukaryotes remains elusive. B-cell lymphoma/leukemia protein 7A/B/C (BCL7A/B/C) have been reported as subunits of SWI/SNF complexes for decades in animals and recently in plants; however, the role of BCL7 subunits in SWI/SNF function remains undefined. Here, we identify a unique role for plant BCL7A and BCL7B homologous subunits in potentiating the genome-wide chromatin remodeling activities of BRAHMA-SWI/SNF complexes in plants. BCL7A/B require the catalytic ATPase BRAHMA (BRM) to assemble with the signature subunits of the BRM-SWI/SNF complexes and for genomic binding at a subset of target genes. Loss of BCL7A and BCL7B diminishes BRM-mediated genome-wide chromatin accessibility without changing the stability and genomic targeting of the BRM-SWI/SNF complex, highlighting the specialized role of BCL7A/B in regulating remodeling activity. We further show that BCL7A/B fine-tunes the remodeling activity of BRM-SWI/SNF complexes to generate accessible chromatin at the juvenility resetting region (JRR) of the microRNAs MIR156A/C for plant juvenile identity maintenance. In summary, our work uncovers the function of previously elusive SWI/SNF subunits in multicellular eukaryotes and provides insights into the mechanisms whereby plants memorize the juvenile identity through SWI/SNF-mediated control of chromatin accessibility.

overall_design: An assay for transposase-accessible chromatin with sequencing (ATAC-seq) comparing the brm and bcl7a bcl7b mutants with WT plants.

first: Chenlong


title: Mosaic loss of Y chromosome is associated with aging and epithelial injury in chronic kidney disease

city: Philadelphia

first: Parker

last: Wilson

address: 422 Curie Blvd

department: Pathology and Laboratory Medicine

institute: University of Pennsylvania

summary:
Background
Mosaic loss of Y chromosome (LOY) is the most common chromosomal alteration in aging men. Here, we use single cell RNA and ATAC sequencing to show that LOY is present in the kidney and increases with age and chronic kidney disease.

Results
The likelihood of a cell having LOY varies depending on its location in the nephron. Cortical epithelial cell types have a greater proportion of LOY than medullary or glomerular cell types, which may reflect their proliferative history. Proximal tubule cells are the most abundant cell type in the cortex and are susceptible to hypoxic injury. A subset of these cells acquires a pro-inflammatory transcription and chromatin accessibility profile associated with expression of HAVCR1, VCAM1 and PROM1. These injured epithelial cells have the greatest proportion of LOY and their presence predicts future kidney function decline. Moreover, proximal tubule cells with LOY are more likely to harbor additional large chromosomal gains and express pro-survival pathways. Spatial transcriptomics localizes injured proximal tubule cells to a pro-fibrotic microenvironment where they adopt a secretory phenotype and likely communicate with infiltrating immune cells.

Conclusions
We hypothesize that LOY is an indicator of increased DNA damage and potential marker of cellular senescence that can be applied to single cell datasets in other tissues.

overall_design: Human kidney cortex was analyzed by 10X Genomics single nucleus multiome ATAC + Gene Expression OR single nucleus ATAC sequencing


title: Heme-TAMs prevent cancer cell apoptosis and drive growth, invasiveness, and metastasis

city: Zurich

first: Florence

last: Vallelian

address: Rämistrasse 100

country: Switzerland

institute: University Hospital Zürich

summary: To study the impact of heme-TAMs on the growth, phenotype, and function of cancer cells, we cultured GFP-MC38 cells either alone or mixed with heme-treated macrophages  in microwell plates, enabling us to collect large numbers of uniform spheroids for scRNA-seq. We observed that after four days, the size of GFP-MC38 spheroids regressed, and the tumor cells underwent apoptosis and decayed metabolically. In contrast, the presence of heme-TAMs within spheroids prevented apoptosis, supporting the extended growth of metabolically viable spheroids beyond 10 days of culture.

overall_design: Spheroids of MC38-GFP and MC38-GFP mixed with BMDMs that were stimulated with heme were grown in microwell plates for 4, 8, and 10 days. 9000 spheroids per condition were pooled and digested into single cells. Single-cell suspensions were directly used for GEM generation.

first: Dominik

last: Schaer

first: Livio

last: Baselgia

first: Elena

last: Duerst


title: Mixed cell-type spheroids endorse the generation and maintenance of heme-TAMs in an experimental tumor microenvironment

summary: To more accurately mimic the tumor microenvironment, we established a three-dimensional culture of MC38 cancer cells mixed with heme-treated BMDMs in microwell plates. This resulted in tumor spheroids with uniformly interspersed macrophages. We profiled the transcriptome of macrophages within these spheroids using scRNA-seq 24 hours after spheroid formation. To test the robustness of the heme effects in this model, we conducted a multiplexed experiment evaluating heme alone and in combination with the classical M1-polarization stimuli INFγ and LPS.

overall_design: We profiled the transcriptome of macrophages within BMDM + MC38-GFP spheroids using scRNA-seq 24 hours after spheroid formation. To test the robustness of the heme effects in this model, we conducted a multiplexed experiment evaluating heme alone and in combination with the classical M1-polarization stimuli INFγ and LPS.


title: Hemorrhage-induced NRF2 signaling in tumor-associated macrophages drives cancer growth, invasion, and immunotherapy resistance

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

overall_design: Refer to individual Series


title: Heme-signaling progresses via NRF2 to educate heme-TAM transformation, tumor cell growth, and invasiveness

summary: Heme-activation of NRF2 is a strong anti-inflammatory signal in macrophages, and analyses in this study indicated that the expressed transcriptome in heme-TAMs was consistently enriched for NRF2 target genes. We have therefore delineated the role of NRF2 in a series of experiments with Nrf2 knockout BMDMs, leading to a locked NRF2-inactive state, and macrophages with a knockout of the cytoplasmic NRF2 capture protein KEAP1, leading to a locked NRF2-active state, irrespective of the presence or absence of heme. To demonstrate that activated NRF2 is sufficient to drive heme-TAM transformation, we analyzed Keap1 knockout macrophages.

overall_design: BMDMs from one Keap1 KO mouse (control) and one WT littermate mouse (control and heme-treated) were resuspended and transferred into 15 ml falcon tubes. 1 x 106 cells per condition were taken for cell staining using lipid tags following 10X Genomics protocol (CG00391; Demonstrated Protocol Cell Multiplexing Oligo Labeling for Samples with >80% Viable Cells; Rev B). Cell Multiplexing Oligos (CMO) labels B308 - B311 (3’ CellPlex Kit Set A) were used to label the 4 samples. After labeling, cell suspensions were counted again and the samples were pooled according to the pooling calculations in appendix of the labeling protocol (CG00391) in equivalent ratios.


title: TDP-43 stable deficiency leads to transcriptional dysregulation and transposable elements activation via global  R-loops and 5hmC landscape alteration [micro-C-Seq]

first: Bing

overall_design: TDP-43 knockdown by shRNA, micro-C-seq


title: Gene regulation in t(6;9) AML resembles that of FLT3-ITD/NPM1 AML with an altered HOX/MEIS axis

city: Nottingham

first: Sophie

last: Kellaway

institute: University of Nottingham


title: RNAseq for WT, mrg1 mrg2, pif4, and mrg1 mrg2 pif4 plants under different temperature

first: Wenhao

last: Xie

summary:
To investigate the effect of the interaction between the MRG1/2 and PIF4, here we analysed differentially expressed genes compared to the mutant and wild-type by RNA-seq under different temperature.
We performed gene expression profiling analysis using data obtained from RNA-seq of WT, mrg1 mrg2, pif4, and mrg1 mrg2 pif4 plants at 22degC and 28degC.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT, mrg1 mrg2, pif4, and mrg1 mrg2 pif4 plants at 22degC and 28degC.

first: Aiwu

last: Dong

first: Nana

last: Zhou

first: Jiachen

last: Wang


title: TDP-43 stable deficiency leads to transcriptional dysregulation and transposable elements activation via global  R-loops and 5hmC landscape alteration

last: Yao

type: Methylation profiling by high throughput sequencing


title: Red blood cell heme drives the generation of TAM-like macrophages with an immunocompromized phenotype

summary: Extravasated RBCs and their main component heme could function as specific microenvironmental drivers of TAM differentiation. We established two subcutaneous matrigel plug models of tissue microhemorrhage, which allow post-experimental recovery of invading cells at high quality for downstream analysis.

overall_design: Macrophage-enriched single-cell suspensions from RBC and RBC-ghost membranes matrigel plugs were used for GEM generation following 10X Genomics protocol (CG000388 Demonstrated Protocol Chromium Next GEM Single Cell 3ʹ v3.1 (Dual Index) with Feature Barcode technology for Cell Multiplexing Rev B) targeting a cell recovery rate of 10’000 cells for each condition.

first: Nadja

last: Schulthess-Lutz


title: TDP-43 stable deficiency leads to transcriptional dysregulation and transposable elements activation via global  R-loops and 5hmC landscape alteration [DRIP-Seq]

overall_design: TDP-43 knockdown by shRNA, DRIP-seq

first: Yingzi

last: Hou

first: Yangping

last: Li


title: TDP-43 stable deficiency leads to transcriptional dysregulation and transposable elements activation via global  R-loops and 5hmC landscape alteration [RNA-Seq]


title: Epigenetic dysregulation in Alzheimer s disease peripheral immunity - ATAC

city: Chicago

first: David

last: Gate

address: 303 E Chicago Ave, Chicago, IL 60611

department: Neurology

institute: Northwestern University

summary: The peripheral immune system in Alzheimer's disease (AD) has not been thoroughly studied with modern sequencing methods. To investigate epigenetic and transcriptional alterations to the AD peripheral immune system, we used single-cell sequencing strategies, including assay for transposase-accessible chromatin and RNA sequencing. We reveal a striking amount of open chromatin in peripheral immune cells in AD. In CD8 T cells, we uncover a cis-regulatory DNA element co-accessible with the CXC motif chemokine receptor 3 gene promoter. In monocytes, we identify a novel AD-specific RELA transcription factor binding site adjacent to an open chromatin region in the nuclear factor kappa B subunit 2 gene. We also demonstrate apolipoprotein E genotype-dependent epigenetic changes in monocytes. Surprisingly, we also identify differentially accessible chromatin regions in genes associated with sporadic AD risk. Our findings provide novel insights into the complex relationship between epigenetics and genetic risk factors in AD peripheral immunity.

overall_design: Peripheral blood mononuclear cells from healthy control and Alzheimer's disease patients, matched for age, sex, and APOE genotype. Samples were thawed, FACS sorted for live cells, lysed to isolate nuclei, and profiled using scATAC-seq.

first: Abhirami

last: Ramakrishnan

first: Natalie

last: Piehl


first: yaoguang

last: yu

overall_design: For ChIP-seq and MNase-seq assays, Arabidopsis seeds were sterilized with 10% sodium hypochlorite solution, washed with sterile water four times, and then stratified at 4 °C in darkness for three days. Seeds were then sown on ½-strength Murashige and Skoog (MS) medium containing 1% sucrose and 0.6% agar.  Seedlings were grown under cold white light (approximately 120 µmol·m-2·s-1) with short-day conditions (10 h light/14 h dark) at 22 °C.  For ChIP-seq, 14-day-old seedlings (∼0.5 g for each biological replicate) grown on ½-strength MS medium under short-day conditions were fixed using 1% formaldehyde under a vacuum for 15 min and then ground into a fine powder in liquid nitrogen. The chromatin was sonicated into ∼300-bp fragments using a Bioruptor sonicator with a 30/30-s on/off cycle (27 total on cycles) at the high setting. Immunoprecipitation was performed using 1 μl of anti-GFP (Abcam, Cat. No. ab290) at 4°C overnight.  For MNase-seq, Approximately 0.4g of seedlings were used per sample. For MNase treatment, the prepared nuclei were resuspended in prewarmed MNase digestion buffer (20 mM Tris-HCl, pH 8.0, 5 mM NaCl, and 2.5 mM CaCl2), followed by the addition of 0.5 units of MNase (Takara, Cat. No. 2910A,) and incubation (10 min at 37◦C with periodic agitation). Finally, the mono-nucleosome DNA was isolated from 2% agarose gels and quantified by the Qubit dsDNA HS assay kit (ThermoFisher, Cat. No. Q32851).

first: Yaoguang

last: Yu

first: Chenlong


title: Spontaneous induction of neural crest cells in human pluripotent stem cell-derived aggregates revealed by single-cell RNA sequencing

city: Sakyo-ku

first: Yusuke

last: Seto

state: Kyoto

address: 53 Shogoin Kawara-cho

institute: Kyoto University

summary: Neural crest cells (NCCs) are a transient population which contributes to the majority of craniofacial tissue. The development of craniofacial tissue involves highly complex intermingled processes of cell fate specification and migration in densely packed mesenchyme. The establishment of appropriate in vitro models would be helpful for understanding these complicated processes. Thus, we developed a new method to induce NCCs in aggregates of human pluripotent stem cells (hPSCs). To gain insight into the process of aggregate development we performed single cell RNA sequencing (scRNA-seq) on day 2, 3, 4, and 5. scRNA-seq analysis revealed that hPSCs in the aggregates acquired neural plate border NPB-like identity before NCC induction. scRNA-seq also revealed that NCCs showed changes in the gene expression associated with epithelial mesenchymal transition as in vivo developmental process.

overall_design: Single-cell RNA sequencing  was performed on human pluripotent stem cell-derived aggregates at differentiation day 2, 3, 4, and 5.

first: Y

first: M

last: Takizawa

last: Eiraku


title: Deciphering hypoxia related glioblastoma-associated macrophages and predicting prognostic markers by single-cell, spatially-resolved transcriptomics and experimental analysis

status: Public on Jan 02 2024

city: Wuhan

first: Haoyu

last: Chen

address: 169 Donghu Rd

institute: Wuhan University

summary: Glioblastomas (GBM), the most common and lethal type of primary brain tumor in adult, currently lack effective treatment and prognostic indicators. Hypoxia，a pathology promoting tumor progression in most solid tumors, has been reported to exhibit a negative correlation with GBM prognosis. Hypoxia related tumor-associated macrophages (hTAMs), the major cellular component of hypoxic regions, have not being systematically studied. We characterized the transcriptomic identity of hTAMs at the single-cell level, simultaneously discovering signatures indicative of poor outcomes. Immunofluorescent staining and spatially resolved transcriptomics revealed pronounced disparities regarding the morphology and distribution of TAMs between hypoxic and non-hypoxic regions of GBMs. We identified ARL4C and HSPA5 as prognostic indicators, exhibiting spatial patterns congruent with hypoxic regions and predicting worse outcomes, verified by qPCR and cell culture. Intercellular communication analysis using single-cell-omics revealed that hypoxia related GBM tumor cells (hGBM cells) exploit the TIMP1/LRP1 ligand-receptor axis to modulate hTAMs, resulting in a worse prognosis. Our study has offered clinically prognostic indicators, along with potentially therapeutic targets for GBMs.

overall_design: The glioma specimens, identified as IDH wild-type grade IV based on postoperative pathology after intraoperative resection, were collected. Subsequently, RNA extraction and concurrent sequencing were performed directly on the obtained tissues.

first: Jincao

first: Fangyu

first: Yigang

last: Yang


title: Red blood cells-heme-exposure and tumor-associated macrophage identity

first: Florence

last: Vallelian

summary: To study TAM phenotypes in hemorrhagic tumor tissues, we treated GFP-MC3 tumor-bearing mice with a single injection of immunotherapeutic anti-CD40 antibody on day 7 after tumor cell injection. Three days later, the tumors were collected for histology and genome-wide spatial analysis of mRNA expression. In both tumor samples, H&E histology visualized extensive tumor necrosis with RBCs extravasated into the necrotic areas of the tumors. Spatial gene-expression analysis allowed us to perform more detailed phenotyping, clearly discriminating zones of remaining viable cancer surrounded by hemorrhagic necrosis as evidenced by the enrichment of RBC-derived RNA (Hbb-bs). In these hemorrhagic regions, we found a Cd68 signal, confirming macrophage infiltration, superimposed by a strong signal for Hmox1 and Arg1. Like CD163 and Spp1 in human cancers, Arg1 is the archetypal marker gene for pro-cancerous and immunosuppressive TAMs in the mouse.

overall_design: Two GFP-MC3 tumor-bearing mice were treated with a single injection of immunotherapeutic anti-CD40 antibody on day 7 after tumor cell injection. Three days later, the tumors were collected for histology and genome-wide spatial analysis of mRNA expression.

first: Livio

last: Baselgia

first: Elena

last: Duerst


title: TDP-43 stable deficiency leads to transcriptional dysregulation and transposable elements activation via global  R-loops and 5hmC landscape alteration [DRIP-Seq II]

first: Bing

last: Yao

first: Yingzi

first: Yangping


title: Lung Epithelial Regnase-1 Dampens Local Immune Response but does not Worsen Susceptibility to Klebsiella pneumoniae

city: Pittsburgh

first: Giraldina

last: Trevejo-Nunez

email: gtrevejomd@gmail.com, gjt9@pitt.edu

state: Pennsylvania

address: 200 Lothrop St. BST-E1056

institute: University of Pittsburgh

summary: Epithelial cells express Reg1, which can ameliorate inflamatory response against KP. We sorted epithelial cells and assessed by scRNAseq the epithelial enrichement of Reg1

overall_design: Infected lungs were harvested at day 2 post KP infection. Lung epithelial  cells were sorted by FACS Aria and sent to scRNAseq.

first: Becky

last: Lin

first: Li

last: Fan

first: Kong


title: Heme-TAMs are resistant to tumoricidal transformation by INFγ

summary: Inflammatory macrophage activation is a critical component of the antitumor immune response and an emerging therapeutic concept. To evaluate the impact of heme exposure on antitumoral macrophage activity, we grew spheroids of mixed GFP-MC38 and BMDMs that were either untreated or pretreated with heme, INFγ, or heme + INFγ, and characterized them by scRNA-seq.

overall_design: Spheroids of MC38-GFP and MC38-GFP mixed with BMDMs that were stimulated with either IFNy, heme, or heme + IFNy were grown in microwell plates for 9 days. 4500 spheroids per condition were pooled and digested into single cells. Cell Multiplexing Oligos (CMO) labels B301 - B305 (3’ CellPlex Kit Set A) were used to label the 5 samples. The 5 samples were pooled and then directly used for GEM generation targeting a cell recovery rate of 10’000 cells.

first: Dominik

last: Schaer


title: RNA-seq Analysis of 14-day-old WT, bcl7a(brip3), bcl7b(brip4), bcl7a bcl7b(brip3 brip4), bcl7a bcl7b brm-3(brip3 brip4 brm-3) and bcl7a bcl7b brm-1(brip3 brip4 brm-1) seedlings under short day condition

first: Yawen

last: Lei

summary:
Purpose：Examination of global gene expression of 14-day-old WT, bcl7a(brip3), bcl7b(brip4), bcl7a bcl7b(brip3 brip4), bcl7a bcl7b brm-3(brip3 brip4 brm-3) and bcl7a bcl7b brm-1(brip3 brip4 brm-1) seedlings under short day condition.
Methods: Total RNA was extracted from 14-day-old Arabidopsis seedlings under short-day conditions using TRIzol reagent (Invitrogen) following the manufacturer's instructions. RNAs from three biological replicates was sequenced. separately at Novogene, using Illumina Hiseq X-Ten (Sequencing method: Hiseq-PE150). The paired-end RNA-seq reads were quantified by Salmon(Patro et al., 2017) (v1.5.1) with parameters -k 31 and --type quasi. Salmon index was generated using AtRTD2.fa as the reference transcriptome. The gene quantification files of all samples were used as inputs (TPM values) for differential gene expression analysis by DESeq2(Love et al., 2014) v3.10. To analyze differential gene expression in all samples, we used “parametric” Fit type, “ratio” method for estimate Size Factors and Wald significance test function provided by DESeq2.
Conclusions: Our study represents the first detailed analysis of bcl7a(brip3), bcl7b(brip4), bcl7a bcl7b(brip3 brip4), bcl7a bcl7b brm-3(brip3 brip4 brm-3) and bcl7a bcl7b brm-1(brip3 brip4 brm-1) transcriptomes, with biologic replicates, generated by RNA-seq technology.

overall_design: Examination of global gene expression in 14-day-old WT, bcl7a(brip3), bcl7b(brip4), bcl7a bcl7b(brip3 brip4), bcl7a bcl7b brm-3(brip3 brip4 brm-3) and bcl7a bcl7b brm-1(brip3 brip4 brm-1) seedlings under short day condition.

first: Chenlong

last: Li


title: BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.

city: Bethesda

last: Pugacheva

state: MD

department: NIAID

institute: NIH

summary: Pervasive usage of alternative promoters leads to deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters. In our present study, we uncovered a novel mechanism by which alternative cancer-testis-specific transcription is activated from the intergenic and intronic clustered CTCF binding sites, which are transcriptionally inert in normal somatic cells. BORIS/CTCFL, a paralog of CTCF with cancer-testis-specific expression forms a heterodimer with CTCF at the clustered binding sites thus triggering epigenetic reprogramming of these sites into units of active transcription. BORIS binding to CTCF sites leads to the recruitment of chromatin-remodeling factor SRCAP, with subsequent replacement of H2A histone with H2A.Z, therefore creating a more relaxed chromatin state in the nucleosomes flanking the clustered binding sites. This facilitates opening of chromatin beyond CTCF/BORIS binding sites and paves the way to the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the CTCF binding sites, epigenetically reprogrammed by ectopic BORIS expression can drive the expression of cancer-testis genes, long-noncoding RNAs, retro-pseudogenes, and dormant transposable elements harbored by activated long transcripts. Taking together, our results reveal that BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for two paralogous proteins, CTCF and BORIS (CTCFL), in combination with gene expression profiling analysis by RNA-seq and mapping of epigenetic marks by ChIP-seq.

middle: M


title: VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms

city: Cleveland

first: Lily

last: Wang

state: OH

address: 9500 Euclid Ave

institute: Cleveland Clinic

summary: To investigate the function of VISTA in regulating the differentiation of BM-derived MDSCs under normoxia conditions

overall_design: BM myeloid progenitor cells were cultured with GM-CSF and IL-6 under normoxia conditions for 4 days. 4 independent biological replicates of  WT vs VISTA  KO BM-derived MDSCs were purified and processed for RNA isolation and  bulk RNA- sequencing analysis.

middle: L


title: Endothelial cells secrete polarized small extracellular vesicles containing distinct cargo to uniquely reprogram luminal and abluminal vascular cells

city: Toronto

first: Sneha

last: Raju

state: ON

address: 101 College Street, PMCRT 3-309

country: Canada

institute: University of Toronto


title: Directional endothelial communication by polarized extracellular vesicle release

summary: EVs were isolated from primary human aortic endothelial cells (ECs) (+/- IL-1b activation), quantified, and analysed by miRNA transcriptomics and proteomics. Compared to quiescent ECs, activated ECs increased EV release, with miRNA and protein cargo that were related to atherosclerosis pathways. RNA sequencing of EV-treated monocytes and vascular smooth muscle cells (VSMCs) revealed that EVs from activated ECs altered pathways that were pro-inflammatory and atherogenic.

overall_design: We performed gene expression profiling analysis on CD14 monocytes and HASMCs treated with media only, quiescent, or activated endothelial extracellular vesicles.

first: Kathryn

last: Howe


title: Uveitic glaucoma-like features in Yap conditional knockout mice

status: Public on Jan 01 2024

city: Saclay

first: Jerome

middle: Eric

last: Roger

state: Please select

address: 151 Route de la Rotonde, Campus CEA Saclay - Bat 151

department: CERTO

country: France

institute: CNRS-Universite Paris Sud

summary: Glaucoma is a multifactorial neurodegenerative disease characterized by the progressive and irreversible degeneration of the optic nerve and retinal ganglion cells. Despite medical advances aiming at slowing degeneration down, around 40% of treated glaucomatous patients will undergo vision loss. It is thus of utmost importance to have a better understanding of the disease and to investigate more deeply its early causes. The transcriptional coactivator YAP, an important regulator of eye homeostasis, has recently drawn attention in the glaucoma research field. Here we show that Yap conditional knockout mice (Yap cKO), in which the deletion of Yap is induced in both Müller glia (i.e. the only retinal YAP-expressing cells) and the non-pigmented epithelial cells of the ciliary body, exhibit a breakdown of the aqueous-blood barrier, accompanied by a progressive collapse of the ciliary body. A similar phenotype is observed in human samples that we obtained from patients presenting with uveitis. In addition, aged Yap cKO mice harbor glaucoma-like features, including deregulation of key homeostatic Müller-derived proteins, retinal vascular defects, optic nerve degeneration and retinal ganglion cell death. Finally, transcriptomic analysis of Yap cKO retinas pointed to early-deregulated genes involved in extracellular matrix organization potentially underlying the onset and/or progression of the observed phenotype. Together, our findings reveal the essential role of YAP in preserving the integrity of the ciliary body and retinal ganglion cells, thereby preventing the onset of uveitic glaucoma-like features.

overall_design: Comparative gene expression analysis of three mouse retinas dissected either from Yapfl/fl wild-type eyes or from Yapfl/fl;RaxCreERT2  to unravel the molecular signature that could explain the uveitic glauoma-like phenotype of Yap cKO mice.

first: Juliette

last: Bitard

first: Elodie-Kim

last: Grellier

first: Sophie

last: Lourdel

first: Helena

last: Prior-Filipe

first: Annaïg

last: Hamon

first: François

last: Fenaille

last: Castelli

first: Emeline

last: Chu-Van

first: Jérôme

first: Morgane

last: Locker

first: Muriel

last: Perron


summary: EVs were isolated from primary human aortic endothelial cells (ECs) (+/- IL-1b activation) grown on transwells, quantified, and analysed by miRNA transcriptomics.

overall_design: EV-microRNA profiles were determined using HTG Molecular Diagnostic EdgeSeq technology (HTG Molecular Diagnostics, Inc, Tucson, AZ, USA).


title: Gene expression profiles of U2OS human osteosarcoma cell lines with ablation of NR1D1 or NR1D2 gene

city: Seoul

first: AHee

last: Yun

address: 52, Ewhayeodae-gil, Seodaemun-gu, Seoul 03760

country: South Korea

institute: Ewha Womans University

summary: We have recently established a panel of mutant U2OS human osteosarcoma cells lacking either REV-ERBα or REV-ERBβ (encoded by NR1D1 and NR1D2 genes, respectively) expression by a CRISPR/Cas9 and dual sgRNA-mediated gene deletion strategy. We then intended to dissect redundant and isoform-specific roles of these circadian nuclear receptors in controlling their target genes by comparing transcriptome profiles among wild-type and mutant U2OS cells.

overall_design: U2OS (ATCC HTB-96) cells were mainly cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution in a humidified incubator containing 5% CO2, at 37°C. To generate mutant cell lines, U2OS cells were transfected with 2 μg of an sgRNA expression vector by use of the Lipofectamin 3000 Transfection Reagent. After 48 h of transfection, cells were selected in the presence of 2 μg/ml puromycin. Cellular colonies were then individually transferred onto 24-well culture plates and deletion of intended genomic regions were confirmed by PCR-based genotyping. A cell line with truncations in both NR1D1 and NR1D2 genes were produced by a sequential transfection of sgRNA-expressing plasmids.

first: Gi

last: Son

first: Hana

last: Cho

first: Ahee

first: Juhee

last: Kim

first: Eunjeong

last: Park

first: Jong-Wha

last: Jung

first: Sooyoung

last: Chung


title: VISTA promotes the metabolism and differentiation of myeloid-derived suppressor cells by STAT3 and polyamine-dependent mechanisms [MDSC_SingleCell]

first: Lily

last: Wang

summary: To investigate the function of VISTA in regulating the differentiation of MC38 tumor-associated MDSCs

overall_design:
WT and VISTA KO BM were used to generate BM chimera mice. After 6 weeks of reconstitution, chimera mice were inoculated with MC38 tumor. Tumor tissues were harvested when tumors reached 9-10 mm diameter. Tumor-associated MDSCs were sorted and 10,000 MDSCs were processed using the 10x genomics platform
Please note that processed data was generated from both *gex and *ab samples, and is linked to the corresponding *gex sample records.

first: Li


title: Heme exposure induces the phenotype of Hmox1high, Marcohigh, and MHC class IIlow macrophages

first: Florence

last: Vallelian

summary: Bulk RNA sequencing revealed that heme exposure induced the phenotype of Hmox1high, Marcohigh, and MHC class IIlow macrophages, demonstrating a strong expression of NRF2-regulated. However, these cells showed weak expression of the TAM marker genes Arg1 and Spp1, indicating that heme treatment alone was insufficient to induce the full heme-TAM phenotype.

overall_design: BMDMs were either treated with heme-albumin or albumin (control). For each condition 3 samples were prepared.

first: Domink

last: Schaer

first: Livio

last: Baselgia

first: Elena

last: Duerst


title: Association of HBsAg levels with differential gene expression in NK, CD8 T, and memory B cells in treated patients with chronic HBV

city: Rotterdam

first: Andre

last: Boonstra

address: Dr. Molewaterplein 40

department: Dept. of Gastroenterology and Hepatology

country: Netherlands

institute: Erasmus MC

summary: Hepatitis B surface antigen (HBsAg) secretion may impact the immune response in chronic hepatitis B virus (HBV) infection. Therapeutic approaches to suppress HBsAg production are being investigated. Our study aims to examine the immunomodulatory effects of high and low levels of circulating HBsAg by analyzing single-cell RNA sequencing data (scRNAseq) from blood and liver fine-needle aspirates (FNA). This will help to better understand anti-HBV immunity.

overall_design: An optimized 10X Genomics scRNAseq-workflow was applied to blood and FNA samples from 18 patients undergoing tenofovir or entecavir treatment (NUC) for chronic HBV infection. They were categorized based on their HBsAg levels: high (920- 12447 IU/mL) or low (1-100 IU/mL). Cluster frequencies, differential gene expression, and phenotypes were analyzed.

first: Zgjim

last: Osmani


title: TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway [scRNA-seq]

city: Iowa City

first: Eric

last: Van Otterloo

state: Iowa

address: 801 Newton Rd

institute: University of Iowa

summary: Cranial neural crest development is governed by positional gene regulatory networks (GRNs). Fine-tuning of the GRN components underly facial shape variation, yet how those in the midface are connected and activated remain poorly understood. Here, we show that concerted inactivation of Tfap2a and Tfap2b in the murine neural crest, even during the late migratory phase, results in a midfacial cleft and skeletal abnormalities. Bulk and single-cell RNA-seq profiling reveal that loss of both Tfap2 members dysregulates numerous midface GRN components involved in midface morphogenesis, patterning, and differentiation. Notably, Alx1/3/4 (Alx) transcript levels are reduced, while ChIP-seq analyses suggest TFAP2 directly and positively regulates Alx gene expression. TFAP2 and ALX co-expression in midfacial neural crest cells of both mouse and zebrafish further implies conservation of this regulatory axis across vertebrates. Consistent with this notion in zebrafish, tfap2a mutants present abnormal alx3 expression patterns, Tfap2a binds alx loci, and tfap2a-alx3 genetic interactions are observed. Together, these data demonstrate TFAP2 paralogs regulate vertebrate midfacial development by activating expression of ALX transcription factors.

overall_design: To investigate the role of TFAP2 (Tfap2a and Tfap2b) in cranial neural crest cell development, we dissociated E11.5 tissue—rostral to the otic placode from 2 Tfap2a;Tfap2b;Wnt1CRE mutants, and 2 sibling matched controls—and conducted FACS based cell sorting of eGFP labeled neural crest cells for single cell RNAseq analysis. The 2 mutant samples were pooled and the 2 control samples pooled, prior to sequencing.

first: Timothy

last: Nguyen