title: Targeted protein degradation reveals BET bromodomains as the cellular target of Hedgehog Pathway Inhibitor-1

city: Geneva

first: Simon

last: Braun

address: rue Michel Servet 1

institute: University of Geneva

summary: Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Illustrative of these are the many screens that have been conducted to find inhibitors for the Hedgehog (Hh) signaling pathway – a major developmental pathway with many implications in health and disease - with many hits but very few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We developed a PROTAC based on the downstream Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using our Hedgehog Pathway PROTAC (HPP) we identified and validated BET bromodomains to be the cellular targets of HPI-1. Furthermore, we found that HPP-9 has a unique mechanism of action as a long-acting Hh pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that has answered the longstanding question of the cellular target of HPI-1 and yielded the first PROTAC that acts on the Hh pathway.

overall_design: 12 samples

first: Sascha

last: Hoogendoorn


title: Lessons from discovery of true ADAR RNA editing sites in a human cell line

status: Public on Jun 05 2023

city: Xiamen

first: Huifen

last: Cao

email: hfcao_bnu@163.com, hfcao@hqu.edu.cn

state: Fujian

address: 668 Jimei Road

department: School of Biomedical Sciences

country: China

institute: Huaqiao University

type: Non-coding RNA profiling by high throughput sequencing

summary: Conversion, or editing, of adenosine (A) into inosine (I) catalyzed by specialized cellular enzymes represents one of the most common naturally occurring post-transcriptional RNA modifications found in multiple species from fruit flies to humans with emerging connection to disease, particularly cancer. The A-to-I conversion can happen at specific sites in certain RNA molecules and lead to mRNA recoding and increase in proteome diversity, changes in RNA stability, splicing and regulation. Such sites could be detected as adenine-to-guanine sequence changes by RNA sequencing.  Thus, not surprisingly, the advent of next-generation sequencing has caused a massive explosion of reported sites detected by a slew of genome-wide studies in different species leading to wide ranges of conclusions about biological implications of the A-to-I editing on the physiology of normal and cancerous cells. Nonetheless, extensive independent validation of the authenticity of the reported sites is often missing in such endeavors which combined with the relatively high error rate of next generation sequence leads to lingering questions about the validity of the current compendiums of the editing sites and conclusions based on them. Therefore, in this work, we provide a comprehensive and an extensively validated list of A-to-I editing sites in a human cancer cell line. Strikingly, we found that the current analytical methods suffer from very high false positive rates and that a significant fraction of sites in the public databases can not be validated.  We provide a potential solution to these problems and discuss their ramifications to the current state of genomics of RNA editing as well as potential biological conclusions drawn from analysis of the compendium of true A-to-I editing sites in a human cancer cell line.

overall_design: Comprehensive identification of authentic ADAR editing sites in a human cancer cell line K562 based on analysis of multiple different RNA-seq samples and extensive Sanger validation.

first: Philipp

last: Kaparnov

first: Fang

last: Wang


title: Mutual antagonism between glucocorticoid and canonical Wnt signaling pathways in B-cell acute lymphoblastic leukemia

status: Public on Jun 06 2023

city: Memphis

first: Daniel

last: Savic

state: Tennessee

address: 262 Danny Thomas Pl

department: Pharmaceutical Sciences

country: USA

institute: St. Jude Children's Research Hospital

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: TREM2 shapes myeloid cell activations in trauma- and autoimmune-mediated neurodegeneration

city: Guangzhou

first: Han

last: Gao

state: Guangdong

address: 600 Tianhe RD

institute: The Third Affiliated Hospital of Sun Yat-sen University

summary: TREM2 signaling in myeloid cells functions as a major pathology-induced immune signaling hub in trauma- and autoimmune-mediated neurodegeneration. Here we compared the influence of TREM2 signaling on single-cell transcriptional profiles of mouse myeloid populations during the pathologies of spinal cord injury (SCI) and multiple sclerosis (MS) models.

overall_design: Single-cell RNA sequencing of myeloid cells in spinal cords from Wt and Trem2-/- mice under naive, SCI (1wk after injury) and experimental autoimmune encephalomyelitis (EAE, peak stage) conditions was performed using BD Rhapsody system. Myeloid cells were enriched using CD11b positive selection from at least two animals in each sample. Different single-cell suspension samples were mixed together using BD Single-cell Multiplexing Kits for high sample throughput and batch effects elimination purposes.


title: The RNA-binding protein PNPase regulates biofilm formation and virulence in Listeria monocytogenes

city: Oeiras

first: José

last: Andrade

address: Av. República

country: Portugal

institute: ITQB Nova/ Universidade Nova de Lisboa

summary: Biofilm-related diseases are typically persistent infections, and a challenge for medical treatment. Biofilms are communities of bacteria that attach to surfaces and are enclosed in an extracellular matrix. These sessile microorganisms can endure external stresses like nutrient deprivation, antibiotic treatments, and immune defences. Therefore, biofilms create conditions favourable for bacterial pathogenesis. The knowledge of novel biofilm regulators may contribute to develop new strategies to fight microbial infections. In this work we study the role of the RNA-binding protein and RNA-degradative enzyme polynucleotide phosphorylase (PNPase) from the human pathogen Listeria monocytogenes. We show that inactivation of Listeria PNPase not only leads to strong defects in biofilm production, but also affects biofilm morphology. RNA-seq analysis of the RNA extracted from biofilms of the wild-type and the PNPase mutant strains revealed major changes in the expression of genes affecting the metabolism of carbon. Lastly, infection assays in eukaryotic cell lines confirmed that PNPase deletion leads to the severe attenuation of Listeria monocytogenes pathogenicity. Overall, our results show that PNPase is a novel regulator of biofilm formation and human cellular invasion of a bacterial pathogen. This work presents PNPase as a new and attractive target for the control of bacterial infection and highlights the expanding role of RNA-binding proteins as critical players in pathogenicity.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT and PNPase deletion mutant.

first: Ana

middle: P

last: Quendera

first: Sandra

middle: N

last: Pinto

first: Wilson

last: Antunes

first: Vânia

last: Pobre

first: Cecília

middle: M

last: Arraiano


title: Transcriptome analysis induced by EV-A71 infection

city: Kunming

first: Jie

last: Song

state: Yunnan

address: Number 935 Jiaoling Load

institute: Institute of Medical Biology, Chinese Academy of Medicine Science and Peking Union Medical College

summary: In this study, we aimed to adopt the transcriptome sequencing technology to obtain the different changes of transcriptome profiles after infecting with EV-A71 in human neuroblastoma cell line SH-SY5Y. And then, through systematic bioinformatics analysis, we hope to find useful clues for the pathogenesis of HFMD.

overall_design: There are 3 samples, including EV-A71-0h（Control group), EV-A71-12h and EV-A71-24h.

first: Longding

last: Liu


title: Transcriptome analysis induced by CVS infection

summary: In this study, we aimed to adopt the transcriptome sequencing technology to obtain the different changes of transcriptome profiles after infecting with CVS in human neuroblastoma cell line SH-SY5Y. And then, through systematic bioinformatics analysis, we hope to find useful clues for the pathogenesis of Rabies.

overall_design: There are 3 samples, including CVS-0h（Control group), CVS-24h and CVS-48h.

first: Shaozhong

last: Dong


title: p53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism (RNA-Seq I)

city: Philadelphia

first: Jonuelle

last: Acosta

state: PA

address: 421 Curie Boulevard

department: Cancer Biology

institute: University of Pennsylvania

summary: The p53 tumor suppressor regulates multiple context-dependent tumor  suppressive programs. Although p53 is mutated in ~90% of small cell lung cancer  (SCLC) tumors, how p53 mediates tumor suppression in this context is unknown. Here,  using a mouse model of SCLC in which endogenous p53 expression can be  conditionally and temporally regulated, we show that SCLC tumors maintain a  requirement for p53 inactivation. However, we identified tumor subtype heterogeneity  between SCLC tumors such that p53 reactivation induces a canonical senescence  response in a subset of tumors, while, in others, p53 induces a non-apoptotic form of  cell death that culminates in necrosis. We pinpointed the cyclophilin family of peptidyl  prolyl cis-trans isomerases as critical determinants of a p53-induced transcriptional  program that is specific to SCLC tumors and cell lines that are poised to undergo p53-  mediated necrosis. Importantly, inhibition of cyclophilin isomerase activity, or genetic  ablation of specific cyclophilin genes, suppresses p53-mediated necrosis by limiting p53  transcriptional output without impacting p53 chromatin binding. Our study demonstrates  that a previously unappreciated intertumoral heterogeneity in SCLC can influence the  biological response to p53 restoration, describes a novel mechanism of p53-regulated  cell death, and uncovers putative mechanisms for the treatment of this most-recalcitrant  tumor type.

overall_design: RNA-sequencing analysis of Type D and Type V SCLC cells after 72hrs of p53 restoration and/or CsA treatment.

first: Qinglan

last: Li

first: Nelson

middle: F

last: Freeburg

first: Nivitha

last: Murali

first: Alexandra

last: Indeglia

first: Grant

last: Grothusen

first: Michelle

last: Cicchini

first: Hung

last: Mai

first: Amy

middle: C

last: Gladstein

first: Keren

last: Adler

first: Katherine

middle: R

last: Doerig

first: Jinyang

first: Miguel

last: Ruiz-Torres

first: Kimberly

middle: L

last: Manning

first: Ben

middle: Z

last: Stanger

first: Luca

last: Busino

first: Maureen

last: Murphy

first: Liling

last: Wan

first: David

last: Feldser


title: Transcriptome analysis induced by CV-A16 infection

first: Jie

last: Song

summary: In this study, we aimed to adopt the transcriptome sequencing technology to obtain the different changes of transcriptome profiles after infecting with CV-A16 in human neuroblastoma cell line SH-SY5Y. And then, through systematic bioinformatics analysis, we hope to find useful clues for the pathogenesis of HFMD.

overall_design: There are 3 samples, including CV-A16-0h（Control group), CV-A16-12h and CV-A16-24h.

first: Yajie

last: Hu


title: TRIM28 modulates progesterone and estrogen signaling in endometrium [mouseD35-PGR-ChIPseq-mm10]

status: Public on Jun 04 2023

city: RTP

first: Franco

last: Demayo

state: NC

address: 111 TW Alexander Drive

institute: NIEHS

summary: TRIM28 interacts with PGR and ESR1 in both human and mouse uterus to modulate estrogen and progesterone signaling. Knocking down of TRIM28 in the human endometrial stromal cells impaired decidualization in vitro. Deletion of TRIM28 from mouse uterus disrupted uterine stromal decidualization leading to infertility. Additionally, TRIM28 deletion caused abnormal accumulation of TRIM28 positive and PGR negative cells in the stroma.

overall_design: Mouse uteri were collected at D3.5 and performed PGR ChIP-seq using sc7208 PGR antibody by Santa Cruz Biotechnology.

first: Rong

last: Li

first: Tianyuan

last: Wang

first: San

last: Wu

first: John

last: Lydon

first: Francesco

middle: J

last: DeMayo


title: Nucleic acid biomarkers of immune response and cell and tissue damage in children with COVID-19 and MIS-C [cf RNA]

city: Ithaca

first: Conor

middle: James

last: Loy

state: NY

address: 237 Tower Road

department: BME

institute: Cornell Univeristy

summary: Differential host responses in coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) remain poorly characterized. Here we use next-generation sequencing to longitudinally analyze blood samples from pediatric patients with acute COVID-19 (n=70) or MIS-C (n=141) across three hospitals. Profiling of plasma cell-free nucleic acids uncovers distinct signatures of cell injury and death between COVID-19 and MIS-C, with increased multi-organ involvement in MIS-C encompassing diverse cell types including endothelial and neuronal cells, and an enrichment of pyroptosis related genes. Whole blood RNA profiling reveals upregulation of similar pro-inflammatory pathways in COVID-19 and MIS-C, but also MIS-C specific downregulation of T cell-associated pathways. Profiling of plasma cell-free RNA and whole blood RNA in paired samples yields different yet complementary signatures for each disease state. Our work provides a systems-level view of immune responses and tissue damage in COVID-19 and MIS-C and informs the future development of new disease biomarkers.

overall_design:
Analysis of plasma cell-free RNA in pediatric patients with COVID-19, MIS-C, and healthy controls

Submitter declares that the raw data have not been deposited due to patient privacy concerns.

first: Alicia

last: Sotomayor-Gonzalez

first: Venice

last: Servellita

first: Jenny

last: Nguyen

first: Joan

last: Lenz

first: Sanchita

last: Bhattacharya

first: Meagan

middle: E

last: Williams

first: Alexandre

last: Cheng

first: Andrew

last: Bliss

first: Prachi

last: Saldhi

first: Noah

last: Brazer

first: Jessica

last: Streithorst

first: William

last: Suslovic

first: Charlotte

last: Hsieh

first: Burak

last: Bahar

first: Nathan

last: Wood

first: Abiodun

last: Foresythe

first: Amelia

last: Gliwa

first: Kushmita

last: Bhakta

first: Maria

middle: A

last: Perez

first: Laila

last: Hussaini

first: Evan

last: Anderson

first: Ann

last: Chahroudi

first: Meghan

last: Delaney

first: Roberta

last: DeBiasi

first: Christina

last: Rostad

first: Iwijn

last: De Vlaminck

first: Charles

middle: Y

last: Chiu


title: Effect of depletion of Notch signaling genes in C2C12 cells on the activation of Notch pathway activity in response to stimulation by plated ligand

status: Public on Jun 02 2023

city: Pasadena

first: Rachael

last: Kuintzle

state: CA

address: 391 S Holliston Ave, Bio 73, MC 140-18

department: Chemistry and Chemical Engineering

institute: California Institute Of Technology

summary:
To obtain a Notch-depleted base cell line into which we could integrate specific ligands or receptors for quantitative analysis of Notch signaling interactions,
we conducted 2 rounds of transfection with RNPs (ribonucleoproteins) consisting of gRNAs targeting Notch2 and Jagged1 complexed with Cas9 protein.
After antibiotic selection and screening of monoclonal colonies, we obtained the C2C12-Nkd clone, which showed loss of Notch2 protein and Jag1 transcript.

overall_design: Here, we analyze the transcriptome of this C2C12-Nkd line compared with wild-type C2C12, and its response to stimulation of Notch signaling by plated recombinant ligands.

first: Kuintzle

last: Rachael

first: Santat

last: Leah

first: Elowitz

last: Michael


title: Zika virus co-opts miRNA networks to persist in placental microenvironments detected by spatial transcriptomics [visium]

status: Public on Jun 03 2023

city: Houston

first: Michael

last: Jochum

state: TX

address: 1 Baylor Plaza

department: Obstetrics and Gynecology

institute: Baylor College of Medicine

summary: We and others have demonstrated Zika virus (ZIKV) congenital infection evades double-stranded RNA detection, and may persist in the placenta for the duration of pregnancy without accompanying overt histopathologic inflammation. We used orthogonal approaches to test the hypothesis that ZIKV disrupts placental miRNAs to enable viral persistence and fetal pathogenesis. In primary human trophoblasts, high-throughput sequencing crosslinking and immunoprecipitation (AGO-HITS-CLIP) demonstrated an unexpected disruption of placental miRNA-regulated TGF-β networks. In gnotobiotic mice, absence of microbes rendered normally resistant mice susceptible to congenital ZIKV infection, and placental spatial transcriptomics revealed distinct microenvironments defined by significant upregulation of complement cascade components. Finally, treatment of ZIKV-infected mice with the RNAi-enhancer enoxacin led to loss of ZIKV placental persistence and rescue of fetal growth restriction. These results collectively suggest that ZIKV co-opts miRNA inflammatory regulatory networks to persist in the placenta reservoir, a conduit of vertical transmission and fetal pathogenesis.

overall_design: Gnotobiotic congenital ZIKV mouse model. Following timed mattings of four-to-six-week-old Swiss/webster mice from the Baylor College of Medicine Gnotobiotics Core, plugged dams were weighed and transferred to separate sterile insulators. Daily intraperitoneal injections of enoxacin (Sigma-Aldrich, Cat. 557304) at 10 mg/kg were previously shown to be safe and effective in a model of obesity32 and were done from embryonic days E1.5-E18.5. Dams were either received mock United States Pharmacopeia (USP)-grade phosphate-buffered saline (PBS) (VWR, Cat. 76081-514) subdermal injections or ZIKV infection with HN16 strain (first passage in Vero-E6 cells) at an MOI of 1x105 PFU on embryonic days E7.5, E8.5, and E9.5. On day 18.5, dams were humanely sacrificed, and uterine horns were dissected. Images of the uterine horns were taken to record resorption events. Individual fetuses were weighed. Placental tissues were stored at -80C in Trizol. All mouse experiments were conducted under the supervision of Baylor College of Medicine Institutional Animal Care and Use Committee approved protocols.

first: Kjersti

last: Aagaard

first: Enrico

last: Barrozo


title: Chronic antigen in solid tumors drives a distinct program of T cell residence

city: Minneapolis

first: David

last: Masopust

state: MN

address: 2101 6th St SE

department: Department of Microbiology and Immunology

institute: University of Minnesota

summary: Analyses of healthy tissue reveal signatures that identify resident memory CD8+ T cells (TRM), which survey tissues without recirculating. The density of TRM-phenotype cells within solid tumors correlates favorably with prognosis, suggesting that intratumoral residents control cancer. However, residence has not been directly tested and intratumoral TRM-phenotype cells could instead reflect aspects of the microenvironment that correlate with prognosis. Using a breast cancer model, we found that conventional TRM markers do not inform the tumor residence of either bystander or tumor-specific cells, which exhibit further distinct phenotypes in the tumor microenvironment and healthy mammary tissue. Rather, tumor-specific, stem-progenitor CD8+ T cells migrate to tumors and become resident while acquiring select markers of exhaustion. These data indicate that tonic antigen stimulation and the tumor environment drive distinct programs of residence compared to healthy tissues and that tumor immunity is sustained by continued migration of tumor-specific stem cells.

overall_design: We performed single-cell RNA sequencing with cellular indexing of transcriptomes and epitopes (CITE-seq) on CD8+ T cells isolated from EO771-ova breast tumors, non-tumor bearing mammary fat pads, and spleens from a pool of nine biologic replicates. Samples include virus-specific Thy1.1+ P14 CD8+ T cells, tumor-specific CD45.1+ OT-I CD8+ T cells, and polyclonal endogenous CD44+CD45.2+CD45.1-Thy1.1- CD8+ T cells.

first: Noah

middle: V

last: Gavil

first: Milcah

last: Scott


title: Gene expression profile at single cell level of mice infected with Mtb (-/+ TLR2)

city: Cambridge

first: Bryan

last: Bryson

state: MA

address: 400 Technology Square

department: Biological Engineering

institute: MIT

summary: The lung is a key site of Mtb infection. We profiled the lung ecosystem of mice following Mtb infection.

overall_design: Murine lungs (-/+ TLR2) were infected with H37Rv Mtb and profiled by 10X genomics,

first: Amy

last: Barczak


title: Zika virus co-opts miRNA networks to persist in placental microenvironments detected by spatial transcriptomics [AGO-HITS-CLIP]

overall_design: Primary human trophoblast isolation and ZIKV infection. Term placentas from 4 healthy patients were subject to cytotrophoblast purification as described previously 11. Cytotrophoblasts were seeded in 60 cm dishes and maintained with DMEM:F12 media (Gibco, Cat. 11320-033) supplemented with + 10% heat-inactivated fetal bovine serum (FBS; Gibco, Cat. 16140-071) + 1% penicillin-streptomycin (P/S; Gibco, Cat. 15140-122). Media was changed daily for 2-4 days until cells differentiated into syncytiotrophoblasts confirmed by peak β-human chorionic gonadotropin expression. At 5 days post-isolation, primary human trophoblasts cells from each patient (105-90 million cells/replicate) were mock-infected with PBS or infected with a contemporary first-passage ZIKV strain HN16 (GenBank accession KY328289.1)10,14 at an MOI of 1 plaque-forming units (PFU) and harvested at 5 days post-infection.


title: microRNA expression profiling in Natural Killer exosomes

city: Rome

first: Andrea

last: Pelosi

state: RM

address: Viale di San Paolo, 15

department: Laboratories

country: Italy

institute: Ospedale Pediatrico Bambino Gesù

summary:
Natural Killer (NK) cells are important components of the immune system in the defense against tumor transformation. They release exosomes containing proteins and nucleic acids, including microRNAs (miRNAs) that play a role in the anti-tumor NK cell functions since they are able to recognize and kill cancer cells.
In this study, we explored the miRNA content of NK exosomes by microarray as compared to their cellular counterpart and we identified a small subset of miRNAs highly expressed in NK exosomes.

overall_design: Exosomes were purified by ultracentrifugation from supernatant of human long-term interleukin-2 activated NK cell cultures. Culture medium was supplemented with exosome-free fetal bovine serum.  We collected 7 samples of NK exosomes and 7 samples of NK cells. After total RNA extraction, samples were hybridized on Affymetrix GeneChip miRNA 4.1 array plate.

first: Anna

last: Di Pace

first: Lorenzo

last: Moretta


title: A genome-wide DNA methylation-based classifier differentiates intrahepatic cholangiocarcinomas from hepatic metastases of pancreatic ductal adenocarcinoma

city: Berlin

first: Mihnea

middle: P.

last: Dragomir

state: Berlin

address: 1 Charitéplatz

department: Pathology

country: Germany

institute: Charité - Universitätsmedizin

summary: Differentiating intrahepatic cholangiocarcinomas (iCCA) from hepatic metastases of pancreatic ductal adenocarcinoma (PAAD) is difficult. We hypothesized that DNA-methylation based machine-learning algorithms may be able to perform this task. We assembled genome-wide DNA-methylation data for iCCA, PAAD, and normal bile from publicly available sources. We used an internal cohort consisting of 72 samples to test our classifier.

overall_design: Internal testing of a classifer using 72 FFPE samples after Infinium HD FFPE DNA Restore Kit and analyed using Illumina Infinium MethylationEPIC BeadChip.

first: Teodor

middle: G

last: Calina


title: ARIH1 activates STING-mediated T-cell activation and sensitizes tumors to immune checkpoint blockade

city: hangzhou

first: liu

last: xiaolan

address: 1369 West Wenyi Road, Hangzhou 311121, China

institute: Zhejiang University Medical Center

summary: To explore the molecular mechanisms how ARIH1 activates CD8+ T cells and enhances PD-L1 blockade therapy, we performed RNA sequencing (RNA-seq) to determine whether Arih1 overexpression affects the gene expression in 4T1 cells.

overall_design: GSEA analysis of the enrichment of top pathways (left) for genes between Arih1-WT-OE and Arih1-C355S-OE 4T1 cells.

first: Xiaolan

last: Liu

first: Hongguang

last: Xia


title: Defining blood-induced microglia functions in neurodegeneration through multiomic profiling [scRNA-Seq II]

city: San Francisco, CA

first: Katerina

last: akassoglou

address: 1650 Owens st

institute: Gladstone Institutes

summary: Blood protein extravasation through a disrupted blood–brain barrier and innate immune activation are hallmarks of neurological diseases and emerging therapeutic targets. However, how blood proteins polarize innate immune cells remains largely unknown. Here, we established an unbiased blood-innate immunity multiomic and genetic loss-of-function pipeline to define the transcriptome and global phosphoproteome of blood-induced innate immune polarization and its role in microglia neurotoxicity. Blood induced widespread microglial transcriptional changes, including changes involving oxidative stress and neurodegenerative genes. Comparative functional multiomics showed that blood proteins induce distinct receptor-mediated transcriptional programs in microglia and macrophages, such as redox, type 1 interferon and lymphocyte recruitment. Deletion of the blood coagulation factor fibrinogen largely reversed blood-induced microglia neurodegenerative signatures. Genetic elimination of the fibrinogen-binding motif to CD11b in Alzheimer’s disease mice reduced microglial lipid metabolism and neurodegenerative signatures that were shared with autoimmune-driven neuroinflammation in multiple sclerosis mice. Our data provide an interactive resource for investigation of the immunology of blood proteins that could support therapeutic targeting of microglia activation by immune and vascular signals.

overall_design: Mouse bone-marrow derived macrophages (BMDMs) or primary mouse microglia were stimulated with fibrin, complement iC3b, LPS, or left unstimulated and then profiled with scRNA-seq.

first: Andrew

middle: S

last: Mendiola

last: Akassoglou


title: A single-cell level comparison of human inner ear organoids with the human cochlea and vestibular organs

city: Leiden

first: Wouter

middle: H

last: van der Valk

address: Albinusdreef 2

department: Department of Otorhinolaryngology and Head & Neck Surgery

country: Netherlands

institute: Leiden University Medical Center

summary: In this study, we generated a human inner ear atlas containing three stages of inner ear development. This atlas was used to evaluate the differentiation approach of human pluripotent stem cells in to complex inner ear tissue, known as inner ear organoids. The primary goal of this single-nucleus RNA-sequencing analysis was to capture the cell type diversity of the human inner ear at different stages of development. The secondary goal was to define the similarity of organoid-derived inner ear cell types with the atlas-derived human inner ear cell types and to determine the developmental stage of the organoid-derived inner ear cell types.

overall_design: Single-nucleus RNA-sequencing data from two human fetal inner ears (fetal age weeks 7.5 and 9.2) and one human adult inner ear.

first: Edward

last: van Beelen

first: Matthew

last: Steinhart

first: Carl

last: Nist-Lund

first: Daniel

last: Osorio

first: John

last: de Groot

first: Lian

last: Sun

first: Peter Paul

last: van Benthem

first: Karl

last: Koehler

first: Heiko

last: Locher


title: TRIM28 modulates progesterone and estrogen signaling in endometrium

first: Franco

last: Demayo

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Defining blood-induced microglia functions in neurodegeneration through multiomic profiling [scRNAseq]

overall_design: CD11b+ brain cells from 6-month old 5XFAD:Fggg390-396A, 5XFAD, Fggg390-396A, and non-transgenic (NTG) control mice (3-4 mice per genotype) were isolated by fluorescence-activated cell sorting (FACS) and used for scRNA-seq. See Methods section of publication for details.


summary: In this study, we used a differentiation approach for guiding human pluripotent stem cells in to complex inner ear tissue, known as inner ear organoids. The primary goal of this single-cell and single-nucleus RNA-sequencing analyses was to define the cellular composition of the inner ear organoids, which we have shown and confirmed by histology. The secondary goal was to determine whether the inner ear organoids generated using different cell lines (WA01 hESCs, two reporter lines from the parental line GM25256, and LUMC0004iCtrl10 hiPSCs) contain similar cell types.

overall_design: Single-cell and single-nucleus RNA-sequencing data from three different timepoints using four different healthy-control stem cell lines.


title: Combined Lineage Tracing and scRNA-seq Reveals Unexpected First Heart Field Predominance of Human iPSC Differentiation

city: Stanford

first: Francisco

middle: Xavier

last: Galdos

address: 265 Campus Dr.

department: Cardiovascular Institute

institute: Stanford University

summary: During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation stage human embryos. Human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.

overall_design: scRNA-seq with oligonucleotide-based sample multiplexing for profiling cardiac differentiation across 12 timepoints and two independent hiPSC lines.

middle: X

first: Sean

last: Wu


title: Defining blood-induced microglia functions in neurodegeneration through multiomic profiling


title: Defining blood-induced microglia functions in neurodegeneration through multiomic profiling [plasma RNAseq]

overall_design: Plasma or artificial cerebrospinal fluid (aCSF) control was bilaterially injected (1.5 uL volume) into the corpus callosum of C57BL/6 mice. 24 h after injection, CD11b+CD45Lo  cells were isolated from mouse brains by fluorescence-activated cell sorting (FACS) and used for bulk RNA-seq. Citrate plasma was isolated from albumin deficient (AlbKO), C3 deficient (C3KO), fibrinogen mutant (Fggg390-396A), fibrinogen deficient (FGNKO) mice. Two mice were pulled per biological replicate, n = 3 - 4 replicates per group. See Methods section of publication for details and related references.


title: Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma

city: La Jolla

first: Shweta

last: Joshi

address: 3855 Health Sciences Drive, MC 0815

department: Department of Pediatrics

institute: UCSD Moores Cancer Center


title: Nucleic acid biomarkers of immune response and cell and tissue damage in children with COVID-19 and MIS-C [wb RNA]

first: Conor

last: Loy

overall_design:
Analysis of whole blood RNA in pediatric patients with COVID-19, MIS-C, and healthy controls

Submitter declares that the raw data have not been deposited due to patient privacy concerns.

first: Alicia

last: Sotomayor-Gonzalez

first: Venice

last: Servellita

first: Jenny

last: Nguyen

first: Joan

last: Lenz

first: Sanchita

last: Bhattacharya

first: Meagan

last: Williams

first: Alexandre

middle: P

last: Cheng

last: Bliss

first: Prachi

last: Saldhi

first: Noah

last: Brazer

first: Jessica

last: Streithorst

first: William

last: Suslovic

first: Charlotte

last: Hsieh

first: Burak

last: Bahar

first: Nathan

last: Wood

first: Abiodun

last: Foresythe

first: Amelia

last: Gliwa

first: Kushmita

last: Bhakta

first: Maria

last: Perez

first: Laila

last: Hussaini

first: Evan

last: Anderson

first: Ann

last: Chahroudi

first: Meghan

last: Delaney

first: Roberta

last: DeBiasi

first: Christina

last: Rostad

first: Iwijn

last: De Vlaminck

first: Charles

last: Chiu


title: Defining blood-induced microglia functions in neurodegeneration through multiomic profiling [5XFAD Toxseq]

first: Katerina

last: akassoglou

overall_design: CD11b+ROS- and CD11b+ROS+ brain cells from 12-month old 5XFAD and non-transgenic (NTG) control mice (3 mice per genotype) were isolated by fluorescence-activated cell sorting (FACS) and used for scRNA-seq. See Methods section of publication for details.

first: Andrew

last: Mendiola

last: Akassoglou


title: Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma: R788 anti-PD-L1 Radiation Experiment

first: Shweta

last: Joshi

summary: Neuroblastoma (NB) is considered an immunologically cold tumor and is usually less responsive to immune checkpoint blockade (ICB). Tumor-associated macrophages (TAMs) are highly infiltrated in NB tumors and promote immune escape and resistance to ICB. Hence therapeutic strategies targeting immunosuppressive TAMs can improve responses to ICB in NB. We recently discovered that spleen tyrosine kinase (Syk) reprograms TAMs toward an immunostimulatory phenotype and enhances T-cell responses in the lung adenocarcinoma model. Here we investigated if Syk is an immune-oncology target in NB and tested whether a novel immunotherapeutic approach utilizing Syk inhibitor together with radiation and ICB could provide a durable antitumor immune response in an MYCN amplified murine model of NB. Myeloid Syk KO mice and syngeneic MYCN-amplified cell lines were used to elucidate the effect of myeloid Syk on the NB tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or combination with anti-PDL1 mAb and radiation was also determined in murine NB models. The underlying mechanism of action of this novel therapeutic combination was also investigated. Herein, we report that Syk is a marker of NB -associated macrophages and plays a crucial role in promoting immunosuppression in the NB TME. We found that the blockade of Syk in NB-bearing mice markedly impairs tumor growth. This effect is facilitated by macrophages that become immunogenic in the absence of Syk, skewing the suppressive TME towards immunostimulation and activating anti-tumor immune responses. Moreover, combining FDA-approved Syk inhibitor, R788 (fostamatinib) along with anti-PDL1 mAb provides a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in mice bearing small tumors (50 mm3) but not larger tumors (250 mm3). However, combining radiation to R788 and anti-PDL1 mAb prolongs the survival of mice bearing large NB9464 tumors.

overall_design: NB9464 tumors were implanted into C57BL/6 WT mice and allowed to grow to a volume of 250 cubic millimeters.  Mice were then treated with vehicle control, R788 SYK inhibitor with anti-PD-L1 antibody, or Radiation with R788 SYK inhibitor with anti-PD-L1 antibody for four doses on days (28, 31, 34, and 37) and the whole tumors' RNA was sequenced to assess transcriptional effects of the different treatments.

first: Deepak

last: Rohila

first: In

last: Park

first: Timothy

last: Pham

first: Riley

last: Jones

first: Elisabette

last: Tapia

first: Kevin

last: Liu

first: Pablo

last: Tamayo

first: Alice

last: Yu

last: Sharabi


title: APEX1 nuclease and redox functions are both essential for adult mouse hematopoietic stem and progenitor cells.

city: Leuven

first: Samantha

last: Zaunz

address: ON4 – Herestraat 49

department: Stem Cell Institute

country: Belgium

institute: KU Leuven

summary: Self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) is carefully controlled by extrinsic and intrinsic factors, to ensure the lifelong process of hematopoiesis. Apurinic/apyrimidinic endonuclease 1 (APEX1) is a multifunctional protein implicated in DNA repair and transcriptional regulation. Although previous studies have emphasized the necessity of studying APEX1 in lineage-specific context and its role in some progenitor cells, no studies have assessed the role of APEX1, nor its two enzymatic domains, in supporting adult HSPC function. In this study, we demonstrated that complete loss of APEX1 from murine bone marrow HSPCs (induced by CRSIPR/Cas9) caused severe hematopoietic failure following transplantation, as well as an ex vivo HSPC expansion defect. Using specific inhibitors against either the nuclease or redox domains of APEX1 in combination with single cell transcriptomics (CITE-seq), we found that both APEX1 nuclease and redox domains are regulating mouse ex vivo HSPC proliferation, differentiation and survival, but through distinct mechanisms. Inhibition of the APEX1 nuclease function resulted in loss of HSPCs accompanied by early activation of differentiation programs and enhanced lineage commitment. By contrast, inhibition of the APEX1 redox function significantly downregulated interferon signaling in expanding HSPCs and their progeny, resulting in dysfunctional megakaryocyte-biased HSPCs, loss of monocytes and lymphoid progenitor cells. In conclusion, we demonstrate that APEX1 is a key regulator for adult regenerative hematopoiesis, and that the APEX1 nuclease and redox domains differentially impact lineage specification and stemness of functional ex vivo cultured HSPCs.

overall_design: We performed CITE-sequencing to understandthe role of APEX1 nuclease and redox function in proliferating hematopoietic stem and progenitor (HSPC) cells. LSK cells were exposed to specific APEX1 inhibitors (E3330, APX2009 and Inhibitor III) during a 7-day culture in medium maintaining HSC function. We combined CITE-sequencing to obtain measurement at the protein (using a panel of 168 antibody derived tags) and transcriptome level and to HASH-sequencing in order to measure all 4 experimental conditions (DMSO, E3330, APX2009, Inhibitor III) simulatanously.

first: Jonathan

last: De Smedt

first: Catherine

last: Verfaillie


title: Oviductal extracellular vesicles from women with endometriosis impair embryo development

city: Wuhan

first: Yuehan

last: Li

address: No. 1095, Jiefang Ave.

institute: Huazhong University of Scicence and Technology

summary:
Objective To investigate the influence that oviductal extracellular vesicles from patients with endometriosis exert on early embryo development.
Design In vitro experimental study
Setting University-affiliated hospital.
Patient(s) Women with and without endometriosis who underwent hysterectomy (n = 15 in total).
Intervention(s) None.
Main Outcome Measure(s) Oviductal extracellular vesicles from patients with endometriosis (oEV-EMT) or without endometriosis (oEV-ctrl) were isolated and co-cultured with two-cell murine embryos for 75 hours. Blastocyst rates were recorded. RNA sequencing was used to identified the differentially expressed genes in blastocysts cultured either with oEV-EMT or with oEV-ctrl. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to identify potential biological processes in embryos that oEV-EMT affect. The functions of oEV on early embryo development were determined by reactive oxygen species (ROS) levels, mitochondrial membrane potentials, total cell numbers and apoptotic cell proportions.
Result(s) Extracellular vesicles were successfully isolated from human Fallopian tubal fluid and their characterizations were described. The blastocyst rates were significantly decreased in the oEV-EMT group. RNA sequencing revealed that oxidative phosphorylation was down-regulated in blastocysts cultured with oEV-EMT. Analysis of oxidative stress and apoptosis at the blastocysts stage showed that embryos cultured with oEV-EMT had increased ROS level, decreased mitochondrial membrane potentials, and increased apoptotic index. Total cell numbers were not influenced.
Conclusion(s) Oviductal extracellular vesicles from patients with endometriosis exert a negative influence on early embryo development.

overall_design: Extracellular vesicles were isolated from human Fallopian tubal fluid. Murine embryos were co-cultured with these extracellular vesicles. Blastocysts were sequenced.

first: Lei

last: Jin

first: Cong

last: Sui


title: Nucleic acid biomarkers of immune response and cell and tissue damage in children with COVID-19 and MIS-C [cf DNA]

first: Conor

last: Loy

overall_design:
Analysis of plasma plasma cell-free DNA in pediatric patients with COVID-19, MIS-C, and healthy controls

Submitter declares that the raw data have not been deposited due to patient privacy concerns.

first: Alicia

last: Sotomayor-Gonzalez

first: Venice

last: Servellita

first: Jenny

last: Nguyen

first: Joan

last: Lenz

first: Sanchita

last: Bhattacharya

first: Meagan

last: Williams

first: Alexandre

last: Cheng

last: Bliss

first: Prachi

last: Saldhi

first: Noah

last: Brazer

first: Jessica

last: Streithorst

first: William

last: Suslovic

first: Charlotte

last: Hsieh

first: Burak

last: Bahar

first: Nathan

last: Wood

first: Abiodun

last: Foresythe

first: Amelia

last: Gliwa

first: Kushmita

last: Bhakta

first: Maria

last: Perez

first: Laila

last: Hussaini

first: Evan

last: Anderson

first: Ann

last: Chahroudi

first: Meghan

last: Delaney

first: Roberta

last: DeBiasi

first: Christina

last: Rostad

first: Iwijn

last: De Vlaminck

first: Charles

last: Chiu


title: Rebalancing liver-infiltrated CCR3+ and CD206+ monocytes improves diet induced NAFLD

city: Beijing

first: YangLu

last: Yang

state: Beijing

address: Beijing Xicheng District Yongan Road NO.95

institute: Beijing Friendship Hospital, Capital Medical University

summary: Melatonin has been reported to improve NAFLD, exploring the underlying mechanisms will be beneficial for better treatment of NAFLD. CDHFD- and MCD-fed mice with melatonin intervention exhibited significantly decreased liver steatosis, lobular inflammation, and focal liver necrosis. Single-cell RNA sequencing revealed melatonin selectively inhibited proinflammatory CCR3+ MoMFs, and upregulated anti-inflammatory CD206+ MoMFs in NAFLD mice. Hepatic infiltrated CCR3+CD14+ MoMFs is also significantly increased in patients with NAFLD. Mechanistically, melatonin receptor independent BTG2-ATF4 signaling plays a vital role in the regulation of CCR3+ MoMFs endoplasmic reticulum stress, survival, and proinflammation by melatonin. In contrast, melatonin upregulated CD206+ MoMFs survival and polarization via MT1/2 receptors. Melatonin stimulation also regulates human CCR3+ MoMFs and CD206+ MoMFs survival and inflammation in vitro. Furthermore, CCR3 depletion antibody monotherapy decreased liver inflammation and improved NAFLD in mice.  Thus, Therapies targeting CCR3+ MoMFs may have potential benefits in NAFLD treatment.

overall_design: Mice were fed with either methionine/choline-deficient diet (MCD), or MCD diet with melatonin. 7AAD-CD45+Ly6G-CD11bintF4/80hi monocytes obtained from MCD-fed mice liver which was treated with or without melatonin, and each treatment was repeated twice.

first: Guangyong

last: Sun

first: Yaning

last: Wang

first: Lu


title: Nucleic acid biomarkers of immune response and cell and tissue damage in children with COVID-19 and MIS-C

first: Conor

last: Loy


title: Analysis of chromatin accessibility in human umbilical artery endothelial cells stimulated with BMP9 and fluid shear stress

status: Public on Jun 01 2023

first: Paul-Lennard

last: Mendez

address: Thielallee 63

department: Institute for Chemistry and Biochemistry

institute: Freie Universität Berlin

summary: Bone morphogenetic protein (BMP) signaling and fluid shear stress (FSS), the frictional force exerted on endothelial cells by blood flowing over them, have complementary functions in vascular homeostasis and disease development. Both induce a wide range of target genes, not only independently  but also in a synergistic or antagonistic manner. Despite thorough research into genetic regulation downstream of BMP9 and FSS, detailed information on how both regulate chromatin accessibility is still missing. Here, we investigated whether BMP9 and FSS act independently, synergistically, or antagonistically in chromatin remodeling. To this end, we employed Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) to analyze arterial endothelial cells stimulated with BMP9 and FSS either individually or in combination.

overall_design: Bulk ATAC-seq of human umbilical artery endothelial cells (HUAECs) was performed in duplicates for a total number of 4 conditions (8 samples in total). Static, unstimulated cells were included as a control.

first: Jerome

last: Jatzlau

first: Lion

last: Raaz

first: Aybuge

last: Altay

first: Petra

last: Knaus


title: Transcriptional profiling of extraintestinal Escherichia coli exposed to cranberry pomace extracts alone or in combination with ceftriaxone

city: Guelph

first: Dion

last: Lepp

address: 93 Stone Rd W

country: Canada

institute: Agriculture and Agri-Food Canada

summary: To investigate the effects of ceftriaxone and cranberry pomace extract on transcriptome of extraintestinal E. coli (ExPEC)

overall_design: Comparative gene expression profiling analysis of RNA-seq data for E. coli O7:H18 (ST38) grown in CAMHB supplemented with sub-inhibitory concentration of CRAN and AXO alone or in combination

first: Moussa

last: Diarra

first: Quail

last: Das


title: Generation of functional posterior spinal motor neurons from hPSCs-derived human spinal cord neural progenitor cells

city: Boston

first: Jiehui

last: Chen

address: 320 Longwood Ave

institute: Boston Children’s Hospital

summary: Spinal motor neurons deficiency results in a series of devastating disorders such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and spinal cord injury (SCI). These devastating disorders are currently incurable, while human pluripotent stem cells (hPSCs) derived spinal motor neurons are promising but suffered by low-efficiency, functional immaturity and lacks of posterior cell identity. In this study, we have established human spinal cord neural progenitor cells (hSCNPCs) via hPSCs differentiated neuromesodermal progenitors (NMPs) and demonstrated the hSCNPCs can be continuously expanded up to 40 passages. hSCNPCs can be rapidly differentiated into posterior spinal motor neurons with high efficiency. The functional maturity has been examined in detail. Moreover, a co-culture scheme which is compatible for both neural and muscular differentiation is developed to mimic the neuromuscular junction (NMJ) formation in vitro. Together, these studies highlight the potential avenues for generating clinically relevant motor neurons and modelling neuromuscular diseases through our defined hSCNPCs.

overall_design: We performed bulk RNA sequencing on samples collected on each day from D0 to D4 and every day at uring NeuDet stage and hSCNPCs at different passages (P5,P10,P20,P30 and P40). We also employed time course RNA-seq analysis on every 6 days from D0 to D30, as well as D42

first: He

last: Jax Xu

first: Xianfa


title: Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma: TCM Macrophage Experiment

first: Shweta

last: Joshi

overall_design: NB9464, a MYCN-amplified murine neuroblastoma cell line, was cultured in media for 48 hours to create tumor conditioned media (TCM).  The TCM was then isolated by centrifuge and Bone Marrow Derived Macrophages (BMDM's) were grown in the media with or without SYK inhibitor (R788).  Transcriptional effects were quantified using RNAseq sequencing.

first: Deepak

last: Rohila

first: In

last: Park

first: Timothy

last: Pham

first: Riley

last: Jones

first: Elisabette

last: Tapia

first: Kevin

last: Liu

first: Pablo

last: Tamayo

first: Alice

last: Yu

first: Andrew

last: Sharabi


title: Single-cell RNA-seq analysis reveals that immune cells induce human nucleus pulposus ossification and degeneration

city: Shanghai

first: Qiang

last: Fu

address: No.100 Haining Road

institute: Department of Spine Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine

summary: Background and aims  Determining the transcriptomes and molecular mechanism underlying human degenerative nucleus pulposus (NP) is of critical importance for treating intervertebral disc degeneration (IDD). Here, we aimed to elucidate the detailed molecular mechanism of NP ossification and IDD using single-cell RNA sequencing.  Methods  Single-cell RNA-seq and bioinformatic analysis were performed to identify NP cell populations with gene signatures, biological processes and pathways, and subpopulation analysis, RNA velocity analysis, and cell-to-cell communication analysis were performed in four IDD patients. We also verified the effects of immune cells on NP ossification using cultured NP cells and a well-established rat IDD model.  Results  We identified five cell populations with gene expression profiles in degenerative NP at single-cell resolution. GO database analysis showed that degenerative NP-associated genes were mainly enriched in extracellular matrix organization, immune response, and ossification. Gene set enrichment analysis showed that rheumatoid arthritis signaling, antigen processing and presentation signaling were activated in the blood cell cluster. We revealed that stromal cells, which are progenitor cells, differentiated toward an ossification phenotype and delineated interactions between immune cells (macrophages and T cells) and stromal cells. Immune factors such as TNF-α, CD74 and CCL-3 promoted the differentiation of stromal cells toward an ossification phenotype in vitro. Blocking TNF-α with a specific inhibitor successfully reversed NP ossification and modified NP morphology in vivo.  Conclusions Our study revealed an increase in macrophages and T cells in degenerative NP, which induced stromal cell differentiation toward an ossification phenotype, and contributed to the identification of a novel therapeutic target to delay IDD.

overall_design: NP samples from four patients in lumbar discectomy surgery were collected and minced with a razor blade into 1 mm3 fragments on a dish under aseptic conditions. And, all the samples were performed single-cell RNA sequencing according to the manufacturer’s protocol.

first: Song

last: Guo

first: Meijun

last: Yan

first: Xinhua

last: Li

first: Shuya

last: Zhang

first: Zhong

first: Kewei

first: Pengcheng

first: Yanbin

first: Guixin

last: Sun


title: Pre-determined diversity in resistant fates emerges from homogenous cells after anti-cancer drug treatment

status: Public on May 31 2023

city: Chicago

first: Yogesh

last: Goyal

state: IL

address: 303 E Superior St

institute: Northwestern University

summary: Even amongst genetically identical cancer cells, therapy resistance often only emerges from a very small subset of those cells. Much effort has gone into uncovering the molecular differences in rare individual cells in the initial population that may allow certain cells to become therapy resistant; however, comparatively little is known about variability in the resistant outcomes themselves. Here, we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically, and functionally distinct fate types. These different resistant types are largely predetermined by molecular differences between cells before addition of drug and not by extrinsic cell-specific microenvironmental factors. Changes in dose and kind of drug can, however, switch the resistant fate type of an initial cell, even resulting in the generation and elimination of certain fate types. Diversity in resistant fates was observed across several single-cell-derived cancer cell lines and types treated with a variety of drugs. Cell fate diversity as a result of variability in intrinsic cell states may be a generic feature of response to external cues.

overall_design:
Isolated and expanded melanoma WM989 resistant clones, isolated and longitudinally profiled melanoma WM989 resistant clones.
We conducted standard bulk paired-end (37:8:8:38) RNA sequencing using RNeasy Micro (Qiagen 74004) for RNA extraction, NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490L), NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB E7770L), NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) oligos (NEB E7600S), and an Illumina NextSeq 550 75 cycle high-output kit (Illumina 20024906), as previously described. Prior to extraction and library preparation, the samples were randomized to avoid any experimental and human biases. As previously described, we aligned RNA-seq reads to the human genome (hg19) with STAR v2.5.2a and counted uniquely mapping reads with HTSeq v0.6.1 and outputs count matrix.

first: Arjun

last: Raj


title: Effect of 30Kc19α-Lin28A protein on gene expression in osteogenesis of urine-derived stem cells

city: Chuncheon

first: Ju Hyun

state: Gangwon-do

address: 1 Kangwondaehak-gil

department: Department of Biomedical Science

country: South Korea

institute: Kangwon National University

summary:
To elucidate the function of 30Kc19α-Lin28A protein in osteogenic differentiation of urine-derived stem cells, we established urine-derived stem cell lines differentiated with or without protein treatment.
We then performed gene expression profiling analysis using data obtained from RNA-seq of osteogenic differentiated urine-derived stem cells with or without 30Kc19α-Lin28A protein treatment, undifferentiated urine-derived stem cells, and human osteoblasts

overall_design: Comparative gene expression profiling analysis of RNA-seq data for urine-derived stem cells differentiated into osteoblasts with or without treatment with 30Kc19α-Lin28A protein.

first: Jinhee


title: Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development

city: Cincinnati

first: Soona

last: Shin

state: Cincinnati

address: 3333 Burnet Ave, OH

institute: Cincinnati Children's Hospital Medical Center

summary: Acute myeloid leukemia (AML) is an aggressive blood cancer that stems from the rapid expansion of immature leukemic blasts in the bone marrow. Mutations in epigenetic factors represent the largest category of genetic drivers of AML. The chromatin assembly factor CHAF1B is a master epigenetic regulator of transcription associated with self-renewal and the undifferentiated state of AML blasts. Upregulation of CHAF1B, as observed in almost all AML samples, promotes leukemic progression by repressing transcription of differentiation factors and tumor suppressors. However, the specific factors regulated by CHAF1B and their contributions to leukemogenesis are unstudied. We analyzed RNAseq from mouse MLL-AF9 leukemic cells and bone marrow aspirates representing a diverse collection of pediatric AML samples and identified the E3 ubiquitin ligase TRIM13 as a target of CHAF1B-mediated transcriptional repression associated with leukemogenesis. We found that CHAF1B binds the promoter of TRIM13, resulting in its transcriptional repression. In turn, TRIM13 suppresses self-renewal of leukemic cells by promoting pernicious entry into the cell cycle through its nuclear localization and catalytic ubiquitination of cell cycle-promoting protein CCNA1. Overexpression of TRIM13 initially prompts a proliferative burst in AML cells that is followed by exhaustion, while loss of total TRIM13 or deletion of its catalytic domain enhanced leukemogenesis in AML cell lines and patient-derived xenografts. These data suggest that CHAF1B promotes leukemic development in part by repressing TRIM13 expression, and that this relationship is necessary for leukemic progression.

overall_design: Examination of the function of CHAF1B in maintaining leukemic cells. We used Chaf1b-floxed MLL-AF9 leukemic cell clones which were transduced with CreERT2. Knockout was induced by treatment with B-estradiol, and cells were lysed 48 hours after induction for analysis. Floxed cells expressing CreERT2 treated with EtOH were used as clonal controls.

first: Aditi

last: Paranjpe

last: Volk


title: CRY1-ChIP-seq in MEFs, PreiPSCs, iPSCs, and ESCs

city: College Station

first: Shogo

last: Sato

state: Texas

address: 3258 TAMU, Biological Science Building West (BSBW) 309

department: Biology

institute: Texas A&M University

summary: Genome-wide occupancy of the circadian clock Cryptochrome1 (CRY1) was compared between somatic differentiated mouse embryonic fibroblasts (MEFs) and pluripotent stem cells including precursor-induced pluripotent stem cells (iPSCs), iPSCs, and embryonic stem cells (ESCs)

overall_design: The library was sequenced on the NovaSeq 6000 Sequencer. Sequencing was performed using paired-end flowcell chemistry and 100 cycles with additional cycles for the index reads. The NovaSeq 6000 real-time analysis software (RTA 3.4.4) converted the images into intensities and base calls. Post-processing of the run to generate the FASTQ files was performed at the Institute for Genomics and Bioinformatics (IGB), UCI.

first: Juan Carlos

middle: I

last: Belmonte

first: Yumei

first: Wei


title: Immune differences in CD4+ T lymphocytes between alloimmunized and non-alloimmunized AML patients.

city: Créteil

first: Mehdi

last: Khelfa

address: 5 Rue Gustave Eiffel

country: France

institute: Institut Mondor de Recherche Biomédicale

summary: Platelet alloimmunized and non-alloimmunized AML patients have differences in their immune system. CD4+ CD45RA- T cells were characterized in scRNAseq.

overall_design: BD Rhapsody technology was used to assess the protein and RNA expression in the memory CD4+ T lymphocytes in AML patients. Up to 7 patients were multiplexed per sample.

first: Benoit

last: Vingert

first: Stephane

last: Kerbrat


title: Swc4 protects nucleosome-free rDNA, tDNA and telomere loci to inhibit genome instability

first: Yue

last: Pan

address: 320 Yue Yang Road

department: Center for Excellence in Molecular Cell Science, CAS

institute: Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

summary: In the baker’s yeast Saccharomyces cerevisiae, NuA4 and SWR1-C, two multisubunit complexes, are involved in histone acetylation and chromatin remodeling, respectively. Eaf1 is the assembly platform subunit of NuA4, Swr1 is the assembly platform and catalytic subunit of SWR1-C, while Swc4, Yaf9, Arp4 and Act1 form a functional module, and is present in both NuA4 and SWR1 complexes. ACT1 and ARP4 are essential for cell survival. Deletion of SWC4, but not YAF9, EAF1 or SWR1 results in a severe growth defect, but the underlying mechanism remains largely unknown. Here, we show that swc4Δ, but not yaf9Δ, eaf1Δ, or swr1Δ cells display defects in DNA ploidy and chromosome segregation, suggesting that the defects observed in swc4Δ cells are independent of NuA4 or SWR1-C integrity. Swc4 is enriched in the nucleosome-free regions (NFRs) of the genome, including characteristic regions of RDN5s, tDNAs and telomeres, independently of Yaf9, Eaf1 or Swr1. In particular, rDNA, tDNA and telomere loci are more unstable and prone to recombination in the swc4Δ cells than in wild-type cells. Taken together, we conclude that the chromatin associated Swc4 protects nucleosome-free chromatin of rDNA, tDNA and telomere loci to ensure genome integrity.

overall_design: Examination of the enrichment of 3Flag tagged Arp4 on chromatin in the budding yeast

first: Yu-Long

last: Chen

first: Lin-Jun

last: Hou

first: Jin-Qiu

last: Zhou


title: Effect of Fusobacterium nucleatum (Fn) infection or Fn-Dps treatment on gene expression of esophageal carcinoma Kyse150 and Kyse30 cells

city: Guangzhou

first: Yiqiu

last: Li

state: State...

address: College of Pharmacy, Sun Yat-sen University

institute: Sun Yat-sen University

summary: Kyse150 and Kyse30 cells were infected by Fn (the MOI of 1:10) or treated with Fn-Dps (1 μM) for 48 h

overall_design: Comparative gene expression profiling analysis and transcription factors prediction factors of RNA-seq data for  K150Con/K30Con (cells without any treatment or infection) and K150Fn/K30Fn (cells were infected by Fn) or K150Nap/K30Nap (cells were treated with Fn-Dps)

first: Ge

first: Wanli

last: Liu


title: PD-L1-Mediated Immune Evasion in Triple-Negative Breast Cancer Is Linked to the Loss of ZNF652

city: beijing

first: cheng

middle: yun

last: liu

address: 北京市海淀区学院路38号

institute: peking university

summary: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for zinc finger protein ZNF652 in MCF-7 cells.

overall_design: Examination of ZNF652 target genes in MCF-7 cell line. We constructed ZNF652 knockout (ZNF652 KO) MCF-7 cells using CRISPR-Cas9 and performed RNA-sequencing (RNA-seq) to examine the ZNF652-regulated transcriptome. Repeat three times for each group.

first: Yuncheng

first: Chunyu

last: Yu


title: Specialized regulatory T cells control venous blood clot resolution through SPARC

city: Mainz

first: Matthias

last: Klein

address: Langenbeckstrasse 1

department: Inst. for Immunology

institute: University Medical Center Mainz

summary: The cells and mechanisms involved in blood clot resorption are only partially known. Regulatory T cells (Treg) accumulate in venous blood clots and regulate thrombolysis by controlling the recruitment, differentiation and matrix metalloproteinase (MMP) activity of monocytes. The clot Treg population is heterogeneous and contains a population of Treg that forms the matricellular acid- and cysteine-rich protein (SPARC). SPARC induces MMP activity in monocytes and SPARC+ Treg are required for clot resorption.

overall_design:
Whole transcriptome analysis (WTA) of CD25+ single cells from day 12  thrombi and vein walls of thrombotic B6 mice (n=10, pooled)

The linked python script can be used to translate cell indexes to raw sequences found in the fastq files. See the linked readme file for further instructions.

first: Fatemeh

last: Shahneh

first: Verena

last: Raker

first: Tobias

last: Bopp

first: Christian

last: Becker


title: Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma: TAM SYK WT/KO Adoptive Transfer Experiment

first: Shweta

last: Joshi

institute: UCSD Moores Cancer Center

summary: Neuroblastoma (NB) is considered an immunologically cold tumor and is usually less responsive to immune checkpoint blockade (ICB). Tumor-associated macrophages (TAMs) are highly infiltrated in NB tumors and promote immune escape and resistance to ICB. Hence therapeutic strategies targeting immunosuppressive TAMs can improve responses to ICB in NB. We recently discovered that spleen tyrosine kinase (Syk) reprograms TAMs toward an immunostimulatory phenotype and enhances T-cell responses in the lung adenocarcinoma model. Here we investigated if Syk is an immune-oncology target in NB and tested whether a novel immunotherapeutic approach utilizing Syk inhibitor together with radiation and ICB could provide a durable antitumor immune response in an MYCN amplified murine model of NB. Myeloid Syk KO mice and syngeneic MYCN-amplified cell lines were used to elucidate the effect of myeloid Syk on the NB tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or combination with anti-PDL1 mAb and radiation was also determined in murine NB models. The underlying mechanism of action of this novel therapeutic combination was also investigated. Herein, we report that Syk is a marker of NB -associated macrophages and plays a crucial role in promoting immunosuppression in the NB TME. We found that the blockade of Syk in NB-bearing mice markedly impairs tumor growth. This effect is facilitated by macrophages that become immunogenic in the absence of Syk, skewing the suppressive TME towards immunostimulation and activating anti-tumor immune responses. Moreover, combining FDA-approved Syk inhibitor, R788 (fostamatinib) along with anti-PDL1 mAb provides a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in mice bearing small tumors (50 mm3) but not larger tumors (250 mm3). However, combining radiation to R788 and anti-PDL1 mAb prolongs the survival of mice bearing large NB9464 tumors.

overall_design: 4 million NB9464 cells were implanted into Myeloid Syk WT and KO mice. The tumors were allowed to grow for 40 days. TAM's were isolated from the implanted tumors and subjected to RNAseq to determine transcriptional effects of Syk KO and to confirm macrophage Syk controls tumor growth and inhibits adaptive immune responses in NB.

first: Deepak

last: Rohila

first: In

last: Park

first: Timothy

last: Pham

first: Riley

last: Jones

first: Elisabette

last: Tapia

first: Kevin

first: Pablo

last: Tamayo

first: Alice

first: Andrew

last: Sharabi


title: Accelerated evolution analysis uncovers PKNOX2 as a key transcription factor in the mammalian cochlea

city: Buenos Aires

first: Lucia

middle: F

last: Franchini

address: Vuelta de Obligado 2490

country: Argentina

institute: INGEBI-CONICET

summary: The genetic bases underlying the evolution of morphological and functional innovations of the mammalian inner ear are poorly understood. Gene regulatory regions are thought to play an important role in the evolution of form and function. To uncover crucial hearing genes whose regulatory machinery evolved specifically in mammalian lineages, we mapped accelerated noncoding sequences (ACNEs) in inner ear transcription factors (TFs) identifying PKNOX2 as the gene displaying the largest amount of ACNEs. To investigate the function of PKNOX2-ACNEs, we tested them using enhancer assays in transgenic zebrafish and unmasked transcriptional enhancers that acquired novel expression patterns as a consequence of the evolutionary process they underwent. Even though PKNOX2 is one of the most highly expressed genes in cochlear hair cells, its function was unknown. Thus, to explore the role of PKNOX2 in mammalian hearing, we generated Pknox2 null mice using CRISPR/Cas9 technology.  Pknox2-/- mice exhibited reduced distortion product otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR) thresholds at high frequencies together with an increase in peak 1 amplitude, consistent with a higher number of IHCs-auditory nerve synapsis observed at the cochlear basal region. A comprehensive cochlear transcriptomic analysis of Pknox2-/- and Pknox2+/+ mice revealed that key auditory genes are under Pknox2 control. Hence, we report, for the first time, that PKNOX2 has a critical function regulating cochlear sensitivity at higher frequencies and that its regulatory machinery underwent lineage-specific evolution, providing novel insight into the contribution of this TF to normal auditory function and to the evolution of high-frequency hearing.

overall_design: Cochleas were extracted from Pknox2+/+ and Pknox2-/-mice at eight days of age (P8) in 5 independent biological samples


title: Transcriptome profiling Zebrafish Sap130b gene mutations in embryogenesis

city: Pittsburgh

first: Michael

last: Tsang

address: 3501 Fifth Ave. BST3-5062

department: Cell Biology

institute: University of Pittsburgh

summary: This study profiled the transcriptome of zebrafish embryos harboring mutation in the Sin3A associated Protein 130b (sap130b) gene at 36 hours post fertilization (hpf). The sap130b mutant was generated via Cas9 mediated gene editing. Zebrafish sap130b mutants are viable and survive as adults with no observable phenotype. Maternal Zygotic sap130b mutants were established and they were also normal and it was reasoned that sap130a was compensating for the loss of sap130b. A transcriptome analysis was performed at 36 hpf to compare with our analysis of sap130a at this stage.

overall_design: Total RNA was extracted from embryos at 36 hours post fertilization from wildetype, MZsap130b mutants.

first: Ricardo

last: DeMoya


title: Transcriptome profiling Zebrafish Sap130a gene mutations in embryogenesis

summary: This study profiled the transcriptome of zebrafish embryos harboring mutation in the Sin3A associated Protein 130a (sap130a) gene at 36 hours post fertilization (hpf). The sap130a mutant was generated via Cas9 mediated gene editing. Zebrafish sap130a mutants are viable presumable due to the presence of maternal RNA. Generation of maternal zygotic (MZ) mutants resulted in an incomplete penetrance of heart ventricle defects that were visible as early as 36 hpf. In this experiment the MZsap130a mutant embryos were further sorted for embryos with smaller ventricle (SV) vs normal size ventricle (NV). To extend on this study and to specifically analyze the heart, sap130a mutant and WT hearts at 48 hpf were extracted for RNA-seq analysis.

overall_design: Total RNA was extracted from embryos at 36 hours post fertilization from wildetype, MZsap130a mutants.


title: RNASeq of Autophagy Mutant BMDMs

city: Cambridge

first: Ramnik

last: Xavier

address: 415 Main Street

institute: Broad Institute of MIT and Harvard

summary: Autophagy is an essential cellular process that is deeply integrated with innate immune signaling; however, studies that examine autophagy in the context of inflammatory conditions are lacking. Here, using mouse models with a constitutively active variant of the autophagy gene Beclin1, we show that increased autophagy dampens cytokine production in models of inflammation and adherent-invasive Escherichia coli (AIEC) infection. We further analyzed primary macrophages from these animals with a combination of transcriptomics and proteomics to identify novel mechanistic targets downstream of autophagy. Our study reveals glutamine metabolism and the RNF128/TBK1 axis as independent regulators of inflammation. Altogether, our work highlights increased autophagic flux as a potential approach to reduce inflammation and defines independent mechanistic cascades involved in this control.

overall_design: BMDMs differentiated from autophagy mutants and their littermates were harvested at 0,6, and 24 hours post-LPS/IFNgamma stimulation

first: Jinjin

last: Xu

first: Lingjia

last: Kong

first: Bihua

last: Li

first: Jacques

last: Deguine


title: Deficiency of GDF11 Accelerates TAC-Induced Heart Failure by impairing cardiac angiogenesis

city: Hangzhou

first: Yun

middle: Jin

last: Zhu

state: zhejiang

address: Jiefang road 88

institute: Department of Cardiology, Second Affiliated Hospital, College of Medicine, Zhejiang University.

summary:
Purpose:The role of GDF11 in aging and cardiovascular diseases is controversial, and its function in cardiomyocytes(CMs) has not been fully explored.
Methods: Total RNA was extracted from CMs using TRIzol® after CMs were transfected with shRNA-GDF11 and sh-Vector for 48h. Preparation of library and sequencing of transcriptome were carried out using Illu-mina HiSeq X Ten (Novogene Bioinformatics Technology Co., Ltd., Beijing, China).
Results: Angiogenesis-related genes were the most downregulated when GDF11 was knockdown in mouse CMs. RNA-seq analysis showed that 3293 genes were downregulated and 3214 genes were upregulated when GDF11 was knockdown with shRNA-GDF11.

overall_design: Total RNA was extracted in accordance with the manual of TRIzol®  from CMs transfected with shRNA-GDF11 and shRNA-NC for 48h. Preparation of library and sequencing of transcriptome were carried out using Illu- mina HiSeq X Ten.

first: Jinyun

first: Ning

last: Zhang


title: Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution

city: Indianapolis

first: Tsubasa

last: Saeki

state: IN

address: 980 W Walnut St

department: Otolaryngology-Head and Neck Surgery

institute: Indiana University School of Medicine

summary: The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human ES cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analysis with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that hair cells arise primarily from supporting cells, rather than bi-fated transitional cells in organoids. Moreover, ion channel- and ion transporter-related gene sets were enriched in supporting cells vs. prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells vs. supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss and balance disorders.

overall_design: Yoshitomo, Ueda

first: Takashi

last: Nakamura

first: Jing

last: Nie

first: Alexander

middle: J

last: Solivais

first: John

middle: R

last: Hoffman

first: Becca

last: Daye

first: Eri

last: Hashino


title: Changes in gene expression of human monocyte derived dendritic cells exposed to live Borrelia burdgorferi or LTA

city: Baltimore

first: Mark

last: Soloski

state: Maryland

address: 5200 Eastern Avenue

department: Medicine

institute: Johns Hopkins University School of Medicine

summary: Dendritic cells bridge the innate and adaptive immune response by serving as sensors of infection and as the primary antigen presenting cells responsible for the initiation of the T cell response against invading pathogens. Initial interactions between B. burgdorferi, the causative agent of Human Lyme disease, and dendritic cells remain largely unexplored.  To address this, we cultured monocyte-derived dendritic cells (moDCs) with and without the Toll-Like Receptor 2 agonist LTA  or live B. burgdorferi and examined changes in gene expression using RNAseq.  We observed that B. burgdorferi exposed moDCs display a distinct and novel gene expression signature that differs from that induced by the Toll-Like Receptor 2 reference agonist.  Collectively these studies indicate that the interaction of live B. burgdorferi with mo-DCs promotes a unique mature DC phenotype that likely impacts the nature of the adaptive T cell response generated in human Lyme disease.

overall_design: We isolated monocytes derived dendritic cells (moDCs) from seven different human subjects.  MoDCs were either left unstimlulated or stimulated them in vitro with either Lipotechoic Acid (LTA) or two different stains of Borrelia burdorferi (A3 and Bb31) .  We also used multiplicities of infection (moi) of 1 and 10 bacteria/cell.  Thre stimulaiton was carreid out for 8 and 24hrs after which cells were harvested and RNA extracted for RNAseq analysis.

first: Maria

last: Gutierrez-Hoffmann

first: Lilianna

last: Florea

first: Jinshiu

last: Fan

first: Corina

last: Antonescu

first: Conover

last: Talbot Jr


title: Direct and indirect consequences of PAB1 deletion in the regulation of translation initiation, translation termination, and mRNA decay [Ribo-seq]

city: Worcester

first: Kotchaphorn

last: Mangkalaphiban

address: 368 Plantation St.

institute: University of Massachusetts Medical School

summary: Cytoplasmic poly(A)-binding protein (PABPC, yeast Pab1) is involved in various stages of mRNA post-transcriptional control, including translation initiation, translation termination, and mRNA decay regulation. Due to the multi-functional properties of PABPC, attempts to define a specific role for PABPC in vivo by deleting, depleting, overexpressing, or mutating the protein has led to conflicting models of PABPC's function. To specifically assess direct and indirect consequences of deleting PABPC, we generated and analyzed RNA-Seq, ribosome profiling, and mass spectrometry data from yeast cells lacking Pab1.

overall_design: RNA-Seq and ribosome profiling of WT, pbp1∆, and pab1∆pbp1∆ cells (pbp1∆ is needed to suppress lethality of pab1∆), 3 replicates for each strain.

first: Allan

last: Jacobson


title: Direct and indirect consequences of PAB1 deletion in the regulation of translation initiation, translation termination, and mRNA decay [RNA-seq]


title: In vivo inhibition of nuclear ACE2 translocation protects against SARS-CoV-2 replication and lung damage through epigenetic imprinting

city: Brisbane

first: Quan

last: Nguyen

state: Queensland

address: 22 Burns Street, Indooroopilly

country: Australia

institute: Institute for Molecular Bioscience

summary:
In vitro, ACE2 translocates to the nucleus to induce SARS-CoV-2 replication. Here, using digital spatial profiling of lung tissues from SARS-CoV-2-infected golden Syrian hamsters, we show that a specific and selective peptide inhibitor of nuclear ACE2 (NACE2i) inhibits viral replication two days after SARS-CoV-2 infection.
NACE2i also prevents inflammation and macrophage infiltration, and increases NK cell infiltration in bronchioles. NACE2i treatment restores host translation in infected hamster bronchiolar cells.

sample_organism:
Mesocricetus auratus
unidentified

platform_organism:
Mesocricetus auratus
unidentified

overall_design: Comparative gene expression profiling analysis of spatial RNA-seq data for the selected regions of interest (ROIs) on the lung tissues of SARS-CoV-2 infected hamster with or without NACE2i treatment .

first: Sudha

last: Rao

first: Wen

last: Tu

first: Michelle

last: Melino

first: Jenny

last: Dunn

first: Robert

middle: D

last: McCuaig

first: Helle

last: Bielefeldt-Ohmann

first: Sofiya

last: Tsimbalyuk

first: Jade

middle: K

last: Forwood

first: Taniya

last: Ahuja

last: Vandermeide

first: Xiao

last: Tan

first: Minh

last: Tran

first: Liang

first: Andy

last: Nam

first: Liuliu

last: Pan

first: Yan

last: Liang

first: Corey

last: Smith

first: Katie

last: Lineburg

first: Tam

first: Julian

last: Sng

first: Zhen

middle: W

last: Tong

first: Keng

last: Chew

first: Kirsty

last: Short

first: Roger

last: Le Grand

first: Nabila

last: Seddiki


title: Effect of vimentin deletion on gene expression in Kras-G12D-mutant, p53-null lung adenocarcinoma cells

first: kishore

last: Anekalla

address: 303 E. Superior

department: Pulmonary and Critical Care

institute: Northwestern University

summary: Tumor initiation was induced in LSL-KrasG12D/+;Tp53flox/flox (KPV+/+) and LSL-KrasG12D/+;Tp53flox/flox;Vim-/- (KPV-/-) mice by Ad-cre delivery. Tumor cells were collected after 6 weeks and maintained in culture. RNA was collected from three independent cell lines per group and subjected to sequencing.

overall_design: Comparative gene expression analysis by RNA-seq for Vim_WT and Vim_KO cells.

first: Alex

last: Berr

first: Kishore

first: Clarissa

last: Koch

first: Hiam

last: Abdala-Valencia

first: Karen

last: Ridge


title: The cholesterol transporter NPC1 is essential for epigenetic regulation and maturation of oligodendrocyte lineage cells ChIP-seq

city: Ann Arbor

first: Andrew

last: Lieberman

state: MI

address: 1150 W Medical Center Drive

institute: University of Michigan

summary: Cholesterol is required for oligodendrocyte maturation and CNS myelination. Here, we demonstrate an essential role for the intracellular cholesterol transporter NPC1 in these processes. NPC1 functions in late endosomes and lysosomes to efflux unesterified cholesterol, and its deficiency causes Niemann-Pick disease Type C, an autosomal recessive lysosomal disorder characterized by progressive neurodegeneration and early death. To identify cell types and pathways affected early in pathogenesis, we performed single nuclear RNA-seq on the forebrain of Npc1-/- mice at P16. This analysis uncovered striking transcriptional changes in the oligodendrocyte lineage during the period of developmental myelination, accompanied by diminished maturation of myelinating oligodendrocytes. Unexpectedly, we identified a significant upregulation of genes associated with neurogenesis and synapse formation in Npc1-/- oligodendrocyte lineage cells, reflecting diminished gene silencing by H3K27me3 and H3K9me3. Npc1-/- oligodendrocyte progenitor cells reproduced impaired maturation in vitro and this phenotype was rescued by treatment with GSK-J4, a small molecule inhibitor of H3K27 demethylases. This study sought to determine the genes being directly impacted by the epigenetic dysfunction at H3K27.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for  the histone modifications H3K27me3 and H3K27ac in oligodendrocyte lineage cells isolated from mouse brain.

first: Thaddeus

last: Kunkel


title: The cholesterol transporter NPC1 is essential for epigenetic regulation and maturation of oligodendrocyte lineage cells bulk RNA-seq of mouse OPCs

overall_design: Oligodendrocyte progenitors were isolated from mouse brains using magnetic-activated cell sorting. RNA-seq was performed on cells from 3 WT and 3 Npc1-null mice.

first: Alice

last: Townsend

first: Kyle

middle: A

last: Sullivan

first: Jean

last: Merlet

first: Daniel

last: Jacobson


title: Transcriptome profiling Zebrafish Sap130a and Sap130b gene mutations in embryogenesis

first: Michael

last: Tsang

state: PA

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

overall_design: Refer to individual Series


title: The cholesterol transporter NPC1 is essential for epigenetic regulation and maturation of oligodendrocyte lineage cells


title: Dynamic single-cell RNA sequencing reveals that intravenous BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation

first: Rulin

last: Wang

address: 1650 Orleans Street

institute: Johns Hopkins University

summary: COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Guérin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG vaccination conferred a modest reduction on lung SCV2 viral load, bronchopneumonia scores, and weight loss. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated local antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

overall_design: Groups of hamsters were intravenously (I.V.) administered saline or were vaccinated (I.V.) with 5×10^6CFU of BCG-Tice. 30 days after vaccination animals were challenged with 5×10^5TCID50units of SARS-CoV-2 (SCV2) (Wuhan-1/2020) by the intranasal route. At D4 and D7 post-challenge animals were sacrificed for analysis. 3 animals (BCG only) were not infected with SCV2 and were sacrificed 30 days post-vaccination to serve as controls

first: Alok

last: Singh

first: Srinivasan

last: Yegnasubramanian

first: William

last: Bishai


title: Opposing transcriptional circuits governing skeletal muscle development and maturation revealed at single nuclear resolution [snRNA-seq]

city: Dallas

first: kenian

last: chen

state: TX

address: 5323 Harry Hines Boulevard

department: Population and Data Science

institute: UTsouthwestern

summary:
To explore the mechanistic basis of the different phases of pre- and postnatal muscle development and the contraction-dependent control of muscle gene expression ,
we generated a multi-omics single nucleus RNAseq (snRNA-seq) and single nucleus ATAC-seq (snATAC-seq) atlas of normal muscle and paralyzed muscle from mice lacking Cacna1s.

overall_design: Matthieu ,Dos Santos

first: Akansha

last: Shah

first: Yichi

last: Zhang

first: Kenian

last: Chen

first: Lin

last: Xu

first: Weichun

last: Lin

first: Rhonda

last: Bassel-Duby

first: Ning

last: Liu

first: Eric

middle: N

last: Olson


title: Opposing transcriptional circuits governing skeletal muscle development and maturation revealed at single nuclear resolution [snATAC-seq]

overall_design: Matthieu, Dos Santos


title: Proteins involved in taking up cyclic peptides

city: Riverside

first: Min

last: Xue

state: CA

address: 501 Big Spring Rd

department: Chemistry

institute: University of California, Riverside

summary: To identify differentially expressed genes that dictates the cell uptake activity

overall_design: We treated U87 cells with the Rhodamine-tagged EPP6 cyclic peptide for 1 hour. Then we used FACS to sort out the top 30% and bottom 30% populations based on Rhodamine fluorescence. The collected cells were used for RNAseq analyses.

first: Xue

last: Min

first: Wang

last: Siwen


title: Opposing transcriptional circuits governing skeletal muscle development and maturation revealed at single nuclear resolution.


title: RNAseq Quantitative Analysis of Arabidopsis Wild Type versus flavonoid 3' hydroxylase tt7 mutant

city: Valencia

first: Jose

last: Gadea

address: Ingeniero Fausto Elio

department: Stress

country: Spain

institute: IBMCP

summary: Transcriptome of seeds at eight days after polinization were compared between wild type and loss-of-function mutant in the flavonoid 3' hydroxylase TT7 gene

overall_design: wild type versus mutant seeds


title: Protein homeostasis is maintained by proteasomes containing PSMB9 induced by EEF1A2 upon mitochondrial stress

city: Warszawa

first: Krzysztof

last: Goryca

address: Banacha 2c

department: CeNT

country: Poland

institute: Uniwersytet Warszawski

summary: Analysis of global gene expression changes in HEK293T cells triggered by mutations in NDUFA11 and NDUFA13 genes

overall_design: RNA sequencing of whole cell RNA in wild-type and 2 complex I-deficient HEK293T cells in quadruplicate, resulting in a total of 3x4=12 samples

first: Minji

last: Kim

first: Agnieszka

last: Chacinska

first: Dorota

last: Adamska


title: Effects of dietary folic acid levles on colitis-associated tumorigenesis.

city: Philadelphia

first: Margie

last: Clapper

address: 333 Cottman Ave

institute: Fox Chase Cancer Center

summary: Folic acid supplementation (8 mg/kg diet) promotes colon tumor formation in mice with established colitis induced by carcinogen azoxymethane (AOM) and dextran sulfate sodium sulfate (DSS).  This induction of colon tumors was associated with hypomethylation of DNA cased by folic acid supplementation.

overall_design: Mice were fed a diet containing folic acid 2 mg/kg chow (basal level), followed by carcinogen azoxymethane injection. Colitis was induced in all mice using 4 cycles of DSS. Immediately after the first DSS treatment, mice were randomized to receive folic acid 0 mg/kg chow (folic acid deficient) or 8 mg/kg chow (folic acid supplemented)  for 16 weeks. At the end of study, mice were euthanized and colons were collected and process for gene expression analyses

first: Wen-Chi

last: Chang

first: Jayashri

last: Ghosh

first: Harry

last: Cooper

first: Lisa

last: Vanderveer

first: Bryant

last: Schultz

first: Yan

last: Zhou

first: Esther

last: Kaunga

first: Karthik

last: Devarajan

first: Yuesheng

last: Li

first: Jaroslav

last: Jelinek

first: Carmen

last: Sapienza


title: Immunohistochemical scoring of LAG-3 in conjunction with CD8 in  the tumor microenvironment predicts response to immunotherapy in  hepatocellular carcinoma

status: Public on May 30 2023

city: Singapore

first: Chun Chau Lawrence

last: Cheung

address: 20 College Road

department: Department of Anatomical Pathology

country: Singapore

institute: Singapore General Hospital

summary: Introduction  Immune checkpoint blockade (ICB) is a systemic therapeutic option for advanced hepatocellular carcinoma (HCC). However, low patient response rates necessitate the development of robust predictive biomarkers that identify individuals who will benefit from ICB. A 4-gene inflammatory signature, comprising CD8, PD-L1, LAG-3, and STAT1, was recently shown to be associated with a better overall response to ICB in various cancer types. Here, we examined whether tissue protein expression of CD8, PD-L1, LAG-3, and STAT1 predicts response to ICB in HCC.   Methods  HCC samples from 191 Asian patients, comprising resection specimens from 124 patients (ICB-naïve) and pre-treatment specimens from 67 advanced HCC patients treated with ICB (ICB-treated), were analyzed for CD8, PD-L1, LAG-3, and STAT1 tissue expression using multiplex immunohistochemistry followed by statistical and survival analyses.   Results  Immunohistochemical and survival analyses of ICB-naïve samples showed that high LAG-3 expression was associated with shorter median progression-free survival (mPFS) and overall survival (mOS). Analysis of ICB-treated samples revealed that high proportions of LAG-3+ and LAG-3+CD8+ cells pre-treatment were most closely associated with longer mPFS and mOS. Using a log-likelihood model, adding the total LAG-3+ cell proportion to the total CD8+ cell proportion significantly increased the predictive values for mPFS and mOS, compared with the total CD8+ cell proportion alone. Moreover, levels of CD8 and STAT1, but not PD-L1, were significantly correlated with better responses to ICB. After analyzing viral-related and non-viral HCC samples separately, only the LAG3+CD8+ cell proportion was significantly associated with responses to ICB regardless of viral status.   Conclusion  Immunohistochemical scoring of pre-treatment levels of LAG-3 and CD8 in the tumor microenvironment may help predict ICB benefits in HCC patients. Furthermore, immunohistochemistry-based techniques offer the advantage of being readily translatable in the clinical setting.

overall_design: PBMCs from 6 ICB-treated HCC patients were extracted for single-cell RNA sequencing

first: Chun

first: Mai

last: Lau

first: Xinru

last: Lim

first: Joe

last: Yeong


title: Kdm6a suppresses the alternative activation of macrophages

city: Shanghai

first: Xu

last: Wang

address: No. 639, Zhizaoju Road

institute: Shanghai Jiao Tong university, School of Medicine

summary: we identified Kdm6a as a critical epigenetic switch that modulates macrophage polarization and further disrupts energy balance

overall_design: BMDM from Kdm6aF/Y mice or Myeloid-specific Kdm6a ablation in Kdm6aF/Y;Lyz2-Cre mice were treat with LPS

last: Wei


title: IL-15 promotes self-renewal of progenitor exhausted CD8 T cells during persistent antigenic stimulation

city: Suwon-si

first: Kyungmin

last: Lee

state: Gyeonggi-do

address: 2066 Seobu-ro, Jangan-gu, Suwon-si, Gyeonggi-do, Republic of Korea

department: Department of Immunology

institute: Sungkyunkwan University School of Medicine

summary: In chronic infections and cancer, exhausted CD8 T cells exhibit heterogeneous subpopulations. TCF1+PD-1+ progenitor exhausted CD8 T cells (Tpex) can self-renew and give rise to Tim-3+PD-1+ terminally differentiated CD8 T cells that retain their effector functions. Tpex cells are thus essential to maintaining a pool of antigen-specific CD8 T cells during persistent antigenic stimulation, and only they respond to PD-1-targeted therapy. Despite their potential as a crucial therapeutic target for immune interventions, the mechanisms controlling the maintenance of virus-specific Tpex cells remain to be determined. We observed approximately 10-fold fewer Tpex cells in the spleens of mice chronically infected with lymphocytic choriomeningitis virus (LCMV) one-year post-infection (p.i.) than at three months p.i. Similar to memory CD8 T cells, Tpex cells have been found to undergo self-renewal in the lymphoid organs, prominently the bone marrow, during chronic LCMV infection. Furthermore, ex vivo treatment with IL-15 preferentially induced the proliferation of Tpex cells rather than the terminally differentiated subsets. Interestingly, single-cell RNA sequencing analysis of LCMV-specific exhausted CD8 T cells after ex vivo IL-15 treatment compared with those before treatment revealed upregulation of ribosome-related genes and downregulation of genes associated with the TCR signaling pathway and apoptosis in both Tpex and Ttex subsets. The exogenous administration of IL-15 to chronically LCMV-infected mice also significantly increased self-renewal of Tpex cells in the spleen and bone marrow. In addition, we assessed the responsiveness of CD8 tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma patients to IL-15. Similar to the data we obtained from chronic viral infection in mice, the expansion of the Tpex subset of PD-1+ CD8 TILs upon ex vivo IL-15 treatment was significantly higher than that of the terminally differentiated subset. These results show that IL-15 could promote self-renewal of Tpex cells, which has important therapeutic implications.

overall_design: Purified CD8 T cells from the spleens of chronically LCMV-infected mice were mixed 1:1 with unlabeled naive splenocytes as feeder cells and cultured with recombinant murine IL-15 (Peprotech) for 3 days. From pre- and post- IL-15-cultured cells, live CD8+PD-1+(GP33+GP276)+ cells were sorted using BD FACSAriaTM III Flow Cytometer (BD Biosciences) of the BIORP. Naïve CD8 T cells (live CD8+PD-1-CD44loCD62L+) were sorted from the spleens of naïve mice and included as a control for analysis.

first: Junghwa

first: Sejin

last: Im


title: Chromatin Accessibility Mapping of Primary Erythroid Cell Populations Leads to Identification and Validation of NFIX As a Novel Fetal Hemoglobin Repressor

first: Mudit

last: Chaand

state: MA

address: 35 Cambridgepark Drive

institute: Syros Pharmaceuticals

summary: Human genetics has validated de-repression of fetal gamma globin (HBG) in adult erythroblasts as a powerful therapeutic paradigm in diseases involving defective adult beta globin (HBB)1. To identify novel factors involved in the switch from HBG to HBB expression, we performed Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq)2 on discrete sorted erythroblast populations derived from bone marrow (BM) or cord blood (CB) progenitors, representing adult and fetal states, respectively.  Comparison of the ATAC-seq profiles revealed enrichment of NFI DNA binding motifs and increased chromatin accessibility at the NFIX promoter in BM populations relative to CB populations, suggesting that NFIX may repress HBG. NFIX knockdown in BM cells increased HBG mRNA and fetal hemoglobin (HbF) protein levels, coincident with increased chromatin accessibility and decreased DNA methylation at the HBG promoter. Conversely, overexpression of NFIX in CB cells reduced HbF levels. Identification of NFIX as a novel target for HbF activation has potential implications in the development of therapeutics for hemoglobinopathies.

overall_design: Examination of chromatin accessibility via ATAC-seq in 1) HUDEP-1 and HUDEP-2 cell lines; 2) Primary bone marrow derived erythroblasts and cord blood derived eyrthroblasts across erythrocyte differention; and 3) Primary bone marrow derived erythroblasts transduced with NFIX shRNA or a non-targeting control.

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