title: single-nucleus RNA sequencing revealed changes in microenvironment of epididymal adipose tissue  during the development of obesity in mouse

city: Jinan

first: Qinglu

state: Shandong

address: 10600 Century Avenue

institute: Shandong Sport University

summary: Obesity causes the change of microenvironment of adipose tissue and induces the occurrence of adipose tissue inflammation. The objective of this study was to analyze the internal mechanism of microenvironment change in mouse parepididymal adipose tissue by single-nucleus RNA sequencing. Nutritional obesity mouse model was established and verified by morphology, blood index and liver oil red O staining. Select Ctrl group, Mid_Ob group and Ob group, and take 8 mice at 3 time points. The changes of cell population, gene and signaling pathway expression in the adipose tissue of the parastriatic muscle in mice during obesity were detected by single-nucleus RNA sequencing and immunohistochemistry. According to the results of single-nucleus RNA sequencing, adipose tissue was divided into fibroblasts, macrophages, neutrophils and endothelial cells. It is found that all cell types have changes during the development of obesity and play an important role in regulating the microenvironment of adipose tissue.

overall_design: The model of obese mice was established by feeding high-fat diet, and then the mice were killed, and the adipose tissue of the epididymis was taken for monocyte sequencing

first: Zhinmin

last: Lu

first: Ling

last: Ding

first: Xuewen

last: Tian


title: A common cis-regulatory variant impacts normal-range and disease-associated human facial shape through regulation of PDKCC during chondrogenesis [ChIP-seq]

status: Public on Jun 23 2023

city: Stanford

first: Jaaved

last: Mohammed

state: CA

address: 265 Campus Drive

department: Chemical and Systems Biology

country: USA

institute: Stanford University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960, a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC - a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, rs6740960 SNP is sufficient to confer a large difference in acetylation of its cognate enhancer preferentially in chondrocytes. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features.

overall_design: We performed H3K27ac ChIP experiments to identify active enhancer in in vitro derived cranial neural crest cells (CNCCs) and cranial chondrocytes.

first: Neha

last: Arora

first: Tomek

last: Swigut

first: Joanna

last: Wysocka


title: Characterization of mouse naive CD4 T cells as a function of age

status: Public on Jun 21 2023

city: Paris

first: sebastien

last: jacques

address: 24 rue du faubourg saint jacques

department: Genomic

country: France

institute: Institut Cochin

summary: Naive CD4 T cells from aged mice exhibit an altered transcriptional signature

overall_design: CD4 T cells from the peripheral lymph-nodes of young ( 3-month-old) or aged  (33-month-old) C57BL/6-Foxp3GFP mice were first purified by depleting B cells and CD8 T cells. Then, naive CD4 T cells were FACS-sorted as CD44-/low GFP- Lin- cells and mRNAs extracted.

first: Bruno

last: Lucas

first: Aurélie

last: Durand


title: Small RNA profiles of follicular fluid-derived small extracellular vesicles

status: Public on Jun 22 2023

city: Nagoya

first: Kosuke

last: Yoshida

state: Aichi

address: 65 Tsuruma-cho

country: Japan

institute: Nagoya University

type: Non-coding RNA profiling by high throughput sequencing

summary: Follicular fluid (FF) provides a complex and suitable environment for oocyte maturation and contains several molecules secreted from oocyte and granulosa, cumulus, and theca cells. In addition, extracellular vesicles (EV) exist in various body fluids and are known as the cargo of several mRNAs, proteins, and miRNAs to communicate from cell to cell. In this study, we investigated the miRNA profiles of FF-derived EVs.

overall_design: EVs were isolated from FF by the serial centrifugation method, and small RNA sequencing was performed.

first: Akira

last: Yokoi

first: Ayako

last: Muraoka

first: Mayuko

last: Murakami

first: Masami

last: Kitagawa


title: Counting and correcting errors within unique molecular identifiers to generate absolute numbers of sequencing molecules [RNA-seq]

status: Public on Jun 01 2023

city: Oxford

first: Adam

last: Cribbs

address: Windmill Road

department: NDORMS

country: United Kingdom

institute: University of Oxford

type: Expression profiling by high throughput sequencing

summary: Unique Molecular Identifiers (UMIs) are random oligonucleotide barcodes sequences? that are critical for the removal of PCR amplification biases within both bulk and single-cell sequencing experiments. However, the impact that PCR and sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We demonstrate that PCR errors and not sequencing errors are the main source of inaccuracy in sequencing data and that the use of UMIs synthesized with homotrimeric nucleoside building blocks provides a solution to pinpoint and remove errors, allowing absolute counting of sequenced molecules.

overall_design: In order to evaluate the accuracy of our homotrimer barcode assignment we processed mixed mixed mouse and human mRNA (at ratio of 50:50) and then performed bulk RNA sequencing.

middle: P


title: Modeling Molecular Pathogenesis of Idiopathic Pulmonary Fibrosis-Associated Lung Cancer in Mice

status: Public on Jun 20 2023

city: Ann Arbor

first: Brian

last: Magnuson

state: MI

address: 1500 East Medical Center Drive

institute: University of Michigan

summary: Idiopathic Pulmonary Fibrosis (IPF) is characterized by progressive, often fatal loss of lung function due to overactive collagen production and tissue scarring. IPF patients have a sevenfold-increased risk of developing lung cancer. The COVID-19 pandemic has increased the number of patients with lung diseases, and infection can worsen prognoses for those with chronic lung diseases and disease-associated cancer. Understanding the molecular pathogenesis of IPFassociated lung cancer is imperative for identifying diagnostic biomarkers and targeted therapies that will facilitate prevention of IPF and progression to lung cancer. To understand how IPF-associated fibroblast activation, matrix remodeling, epithelial-mesenchymal transition, and immune modulation influences lung cancer predisposition, we developed a mouse model to recapitulate the molecular pathogenesis of pulmonary fibrosis-associated lung cancer using the bleomycin and Lewis Lung Carcinoma models. We demonstrate that development of pulmonary fibrosis-associated lung cancer is likely linked to increased abundance of tumor-associated macrophages and a unique gene signature that supports an immune-suppressive microenvironment through secreted factors. Not surprisingly, pre-existing fibrosis provides a pre-metastatic niche and results in augmented tumor growth, and tumors associated with bleomycin-induced fibrosis are characterized by a dramatic loss of cytokeratin expression, indicative of epithelial-to-mesenchymal transition. Implications: This characterization of tumors associated with lung diseases provides new therapeutic targets that may aid in the development of treatment paradigms for lung cancer patients with pre-existing pulmonary diseases.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for lung tissues from control mice (3), IPF only mice (3), LC only mice (3), and IPF-LC mice (3).

first: Stefanie

last: Galban


title: Counting and correcting errors within unique molecular identifiers to generate absolute numbers of sequencing molecules

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

overall_design: Refer to individual Series


title: Identification of the RNAs that bind with MDM2 by high-throughput RIP-seq in MDA-MB-231 and MCF-7 cells

status: Public on Jun 19 2023

city: Guangzhou

first: Man-Li

last: Luo

address: No.107 Yanjiang West Road, Yuexiu District, Guangzhou

country: China

institute: Sun Yat-sen Memorial Hospital

summary: The E3 ligase MDM2 promotes tumor growth and progression by inducing ubiquitin-mediated degradation of P53 and other tumor suppressing proteins. Here, we identified an MDM2-interacting lncRNA NRON, which promotes tumor formation by suppressing both P53-dependent and independent pathways. NRON binds to MDM2 and MDMX (MDM4) via two different stem-loops respectively and induces their heterogenous dimerization, thereby enhancing the E3 ligase activity of MDM2 towards its tumor suppressing substrates, including P53, RBI and NFATI, etc.

overall_design: To identify those lncRNAs that interact with MDM2 and function in both P53-dependent and -independent ways, MDA-MB-231 and MCF-7 cells with exogenous Flag-tagged MDM2 were established and subjected to RNA immunoprecipitation (RIP) using negative control normal mouse IgG and anti-FLAG antibody respectively. RIP–sequencing (RIP-seq) was then performed to identify the lncRNAs that specifically bind to FLAG-tagged MDM2 but not to normal mouse IgG control.

first: Yunmei

last: Zhang

first: Yihui

last: Li

first: Qiannan

last: Guo


title: RNA-Seq and RNC-Seq of HeLa cells stably expressing NDV NP protein

city: Yangzhou

first: Liao

last: Tianxing

state: Jiangsu province

address: 48 Wenhui East Road, Hanjiang District, Yangzhou City, Jiangsu Province

department: College of Veterinary Medicine

institute: Yangzhou University

summary: The oncolytic potential of NDV has gained significant attention in the context of clinical trials. After systematically comparing the oncolytic activities of different NDV subtypes,we found that all subtypes, except for highly pathogenic genotype VII NDVs, display remarkable infectivity on tumor cell lines.A single amino acid mutation (F450L) in the NP protein at position 450 leads to an approximate 1000000-fold increase in the TCID50 value of genotype VII NDVs on tumor cells.Mechanistic inquiries unveiled that the amino acid at position 450 of the NP protein governs preferential translation of NDV mRNA.The preferential translation that regulates viral mRNA translation is often accompanied by modulation of the host translation machinery. Therefore, we established HeLa cell lines stably expressing NP protein from different NDV strains. Ribosome-bound mRNA was enriched to perform Ribosome Nascent-chain Complex sequencing(RNC-seq), while total cellular mRNA was enriched to conducted RNA-Seq . Untreated cells were used as controls. These experiments aimed to investigate the impact of NP on the host translation system.

overall_design: We generated HeLa cell lines that stably expressed the NP protein from different NDV strains using a lentiviral packaging system. The NP protein derived from the oncolytic Herts/33 strain was designated as HNP, while the NP protein from the non-oncolytic I4 strain was referred to as INP. Subsequently, ribosomes were isolated, and the RNA bound to the ribosomes was extracted from the ribosomal fractions for Ribosome Nascent-chain Complex sequencing (RNC-seq). Total RNA was used for RNA-Seq analysis, serving as a control for changes in RNA abundance. The key distinction between RNC-Seq and RNA-Seq lies in the source of RNA, while the subsequent library preparation and sequencing methods were consistent.

first: Tianxing

last: Liao

first: Yu

last: Chen

first: Lili


title: Cell-cell interaction between cardiomyocytes and cardiac fibroblasts regulates ferroptosis and fibrosis after myocardial injury

status: Public on Jun 02 2023

city: Charleston

first: Allison

last: Trouten

state: SC

address: 173 Ashley Ave Room BSB 642

department: Regenerative Medicine and Cell Biology

institute: Medical University of South Carolina

summary: The heart suffers from a severe loss of cardiomyocyte after myocardial infarction (MI). However, it is not known which type of cell death is the major contributor of the loss of cardiomyocytes. By studying a mouse heart MI model at a regenerative and a non-regenerative stage, we demonstrate that ferroptosis, not apoptosis or necroptosis, is the major contributor to cardiomyocyte death starting at 1 day-post-MI. Intrinsic Pitx2 signaling in cardiomyocyte prevents ferroptosis. Meanwhile, cardiac fibroblasts expressing high level of Fth1 interact with cardiomyocytes to share the iron burden, therefore inhibit cardiomyocyte ferroptosis. Cardiomyocyte Pitx2 also inhibits Tsp1 expression, therefore negatively regulate fibroblast-to-myofibroblast activation to limit fibrosis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from induced cardiomyocyte (iCMs) treated with siRNA targeting Pitx2 or a scramble control at various doses. Some cultures were exposed to oxidative stress via H2O2 treatment.

first: Ge

last: Tao

first: Shuang

first: Mary

last: Mohr

first: Rebecca

last: Stairley

first: Patrick

last: Roddy

first: Chun

last: Liu

first: Min

first: Henry

middle: M

last: Sucov


title: Effect of D419N mutation on STAT6 transcription factor activity

city: Montreal

first: Koren

middle: Kathleen

last: Mann

state: QC

address: 3755 Chemin de la Cote Sainte Catherine

country: Canada

institute: Lady Davis Institute

summary: To investigate how mutation of STAT6 at residue D419 impacts gene expression in response to acute (3hr) and chronic (72hr) IL-4 stimulation

overall_design: OCI-Ly8 cells which are STAT6 WT or D419N mutant, treated with acute or chronic IL-4

first: Mann

last: Koren

first: Beniot

last: Alexandre

first: Abraham

last: Madelyn


title: Activation of adult human pancreatic ductal and ductal progenitor-like cells requires extracellular matrix protein signaling [bulkRNA-seq]

status: Public on May 31 2023

city: Duarte

first: Hsun

middle: Teresa

last: Ku

address: 1500 E. Duarte Road

department: Translational Research and Cellular Therapeutics

institute: City of Hope

summary: Ductal progenitor-like cells are a subpopulation of ductal cells in the adult human pancreas that have the potential to contribute to regenerative medicine. However, the microenvironmental cues that regulate their activation are poorly understood. Here, we establish a 3-dimensional suspension culture system containing six defined soluble factors in which primary human ductal and ductal progenitor-like cells can survive but do not proliferate. Expansion and polarization occur when cells are provided with a low concentration (5% v/v) of Matrigel, a sarcoma cell product enriched in many extracellular matrix (ECM) proteins. Screening of ECM proteins identified that collagen IV is capable of partially recapitulating the effects of Matrigel. Inhibition of integrin α1β1, a major collagen IV receptor, negates collagen IV- and Matrigel-stimulated effects. These results demonstrate that collagen IV is a key ECM protein that stimulates the expansion and polarization of human ductal and ductal progenitor-like cells via integrin α1β1 receptor signaling.

overall_design: Bulk mRNA-sequencing of adult human pancreatic exocrine cells cultured in 3D suspension for 7 days in the presence or absence of 5% v/v Matrigel. The cells were derived from cryopreserved exocrine tissue that had been dissociated into a single-cell suspension.

first: Heather

middle: N

last: Zook

first: Janine

middle: C

last: Quijano

first: Min-Hsuan

middle: T


title: Maternal to paternal epigenome reprogramming during early sea lamprey (Petromyzon marinus) development (Bisulfite-Seq)

status: Public on May 26 2023

city: Sydney

first: Allegra

last: Angeloni

state: NSW

address: 384 Victoria St Darlinghurst

department: Genomics and Epigenetics Division

country: Australia

institute: Garvan Institute of Medical Research

type: Methylation profiling by high throughput sequencing

overall_design: Whole-genome bisulfite sequencing in lamprey adult brain, muscle, PBMCs, egg, and sperm, and embryonic stages Day 1 and Day 2.

first: Ozren

last: Bogdanovic


title: Intratumoral mregDC and Tfh-like niches enable local reinvigoration of CD8 T cells following PD-1 blockade

status: Public on Jun 16 2023

city: New York

first: Darwin

last: D'Souza

state: NY

address: 1470 Madison Avenue

department: ImmunoBio

institute: Human Immune Monitoring Center

summary: While T cell accumulation in tumors is associated with response to immune checkpoint blockade (ICB), many T cell-rich tumors fail to respond to ICB. Here we leveraged a large neoadjuvant PD-1 blockade trial in hepatocellular carcinoma (HCC) to search for correlates of ICB response within T cell-rich tumors. Paired scRNAseq/TCRseq of nearly one million immune cells revealed that tumor responses to ICB correlated with significant clonal expansion of intratumoral CH25H+CXCL13+IL-21+CD4 T cells and GranzymeK+PD-1+CD8 effector T cells, whereas terminally exhausted CD39hiToxhiPD-1hi CD8 clones dominated in non-responders. Notably, proliferating and progenitor CD8 T cells clones were found in tumors of responders and non-responders. However, tumors from responders were highly enriched in mregDCs, a DC state triggered by capture of tumor debris, which formed physically interacting cellular triads with Tfh-like CD4 and progenitor-like CD8 T cells. Receptor-ligand analysis revealed unique interactions within these triads that may promote progenitor CD8 T cell differentiation into effector cells upon PD-1 blockade. These results suggest that discrete cellular niches that include mregDCs and Tfh-like CD4 T cells might control the differentiation of tumor-specific progenitor CD8 T cell clones into effective anti-tumor T cells in human tumors.

overall_design:
Samples of tumor and non-involved liver were obtained from surgical specimens of patients undergoing resection at Mount Sinai Hospital (New York, NY) after obtaining informed consent in accordance with a protocol reviewed and approved by the Institutional Review Board at the Icahn School of Medicine at Mount Sinai. Cells were counted using the Nexcelom Cellaca. An aliquot of 400,000 cells from each sample were centrifuged at 350g for 5 minutes at 4°C. The supernatant was discarded and the pellets were resuspended in a hashtag antibody solution and incubated on ice for 20 minutes. Hashtag antibodies were conjugated as per New York Genome Center hashing protocol (https://citeseq.files.wordpress.com/2019/02/cell_hashing_protocol_190213.pdf). Stained cells were washed three times in 1mL wash buffer (PBS + 0.5% BSA) by 4°C centrifugation at 350g to remove unbound antibodies. Washed cells were resuspended in 150µl wash buffer and counted using a Nexcelom Cellaca. Hashed samples were pooled and centrifuged at 350g for 5 minutes at 4°C. Supernatant was removed and pellet was resuspended in 100µl of antibody cocktail (Table S4) and incubated at 4°C for 30 minutes. Stained cells were washed three times in 1mL PBS + 0.5% BSA by 4°C centrifugation at 350g to remove unbound antibodies. The washed cells were resuspended in wash buffer to achieve a target concentration of 4 million cells/mL. Sample pool was counted and loaded on the 10x Genomics NextGem 5’v1.1 assay as per the manufacturer’s protocol with a targeted cell recovery of 25,000 cells per lane (CITEseq) or 10,000 cell per lane (direct load). Gene expression and Feature Barcode libraries were made as per the 10x Genomics demonstrated protocol. Hashtag oligonucleotides (HTO) were enriched during cDNA amplification with the addition of 3 pmol of a HTO Additive primer (5’GTGACTGGAGTTCAGACGTGTGCTC). This PCR product was isolated from the mRNA-derived cDNA via SPRISelect size selection, and libraries were made as per the New York Genome Center Hashing protocol. All libraries were quantified via Agilent 2100 hsDNA Bioanalyzer and KAPA library quantification kit (Roche) Gene expression libraries were sequenced at a targeted depth of 25,000 reads per cells. ADT libraries were sequenced at a targeted read depth of 10,000 reads per cell. HTO libraries were sequenced at a targeted read depth of 1,000 reads per cell. Paired-end sequencing was performed on Illumina NovaSeq 6000 for RNA-seq and CITE-seq libraries (for 3’ libraries, used Read 1 28 bp for UMI and cell barcode, Read 2 80-bp for transcript read, with 8-bp i7 and 0-bp i5 reads; for 5’ libraries, used Read 1 26 bp for UMI and cell barcode, Read 2 80-bp for transcript read, with 8-bp i7 and 0-bp i5 reads). V(D)J libraries were also sequenced on Illumina NovaSeq 6000 (Read 1 150-bp, 8-bp i7, 0-bp i5, Read 2 150-bp). Hashtag libraries were sequenced on Illumina NextSeq500.

Human liver tumor samples were collected at baseline biopsy without treatment and after PD-1 Antibody (Cemiplimab) treatment at resection. Tumor tissus were sequenced for TCR.

first: Miriam

last: Merad

first: Assaf

last: Magen

first: Pauline

last: Hamon

first: Gavin

last: Thurston


title: PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve tumor

city: Okayama

first: Takeshi

last: Takarada

institute: Okayama University

summary: Malignant nerve sheath tumors (MPNST) are rare types of malignant soft tissue sarcomas and characterized by high resistance for current chemotherapeutic strategies, including topoisomerase 2a (TOP2A) inhibitor, etoposide. Although the poor therapeutic or severe side effects of etoposide in MPNST patients are demonstrated, novel molecular targets that promote tumor malignancy have not been identified. Previously, we reported that paired related-homeobox 1 (PRRX1) serves as a malignant factor in human osteosarcoma. Here, we found that expression level of PRRX1 in tumor tissues, especially sarcomas, are higher than in normal tissues using database analysis. PRRX1 was expressed in various human sarcoma tissues, and its level increased during malignant progression from schwannoma or neurofibroma to MPNST. High expression of PRRX1 was also associated with poor prognosis of human MPNST patients, and PRRX1 knockdown suppressed proliferation, invasion and tumorigenic potential of human MPNST cell line, HS-PSS. Immunoprecipitation and mass spectrometry analysis revealed that PRRX1 interacts with TOP2A, and human MPNST patients with high expression of TOP2A showed poor prognosis. Interestingly, database analysis revealed that expression level of TOP2A in tumor tissues were positively correlated with PRRX1. TOP2A knockdown in HS-PSS suppressed proliferation, and inhibited the migration induced by PRRX1A overexpression. Overexpression of PRRX1 and TOP2A cooperatively increased migration and expression of tumor-malignancy associated gene sets, including EMT, mTORC1, KRAS and SRC signaling pathways. Inhibition of PRRX1-TOP2A interaction would be a novel tumor-selective therapeutic strategy that has less severe side effects than current chemotherapies.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for Limb Bud Mesenchymal cells (3xFLAG-PRRX1A, 3xHA-TOP2A, and HS-PSS/3xFLAG-PRRX1A/3xHA-TOP2A).

first: Shota

last: Takihira

first: Daisuke

last: Yamada


title: Cell type-specific cis-regulatory divergence in gene expression and chromatin accessibility revealed by human-chimpanzee hybrid cells [ATAC-seq]

status: Public on May 23 2023

first: Hunter

last: Fraser

address: 327 Campus Drive

summary: Although gene expression divergence has long been postulated to be the primary driver of human evolution, identifying the genes and genetic variants underlying uniquely human traits has proven to be quite challenging. Theory suggests that cell type-specific cis-regulatory variants may fuel evolutionary adaptation due to the specificity of their effects. These variants can precisely tune the expression of a single gene in a single cell type, avoiding the potentially deleterious consequences of trans-acting changes and non-cell type-specific changes that can impact many genes and cell types, respectively. It has recently become possible to quantify human-specific cis-acting regulatory divergence by measuring allele-specific expression in human-chimpanzee hybrid cells—the product of fusing induced pluripotent stem (iPS) cells of each species in vitro. However, these cis-regulatory changes have only been explored in a limited number of tissues and cell types. Here, we quantify human-chimpanzee cis-regulatory divergence in gene expression and chromatin accessibility across six cell types, enabling the identification of highly cell type-specific cis-regulatory changes. We find that cell type-specific genes and regulatory elements evolve faster than those shared across cell types. Furthermore, we identify several instances of lineage-specific natural selection that may have played key roles in specific cell types, such as coordinated changes in the cis-regulation of dozens of genes involved in neuronal firing in motor neurons. Finally, using novel metrics and a machine learning model, we identify genetic variants that likely alter chromatin accessibility and transcription factor binding, leading to neuron-specific changes in the expression of the neurodevelopmentally important genes FABP7 and GAD1. Overall, our results demonstrate that integrative analysis of cis-regulatory divergence in chromatin accessibility and gene expression across cell types is a promising approach to identify the specific genes and genetic variants that make us human.

sample_organism:
Pan troglodytes
Homo sapiens

platform_organism:
Pan troglodytes
Homo sapiens

overall_design: ATAC-seq of human-chimpanzee hybrid iPS cells derived five cell types

first: Ban

last: Wang

first: Alexander

middle: L

last: Starr

middle: B


title: Cell type-specific cis-regulatory divergence in gene expression and chromatin accessibility revealed by human-chimpanzee hybrid cells [RNA-seq]

overall_design: RNA sequencing of human-chimpanzee hybrid iPS cells derived six cell types


title: Maternal to paternal epigenome reprogramming during early sea lamprey (Petromyzon marinus) development

status: Public on May 25 2023


title: Cell type-specific cis-regulatory divergence in gene expression and chromatin accessibility revealed by human-chimpanzee hybrid cells


title: Lactylation-driven FTO-mediated vascular endothelial tip cell formation and its crosstalk with pericyte and microglia aggravate diabetes-induced microvascular dysfunction

status: Public on May 15 2023

city: Nanjing

first: Ruxu

last: Sun

state: Jiangsu

address: 140 Hanzhong Road

institute: Nanjing medical university

summary:
Diabetic retinopathy (DR), a leading cause of irreversible vision loss in the working-age population, is an inflammatory, neuro-vascular complication of diabetes with poorly understood mechanism. N6-methyladenosine (m6A) modification plays crucial roles in biological and pathological events, while its role in DR remain elusive. Herein, we identified the pathological involvement of m6A demethylase FTO in DR. FTO expression was elevated in proliferative membranes of DR patients, endothelial cells (EC) under diabetic stress, and retinal vessels of diabetic murine models. FTO overexpression in EC promoted EC cycle progression and tip cell formation to facilitate angiogenesis in vitro, in mice and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetes-induced microvascular leakage, and mediated EC-microglia crosstalk to induce retinal inflammation and subsequent neurodegeneration in vivo and in vitro. Mechanistically, FTO regulated EC features depending on its demethylation activity via modulating CDK2 mRNA stability with YTHDF2 as the reader. Moreover, FTO up-regulation in EC under diabetic conditions was driven by lactic acid mediated histone lactylation. FB23-2, which inhibits FTO’s m6A demethylase activity, suppressed diabetes associated endothelial phenotypes in vitro and in vivo. Noteworthy, we developed a novel macrophage membrane coated and PLGA-Dil based nanoplatform encapsulating FB23-2 for systemic administration, and confirmed its targeting and therapeutic efficacy in mice. Collectively, our study demonstrated an FTO-mediated regulatory network that coordinates EC biology and retinal homeostasis in DR, providing a promising nanotherapeutic approach for DR treatment.
Diabetic retinopathy (DR), a leading cause of irreversible vision loss in the working-age population, is an inflammatory, neuro-vascular complication of diabetes with poorly understood mechanism. N6-methyladenosine (m6A) modification plays crucial roles in biological and pathological events, while its role in DR remain elusive. Herein, we identified the pathological involvement of m6A demethylase FTO in DR. FTO expression was elevated in proliferative membranes of DR patients, endothelial cells (EC) under diabetic stress, and retinal vessels of diabetic murine models. FTO overexpression in EC promoted EC cycle progression and tip cell formation to facilitate angiogenesis in vitro, in mice and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetes-induced microvascular leakage, and mediated EC-microglia crosstalk to induce retinal inflammation and subsequent neurodegeneration in vivo and in vitro. Mechanistically, FTO regulated EC features depending on its demethylation activity via modulating CDK2 mRNA stability with YTHDF2 as the reader. Moreover, FTO up-regulation in EC under diabetic conditions was driven by lactic acid mediated histone lactylation. FB23-2, which inhibits FTO’s m6A demethylase activity, suppressed diabetes associated endothelial phenotypes in vitro and in vivo. Noteworthy, we developed a novel macrophage membrane coated and PLGA-Dil based nanoplatform encapsulating FB23-2 for systemic administration, and confirmed its targeting and therapeutic efficacy in mice. Collectively, our study demonstrated an FTO-mediated regulatory network that coordinates EC biology and retinal homeostasis in DR, providing a promising nanotherapeutic approach for DR treatment.

overall_design: MeRIP-seq and RNA-seq for cells transduced with L-EV (GFP group) or L-FTO (FTO group).

first: Xue

last: Chen

first: Xinyu


title: Maternal to paternal epigenome reprogramming during early sea lamprey (Petromyzon marinus) development (BioCAP)

overall_design: Biotinylated CxxC affinity purification DNA-sequencing (BioCAP-seq) in lamprey adult brain, muscle, PBMCs, and sperm, and embryonic stages Day 1 and Day 2.


title: The Upstream Sequence Transcription Complex Dictates Nucleosome Positioning and Promoter Accessibility at piRNA Genes in the C. elegans germ line [ATAC-Seq]

status: Public on May 14 2023

city: New Haven

first: Valerie

last: Reinke

state: CT

address: 333 Cedar St

department: Genetics

institute: Yale University School of Medicine

summary: The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility.  In C. elegans and other nematodes, Type-I piRNA precursor transcripts are expressed from >10,000 small, independently regulated genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. These large domains likely play a significant role in promoting germline-specific expression of piRNAs, but the underlying mechanisms are unclear.  By examining the chromatin environment specifically in isolated germ nuclei, we demonstrate that piRNA clusters are located in closed chromatin, and confirm the enrichment for the inactive histone modification H3K27me3.  The piRNA biogenesis factor USTC (Upstream Sequence Transcription Complex) plays two roles – it promotes a strong association of nucleosomes throughout the domain, and it organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Overall, this work reveals new insight into how chromatin state coordinates transcriptional regulation over large genomic domains, which has implications for understanding global genome organization in the germ line.

overall_design: Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) of isolated germline nuclei off wild-type and prde-1(mj207)V in C. elegans.

first: Nancy

last: Sanchez

first: Lauren

last: Gonzalez


title: TET1-JMJD3-H3K27me3 targets and regulates Pramel3 to affect spermatogonia self-renewal and proliferation and differentiation

status: Public on May 24 2023

city: Hefei

first: wang

last: jin

state: Anhui

address: Shushan District

department: School of Basic Medicine

institute: Anhui Medical University

summary: Histone modification and DNA methylation among epigenetic modifications play a coordinated role in the regulation of spermatogonia. Tet methylcytosine dioxygenase 1 (TET1) can either act as a DNA demethylase, converting 5-methylcytosine to 5-hydroxymethylcytosine or regulate gene expression by forming a complex with other proteins. Increased levels of H3K27me3 will result in transcriptional repression and reduced or silenced protein expression. However, the mechanism by which TET1 mediates the regulation of H3K27me3 in spermatogonia and the target genes that act is not clear. The results of this study showed that the expression of self-renewal-related gene GFRA1 and proliferation-related gene PCNA were up-regulated after JMJD3 overexpression. Moreover, it was demonstrated that TET1 and JMJD3 can interact with H3K27me3 by forming a protein complex, and the CHIP-Seq sequencing results analyzed that TET1 targets and regulates the Pramel3 gene when interacting with H3K27me3, and TET1 overexpression promotes the up-regulation of Pramel3 expression. Our study identified a novel link between TET1 and H3K27me3 and established a TET1-JMJD3-H3K27me3-Pramel3 regulatory axis to regulate spermatogonia self-renewal, proliferation and differentiation. It provides a new idea to further investigate the mechanism of spermatogenesis and spermatogenic disorders from the epigenetic level.

overall_design: Chromatin immunoprecipitation DNA sequencing (CHIP-seq) of histone modified H3K27me3 in GC-1 cells

first: jin


title: Whole-genome CRISPR screen of etoposide-treated U2OS and U2OS p53KO cells, and RNAseq of U2OS cells incubated in media conditioned by etoposide-treated U2OS or U2OS p53KO cells.

status: Public on May 17 2023

city: Worcester

first: Michael

middle: J

last: Lee

state: MA

address: 368 Plantation Street

institute: UMass Chan Medical School

summary:
CRISPR screen: U2OS or U2OS p53KO cells expressing Cas9 were transduced with a whole-genome library of CRISPR sgRNAs, then treated with either DMSO or etoposide. Differential sgRNA abundances were calculated for each condition and used to determine the effect of each single gene knockout on fitness and the drug-induced death rate.

RNA-Seq: U2OS or U2OS p53KO cells were cultured with either DMSO or etoposide for 48 hours, and then U2OS cells were incubated in this conditioned media for 8 hours. RNA was collected and use to observe differential expression changes between conditions.

overall_design:
CRISPR screen: Pooled library of U2OS or U2OS p53KO cells was treated with DMSO or etoposide for 96 hours, gDNA was isolated using phenol-chloroform extraction, sgRNA libraries were amplified and barcoded using PCR, whole-genome libraries were sequenced on an Illumina HiSeq platform.

RNA-Seq: U2OS cells were cultured in media conditioned by U2OS or U2OS p53KO cells for 8 hours, RNA was extracted using a Qiagen RNeasy Mini Kit, total RNA was sequenced by BGI Genomics.

first: Megan

middle: E

last: Honeywell


title: Transcriptome measurements of Nannochloropsis wide-type and 6mA mutants under high light

city: Qingdao

first: Yanhai

last: Gong

address: No.189 Songling Road

department: single cell center

institute: qibebt, cas

summary: N. oceanica IMET1 was inoculated into modified f/2 liquid medium, which was prepared with 35 g L−1 sea salt, 1 g L−1 NaNO3, 67 mg L−1 NaH2PO4*H2O, 3.65 mg L−1 FeCl3*6H2O, 4.37 mg L−1 Na2EDTA*2H2O, trace metal mix (0.0196 mg L−1 CuSO4*5H2O, 0.0126 mg L−1 NaMoO4*2H2O, 0.044 mg L−1 ZnSO4*7H2O, 0.01 mg L−1 CoCl2, and 0.36 mg L−1 MnCl2*4H2O), and vitamin mix (2.5 µg L−1 VB12, 2.5 µg L−1 biotin, and 0.5 µg L−1 thiamine HCl). The algal cells were grown in liquid cultures under continuous light (∼500 µmol photons m−2 s−1) at 25°C and aerated by bubbling with a mixture of 1.5% CO2 in air.

overall_design: 15 samples

first: Qintao

first: Jian

last: Xu


title: Self-renewal and differentiation dynamics of native hematopoietic stem and progenitor cells at single-cell resolution

city: Cambridge

first: Bertie

last: Gottgens

address: Jeffrey Cheah Biomedical Centre

institute: University of Cambridge

summary: Hematopoiesis has a long track record of embracing single-cell functional and molecular profiling technologies. However, a single-cell resolution quantitative model of native hematopoiesis has never been reported. Here, we employ persistent labelling of stem cells combined with time-series single-cell RNA-Seq (scRNA-Seq) to build the first quantitative model of the native blood stem and progenitor dynamics in the mouse bone marrow at single-cell resolution. We capture compartment-wide flux and net proliferation rates in real-time, thus revealing the distinct ways in which different blood lineages maintain their cellular output. By exploring the molecular layers of scRNA-Seq, we pinpoint molecular changes associated with changes in cell behavior, such as increased differentiation or growth rates. Finally, unlike immunophenotyping data, our model can easily be integrated with external data. This enabled quantitative comparison between homeostatic and transplantation conditions which highlighted lineages and specific stages within them with up-regulated differentiation.

overall_design: Time-course experiment tracking descendants of labelled hematopoietic stem and progenitor cells in mouse bone marrow.

first: Iwo

last: Kucinski

first: Melania

last: Barile

first: Rebecca

last: Hannah

first: Berthold


title: Single-cell RNA sequencing of ILCs and T cells reveals enrichment of NK-like T cells in pediatric and adult inflammatory bowel disease

status: Public on May 11 2023

city: Huddinge

first: Jenny

last: Mjösberg

state: Stockholm

address: Alfred Nobels allé 8

department: Department of Medicine

country: Sweden

institute: Karolinska Institutet

summary:
Background and aims:  Innate lymphoid cells (ILCs) are the innate counterparts of adaptive T cells, which together execute specialized effector programs in the intestinal mucosa. Understanding how unique and common dysregulation of the innate and adaptive lymphocytes contribute to inflammatory bowel disease (IBD) pathogenesis is crucial for development of novel and efficient therapies.
Methods: We used flow cytometry to extensively characterize ILCs in pediatric (p)IBD patients (n=12) and controls (n=9), as well as single-cell RNA sequencing to determine the heterogeneity and inflammation-specific imprinting of ILCs, NK cells and T cells (n=6). Key findings were extended to adult IBD colonic mucosa (n=8) using flow cytometry.
Results: We report a dysregulated mucosal ILC composition in pIBD that correlates to inflammatory activity. ILCs show pronounced and unique transcriptional changes in inflammation, particularly those related to antigen-presentation, while several interferon-associated transcripts are upregulated across lymphocyte lineages in inflammation. Furthermore, we identify two transcriptionally distinct T cell subsets, which are dysregulated in inflammation. This includes a subset transcribing KLRF1 and other NK-associated transcripts, that were enriched in inflamed pIBD mucosa. A corresponding NK-like NKp80+ T cell population, expressing high levels of NK cell receptors and cytotoxic molecules, are also accumulated in adult IBD.
Conclusions: Using a combination of high-dimensional single-cell analysis on the transcriptional and protein level, we provide a comprehensive characterization of ILCs and T cells in pediatric and adult IBD. Our findings contribute to increased mechanistic understanding of intestinal inflammation and identify promising targets for IBD therapy, including the NK cell receptor NKp80.

overall_design:
Single-cell RNA sequencing of ILCs and T-cells in matched noninflamed and inflamed colonic tissues of 6 pediatric IBD patients
**The submitter declares that the raw data cannot be deposited due to patient privacy concerns.**

first: Efthymia

last: Kokkinou

first: Tea

last: Soini

first: Ram Vinay

last: Pandey

first: Aline

last: van Acker

first: Egle

last: Kvedaraite

first: Niels

last: Vandamme

first: Chiara

last: Sorini

first: Magdalini

last: Lourda

first: Heinrich

last: Schlums

first: Whitney

last: Weigel

first: Christopher

middle: A

last: Tibbitt

first: Ulrik

last: Lindforss

first: Caroline

last: Nordenvall

first: Malin

last: Ljunggren

first: Maja

last: Ideström

first: Mattias

last: Svensson

first: Jan-Inge

last: Henter

first: Eduardo

last: Villablanca

first: Yenan

last: Bryceson

first: Helena

last: Rolandsdotter


title: Transcriptome analysis of S. maltophilia in the exponential and stationary phases and after a treatment with quorum sensing autoinducers.

status: Public on May 16 2023

city: Bellaterra

first: Isidre

last: Gibert

state: Barcelona

address: Carrer de la Vinya, s/n

department: Institut de Biotecnologia i de Biomedicina (IBB)

country: Spain

institute: Universitat Autònoma de Barcelona (UAB)

summary: Stenotrophomonas maltophilia is an emerging opportunistic multidrug-resistant pathogen frequently co-isolated with other relevant nosocomial pathogens in respiratory tract infections. S. maltophilia uses the endogenous DSF quorum sensing (QS) system to regulate virulence processes but can also respond to exogenous AHL signals produced by neighboring bacteria. A whole-transcriptome sequencing analysis was performed for S. maltophilia strain K279a in the exponential and stationary phases and in exponential cultures after a treatment with exogenous DSF or AHLs. Our first analysis reveals that during the beginning of the stationary phase 1673 genes are differentially expressed representing 37% of total genomic content. COG analysis showed that most of these genes were enriched for energetic metabolism processes and regulation of gene expression. In a second analysis, and after adding DSF or AHLs to cultures in exponential phase, 82 and 28 were found deregulated respectively, 22 of which were commonly upregulated by both autoinducers. Interestingly, among these genes affected by both DSFs and AHLs, 14 functional genes were also upregulated in the stationary phase. Gene functions regulated by all tested conditions include lipid and amino acid metabolism, stress response and signal transduction mechanisms, nitrogen and iron metabolism, and adaptation to microoxic conditions.  Overall, our findings provide clues on the role that the QS could have in the transition from the exponential to the stationary phase in S. maltophilia and for the bacterial fitness for high-density growth.

overall_design: In total 14 samples were analyzed (5 different conditions with two or three independent samples), and all cultures were done in LB at 37C. Condition K279a early-stationary phase: S. maltophilia K279a grown on LB to an OD550 of 5.0 (24 h); condition K279a mid/late-exponential phase: S. maltophilia K279a grown on LB to an OD550 of 1.5 (4.5 h); condition K279a treated with DMSO: S. maltophilia K279a culture supplemented at the early-exponential phase with DMSO and cells collected 3 hours later as a control; condition K279a treated with DSF: S. maltophilia K279a culture supplemented at the early-exponential phase with DSF at 10 uM and cells collected 3 hours later; condition K279a treated with AHLs: S. maltophilia K279a culture supplemented at the early-exponential phase with a combo of C8-HSL, 3OC8-HSL and C10-HSL, at 10 µM each and cells collected 3 hours later.

first: Xavier

last: Coves

first: Pol

last: Huedo

first: Marc

last: Bravo Bravo

first: Òscar

last: Conchillo-Solé

first: Andromeda-Celeste

last: Gomez

first: Anna

last: Esteve-Codina

last: Dabad

last: Daura

first: Daniel

last: Yero


title: Genome-wide analysis of gene expression by B. subtilis biofilms grown in buffered or minimally buffered conditions,

status: Public on May 12 2023

city: Chicago

first: Peter

last: Tran

state: Illinois

address: impson Querrey Research Bldg 302 E Huron St, Room 11-500

institute: Prindle Lab, Northwestern University

summary: Analysis of B. subtilis biofilm metabolism at gene expression level. The hypothesis tested in the present study was that extracellular pH and environmental buffer influence the biofilm development. Results provide important information of the response of biofilms to changes in extracellular pH.

overall_design: Total RNA obtained from B. subtilis NCIB 3610 WT or ∆alsS mutant biofoilms grown on MSgg with 1 mM MOPS (minimally buffered) at 24 or 36 h compared to NCIB 3610 WT or ∆alsS mutant biofoilms grown on MSgg with 100 mM MOPS (buffered) at 24 or 36 h

first: Arthur

last: Prindle


title: Activation of adult human pancreatic ductal and ductal progenitor-like cells requires extracellular matrix protein signaling

first: Hsun

last: Ku

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

overall_design: Refer to individual Series


title: The impact of extracellular matrix on the precision medicine utility of pancreatic cancer patient-derived organoids

status: Public on May 10 2023

city: La Jolla

first: Kathryn

last: Lande

address: 10010 N Torrey Pines Rd

department: IGC

institute: Salk Institute

summary:
Organoids are cultivated in extracellular matrix from different basement membrane extracts (BMEs) that are most commonly acquired commercially. However, the impact of different sources and lots of BMEs on organoid drug response is unknown. Here, we tested the impact of BME source and lot on proliferation, chemotherapy and targeted therapy drug response, and global gene expression in mouse and human pancreatic ductal adenocarcinoma organoids.
Both mouse and human organoids displayed increased proliferation in Matrigel (Corning) compared to Cultrex (RnD) and UltiMatrix (RnD). However, we observed no substantial impact on drug response when organoids were cultured in Matrigel, Cultrex, or UltiMatrix. We also did not observe major shifts in gene expression across the different BME sources.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Comparative gene expression profiling analysis of RNA-seq data for three human pancreas organoids and three mouse pancreas organoids grown in five different basement membrane extracts.

first: Jan

last: Lumibao

first: Shira

middle: R

last: Okhovat

first: Kristina

last: Peck

first: Xiaoxue

last: Lin

first: Jingjing

last: Zou

first: Dannielle

middle: D

last: Engle


title: Effect of depletion of RFX4 on gene expression during neuronal differentiation of ESC to NPC

status: Public on May 27 2023

city: Seoul

first: Su Min

last: Kim

state: Gangnam-gu

address: Dogok-ro 237

country: South Korea

institute: Severance Hospital

summary: To investigate the function of RFX4 in the regulation of neurogenic differentiation, we extablished RFX4 K/O ESC cell lines by using CRISPR-Cas9 system.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for ESC cells and NPS cells with/without RFX4 K/O

first: Won-Young

last: Choi

first: Mu

middle: S

last: Choe

first: Su

first: Min-Young

last: Lee

first: Lark

middle: K


title: Modified live attenuated influenza B virus vaccines and sex-driven differences on humoral immune responses in the DBA/2J mouse model

status: Public on May 01 2023

city: Athens

first: Daniel

middle: Roberto

last: Perez

state: GA

address: 953 College Station Road

institute: University of Georgia

summary: Influenza B virus (FLUBV) is a major respiratory pathogen of humans. Seasonal influenza vaccines include either one or both FLUBV lineage strains, Victoria, and Yamagata. Vaccine mismatch occurs frequently, particularly in countries where vaccines contain only one of the lineages. We have previously described the safety and efficacy of modified live attenuated FLUBV vaccines based on either virus with rearranged genomes (FluB-RAM and FluB-RANS) or carrying a PB1 segment with a combination of temperature sensitive mutations and a C-terminal HA tag (FluB-att). We compared side by side the immunological responses in female and male DBA/2J mice vaccinated with either one of these vaccines and those with isogenic backgrounds encoding a chimeric HA segment carrying an N-terminal peptide encoding the IgA inducing peptide (IGIP).  Recombinant viruses with or without the IGIP modification were genetically stable over multiple passages in eggs. In mice, introduction of IGIP improved attenuation of the vaccine candidates, particularly for the FluB-RAM/IGIP compared with the non-IGIP FluB-RAM counterpart. In a prime-boost regime, mice were completely protected against lethal challenge with a homologous FLUBV strain. Recombinant viruses induced antibodies against HA considered of protective value. Antibodies against NA and NP were readily detected. Compared to male mice and regardless of the vaccine used, female mice showed a clear trend towards enhanced humoral and cross-reactive IgG and IgA anti-HA responses as well as against NA and NP. The presence of IGIP in the vaccine resulted in an overall trend towards reduced anti-HA responses but enhanced anti-NA and anti-NP responses, particularly of the IgA isotype. Mucosal and serological responses two weeks after challenge showed similar trends with clear differences observed based on sex, vaccine backbone, and whether the vaccine carried the IGIP modification.  These findings are significant for the development of universal influenza vaccines.

platform_organism:
Influenza A virus
Influenza B virus

overall_design: We developed three live attenuated vaccine candidates with re-arranged genome expressing the M2 or NS2 from the PB1 gene segment, and the IgA inducing peptide (IGIP) from the HA gene segment. DBAJ/2 mice were primed and boosted (3 weeks ppart) with the modified viruses to test safety and protection efficacy. Body weight was recoreded for up to 12 days post vaccination and clinical signs were monitored daily throughout the length of the study. Blood samples were collected at 19 days post-boost (dpb) to assess neutralizing antibody responses through HI and virus neutralization. In addition, sera were used to performa microarrays against multiple influenza A and B protein antigens. Three weeks after boost, mice were challenged with a lethal dose of B/Brisbane/60/2008 PB2 F406Y. Body weigh was recorded for up to 12 days post-challenge (dpc) and clinical signs for up to 14 dpc. Blood and nasal wash samples were collected at 14 dpc for serology and to determine antigen-specific IgG and IgA responses through microarray analysis.

first: Stivalis

last: Cardenas-Garcia

first: C

last: Cáceres

first: Aarti

last: Jain

first: Ginger

last: Geiger

first: Jongsuk

last: Mo

first: L

last: Gay

first: Brittany

last: Seibert

first: Algimantas

last: Jasinskas

first: Rie

last: Nakajima

first: Daniela

last: Rajao

first: D

middle: H

last: Davies


title: RNA-Seq of a panel of human lymphoma cell lines (B cells)

city: Bellinzona

first: Luciano

last: Cascione

state: Ticino

address: Via Chiesa 5

department: Lymphoma & Genomics Research Program

country: Switzerland

institute: IOR

summary: We invastigated the baseline transcriptomic profiles of a panel of B-cell lymphoma cell lines

overall_design: Cell lines were deep sequenced on a Illumina NextSeq using High Output, PhiX was spiked into the library at 5%.

first: Francesco

last: Bertoni


title: The role of BFGF-related transcriptional regulation  in calcification model of valve interstitial cells induced by osteoblastic medium

city: shanghai

first: YUAN

last: GAO

address: No. 168, Changhai Road, Yangpu District, Shanghai

department: Cardiovascular surgery

institute: Changhai Hospital

summary:
Purpose:To explore the  the function and underlying mechanisms of basic fibroblast growth factor (BFGF) on  calcific aortic valve disease (CAVD) a.
Methods: The valvular interstitial cells were isolated from porcine aortic valve leaflets. To investigate the effect of BFGF on osteogenic differentiation of VICs, the osteogenic induced medium (OIM) and BFGF were added. RNA sequencing is used   to identify changes in gene proffles.
Results:This analysis completed the full transcriptome sequencing of 9 samples, with a total of 2 differentially expressed gene groups. The number of differentially expressed genes detected was 2526, 159. 6596 lncRNAs were identified, and the number of differentially expressed lncRNAs detected was 349,100. 6179 new circRNAs were predicted, and  the number of differentially expressed circRNAs detected was 4,243.
Conclusions:This study  completed for the first time to the sequencing of the full transcriptome expression of valve interstitial cells under the action of BFGF, and has proved that BFGF can inhibit the calcification process of valve interstitial cells,  our study suggests that bFGF has important value in the prevention and treatment of CAVD.

overall_design: We used porcine valvular interstitial cells (PVIC) as the model. We used OIM differentiation medium to induce calcification as well as bFGF treatment to inhibit the calcification for 3 days. The PVICs expression profiles of mRNA and its related transcriptional regulation were investigated by transcriptome sequencing.

first: Gao

last: Yuan


title: Role of PIP5K1a in human Th17 gene expression

city: Pittsburgh

first: Dhivyaa

last: Rajasundaram

state: PA

address: 4401 Penn Avenue

department: Pediatrics

institute: University of Pittsburgh

summary: This data was performed as part of Revu et al, "Human IL-17A protein expression is controlled through a PIP5K1a-dependent ARS2 checkpoint". IL-17 secreted by TH17 cells is beneficial for microbial control, but causes inflammation and pathological tissue remodeling in autoimmunity. Hence TH17 differentiation and IL-17 production must be tightly regulated, but to date this has been defined only in terms of transcriptional control.  Phosphatidyinositols are second messengers activated during T cell activation that transduce signals from the TCR and costimulatory receptors at the cell membrane. Here we show phosphatidylinositol(4,5)bisphosphate (PIP2) was unexpectedly enriched in the nucleus of human TH17 cells, dependent on the kinase PIP5K1, and that blockade of PIP5K1 impaired IL-17A production. In contrast, nuclear PIP2 enrichment was not observed in Th1 or Th2 cells and these subsets did not require PIP5K1 for cytokine production. In multiple sclerosis patient T cells, IL-17 production elicited by myelin basic protein could be inhibited by PIP5K1 blockade. Surprisingly, PIP5K1 blockade did not alter IL17A mRNA levels or stability in TH17 cells. Instead, analysis of PIP5K1-interacting proteins revealed that PIP5K1 targets ARS2, a nuclear cap binding complex scaffold protein, to facilitate ARS2 binding to IL17A mRNA and subsequent IL-17 protein expression, uncovering a new mechanism for regulating IL-17A protein production and suggesting a novel mechanism of translation control of cytokines.

overall_design: Human naïve T cells were isolated and stimulated with anti-CD3, IL-23 and IL-1b for 5 days. On day 3 PIP5K1a inhibitor was added to cultures. On day 5 RNA was isolated and RNA sequencing performed.

first: Mandy

last: McGeachy

first: Dhivya

first: Shankar

last: Revu


title: CUT&Tag analysis uncovered Pou4f1-bound DNA elements in E16.5 retinas

status: Public on Apr 30 2023

city: Houston

first: Chai-An

last: Mao

state: Texas

address: 6431 Fannin Street, MSB7.242

department: Ophthalmology And Visual Scien

institute: University of Texas McGovern Medical School

summary: To understand how Pou4f1 functions in RGC lineage specification and subtype formation, we performed “Cleavage Under Targets & Tagmentation” (CUT&Tag) analysis using a rabbit anti-Pou4f1 antibody and embryonic 16.5 (E16.5) retinal cells to generate barcoded PCR libraries that are enriched for Pou4f1-mediated binding. In parallel, rabbit IgG was used as a negative control for peak calling analysis, and rabbit anti-H3K9ac antibody was used to mark active enhancers and promoters.

overall_design: Four retinas isolated from wildtype mouse embryos at E16.5 were pooled, and then dissociated using papain dissociation system (#LK003150, Worthington Biochemical Corporation, Lakewood, NJ). The CUT&Tag library was prepared using CUT&TAG-IT Assay Kit (#53160, Active Motif, Carlsbad, CA) following manufacturer's protocols. The libraries were pooled evenly and subjected to the paired-end 75-cycle sequencing on an Illumina NextSeq 550 System using High Output Kit v2.5 (#20024907, Illumina, San Diego, CA).

first: Takae

last: Kiyama


title: DNA methylation changes in mice induced by parabiosis and recovery [RRBS]

status: Public on Apr 21 2023

city: Boston

first: Jesse

last: Poganik

address: 77 Avenue Louis Pasteur

department: Medicine/Genetics

institute: Brigham and Women's Hospital, Harvard Medical School

summary: Aging is classically conceptualized as an ever-increasing trajectory of damage accumulation and loss of function, leading to increases in morbidity and mortality. However, recent in vitro studies have raised the possibility of age reversal. We characterized several models in which biological age is perturbed. Heterochronic parabiosis and recovery from this procedure is one such example.

overall_design: We studied a mouse model of heterochronic parabiosis. Mouse pairs (either age-mismatched, heterochronic; or age-matched, isochronic) were surgically joined and maintained for 3 months. After this period, a subset of mice were euthanized and tissues were collected for analysis, and another subset of pairs was surgically detached and allowed to recover for 2 months, after which tissues were collected. We subjected DNA isolated from these samples to reduced representation bisulfite sequencing (RRBS).

first: Bohan

last: Zhang

first: Gurpreet

last: Baht

first: Alexander

last: Tyshkovskiy

first: Amy

last: Deik

first: Csaba

last: Kerepesi

first: Sun

last: Yim

first: Ake

last: Lu

first: Amin

last: Haghani

first: Tong

last: Gong

first: Anna

middle: M

last: Hedman

first: Ellika

last: Andolf

first: David

last: Lee

first: Alexandre

last: Trapp

first: Akshay

last: Bareja

first: Lauren

last: McKay

first: Anastasia

middle: V

last: Shindyapina

first: Sergey

last: Dmitriev

first: Gören

last: Pershagen

first: Catarina

last: Almqvist

first: Clary

last: Clish

first: Steve

last: Horvath

first: James

middle: P

last: White

first: Vadim

last: Gladyshev


title: Gene expression changes in mice induced by parabiosis and recovery [RNA-seq]

first: Jesse

last: Poganik

overall_design: We studied a mouse model of heterochronic parabiosis. Mouse pairs (either age-mismatched, heterochronic; or age-matched, isochronic) were surgically joined and maintained for 1-3 months. After this period, a subset of mice were euthanized and tissues were collected for analysis, and another subset of pairs was surgically detached and allowed to recover for 2 months, after which tissues were collected. We sequenced RNA isolated from these samples to analyze gene expression changes.

first: Bohan

last: Zhang

first: Gurpreet

last: Baht

first: Alexander

last: Tyshkovskiy

first: Amy

last: Deik

first: Csaba

last: Kerepesi

first: Sun

last: Yim

first: Ake

last: Lu

middle: N


title: DNA methylation and gene expression changes in mice induced by parabiosis and recovery

platform_organism:
Macaca mulatta
Homo sapiens
Mus musculus
Rattus
Rattus norvegicus


title: DNA methylation changes in mice induced by parabiosis and recovery [HorvathMammalMethylChip40]

summary: Aging is classically conceptualized as an ever-increasing trajectory of damage accumulation and loss of function, leading to increases in morbidity and mortality. However, recent in vitro studies have raised the possibility of age reversal. We characterized several models in which biological age, assessed primarily through analysis of DNA methylation, undergoes reversible changes. Heterochronic parabiosis and recovery from this procedure is one such example.

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: We studied a mouse model of heterochronic parabiosis. Mouse pairs (either age-mismatched, heterochronic; or age-matched, isochronic) were surgically joined and maintained for 3 months. After this period, a subset of mice were euthanized and tissues were collected for analysis, and another subset of pairs was surgically detached and allowed to recover for 2 months, after which tissues were collected. We subjected DNA isolated from these samples to the HorvathMammalMethylChip40 for methylation profiling.

first: Amin

last: Haghani

first: Tong

last: Gong

first: Anna

last: Hedman

first: Ellika

last: Andolf

first: David

last: Lee

first: Alexandre

last: Trapp

first: Akshay

last: Bareja

first: Lauren

last: McKay


title: Integrated systems immunology approach identifies impaired effector T cell memory responses as a feature of progression to severe dengue fever

status: Public on Apr 13 2023

city: Clayton

first: Stephanie

middle: Irene

last: Studniberg

state: VIC

address: 19 Innovation Walk

department: Microbiology

institute: Infection Discovery Program, Monash Biomedicine Discovery Institute

summary:
Typical symptoms of uncomplicated dengue fever (DF) include headache, muscle pains, rash, cough, and vomiting. A proportion of cases progress to severe dengue hemorrhagic fever (DHF), associated with vascular permeability, thrombocytopenia, and hemorrhages. Progression to severe dengue is difficult to diagnose at the onset of fever, which complicates patient triage, posing a socio-economic burden on health systems.

To identify parameters associated with protection and susceptibility to DHF, we pursued a systems immunology approach integrating plasma chemokine profiling, high-dimensional mass cytometry and peripheral blood mononuclear cell transcriptomic analysis at the onset of fever in a prospective study conducted in Indonesia.

overall_design:
RNA-seq analysis was performed using bulk PBMCs from study participants with either DF (n=5) or DHF (n=6).

>>>Submitter states that raw data are unavailable due to patient privacy concerns<<<

first: Lisa

last: Ioannidis

middle: I

first: Emily

last: Eriksson

first: Suhendro

last: Suwarto

first: Dionisius

last: Denis

first: Yang

last: Liao

first: Wei

last: Shi

first: Alexandra

last: Garnham

first: R. Tedjo

last: Sasmono

first: Diana

last: Hansen


title: Mettl3-mediated m6A Methylation Determines the Size of Endocrine Progenitor Pool and Postnatal Islet Mass

status: Public on Apr 16 2023

city: Shanghai

first: Jiajun

last: Sun

state: Shanghai City

address: 197 Rui Jing Er Rd

institute: Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital

summary: To investigate the role of RNA methyltransferase-like 3 (Mettl3)-mediated m6A modification in pancreatic endocrine differentiation in early embryonic period. We then performed m6A MeRIP-seq (GenSeq® m6A MeRIP Kit) at E15.5 Mettl3pKO  pancreas group and E15.5 WT pancreas group

overall_design: We generated transgenic mice lacking Mettl3 specifically in Pdx1+ pancreatic progenitor cells(Mettl3pKO) and  dissect E15.5 Mettl3pKO and WT pancreata for m6A MeRIP-seq.


title: A genome-wide CRISPR screen for genes regulating ferroptosis sensitivity

status: Public on Apr 17 2023

city: Simotsuke

first: Tadayoshi

last: Karasawa

state: Tochigi

address: 3311-1 Yakushiji

institute: Jichi Medical University

summary: Genome-wide CRISPR screens were performed in Huh-7 hepatoma cell line to identify genes regulating ferroptosis sensitivity.  Huh-7 cells were transduced with Human GeCKO library and selected by RSL-3.

overall_design: Genome-wide CRISPR screen (GeCKOv2 library) in Huh-7 cells teather reated with RSL-3 or linoleic acid and RSL-3

first: Naoya

last: Yamada

first: Masafumi

last: Takahashi


title: Buffered hypercapnia alters the transcriptional profile in monocytes.

status: Public on Apr 07 2023

city: Dublin

first: Eoin

last: Cummins

state: Dublin

address: UCD Conway Institute of Biomolecular and Biomedical Research, UCD Belfield

department: Conway Institute/School of Medicine

country: Ireland

institute: University College Dublin

summary: In monocytes we used RNA-sequencing to investigate the effect of 4hrs of exposure to buffered hypercapnia (10% CO2) compared to normocapnia (5% CO2) with and without the pro-inflammatory stimulus LPS (2.5ug/ml) for the final 2hrs. Buffered hypercapnia causes transcriptional  changes associated with altered metabolic function in both the basal and stimulated states.

first: David

last: Phelan


title: Polycomb Ezh1 maintains muscle stem cells quiescence through non-canonical regulation of the Notch signaling

status: Public on Apr 04 2023

city: Bethsda

first: Vittorio

last: Sartorelli

state: MD

address: 50 South Dr.

department: NIAMS

institute: NIH

summary: Organismal homeostasis and regeneration are predicated on committed stem cells which, in tissues and organs with low turnover, reside for long periods in a reversible cell cycle arrest, defined as quiescence. Inability to exit or premature escape from quiescence, as occurring in pathological conditions and aging, is detrimental as it results in either lack of stem cell mobilization or pool depletion with consequent defective tissue homeostatis and regeneration. It is therefore unsurprising that quiescence is safeguarded by multilayered regulatory mechanisms. Here, we report that Polycomb Ezh1 confers quiescence to muscle stem cells (MuSCs) through a non-canonical function. In the absence of Ezh1, MuSCs spontaneously exit quiescence and, following repeated injuries, the stem cell pool is depleted resulting in failure to sustain appropriate muscle regeneration. Rather than regulating repressive Polycomb-dependent histone H3K27 methylation, Ezh1 actively maintains the Notch signaling pathway in MuSCs. Accordingly, selective genetic reconstitution of the Notch signaling corrects stem cell number and re-establishes quiescence of Ezh1-/-  MuSCs.

overall_design:
To establish the role of polycomb Ezh1 in MuSCs biology, we performed RNASeq, ATACSeq, and H3K27me3 ChIPseq in FACS-purified MuSCs from the hindlimbs of wild-type (WT) or Ezh1-/- mice in homeostatic conditions.
To further evaluate potential differences between WT and Ezh1-/- mice, tibialis anterior (TA) muscles of WT and Ezh1-/- mice were injured with notexin (NTX), and an additional NTX injection was performed twenty-eight days after the first injury. We conducted scRNAseq experiments in uninjured and regenerating MuSCs (collected three days after the second muscle injury).

first: Feng

last: Xuesong

first: Wang

last: A Hongjun

first: Juan

last: Aster H

first: KyungDae

last: Ko

first: Kan

last: Jiang

first: Riparini

last: Giulia

first: Ciuffoli

last: Veronica

first: Veronica

last: Ciuffoli

first: Kaba

last: Aissah

first: Lopez

last: Cristopher

first: Naz

last: Faiza

first: Jarnik

last: Michal

first: Aliberti

last: Elizabeth

first: Hu

last: Shenyuan

first: Segalés

last: Jessica

first: Khateb

last: Mamduh

first: Acevedo-Luna

last: Natalia

first: Randazzo

last: Davide

first: Cheung

last: Tom

first: Muñoz-Cánoves

last: Pura

first: Stefania

last: Dell'Orso


title: Striatal astrocyte Crym KO transcriptome

status: Public on Apr 03 2023

city: Los Angeles

first: Riki

last: Kawaguchi

state: CA

address: 760 Westwood Plaza, Room 37-420

department: Department of Psychiatry and Neurology Department of Psychiatry

institute: University of California, Los Angeles

summary: We overexpressed Rpl22-HA in astrocytes of the mouse striatum in both wild type (WT) mice and in Crym KO mice at five months of age. Crym has been shown to be expressed predominantly in striatum and is highly expressed in astrocytes, however the function of this protein in astrocytes is unknown.

overall_design: RNA was immunoprecipitated using Rpl22-HA and therefore, the IP RNA samples are astrocyte enriched while the input RNA samples contain RNAs from all cells types.

first: Matthias

last: Olivier

first: Joselyn

last: Soto

first: Baljit

last: Khakh


title: STING agonism leads to durable clinical response in an anti-PD-L1 refractory patient: Tumor antigen-specific, single cell level biomarker analyses.

status: Public on Apr 01 2023

city: Seattle

first: Thomas

middle: Heyward

last: Pulliam

state: WA

address: 850 Republican St.

institute: University of Washington

summary:
Background: PD-1 pathway blockade has revolutionized cancer care, but many patients do not durably benefit from these therapies. Novel treatments to engage anti-tumor immunity in this setting are needed. The stimulator of interferon genes (STING) protein, an innate sensor of cytoplasmic DNA, is a promising target with several agonists in development; however, recent clinical trials of these agents have not demonstrated efficacy for most patients. Here, we present detailed biomarker analyses of a patient with anti-PD-L1 refractory Merkel cell carcinoma that had a durable (~1 year), abscopal response to ADU-S100 (intralesional STING agonist) combined with anti-PD-1 blockade.
Methods: Tumor biopsies and peripheral blood acquired pre- and post-treatment were analyzed with single cell RNA sequencing, 27-color flow cytometry, T cell receptor sequencing and multiplexed immunohistochemistry (mIHC; post treatment tumor specimen only). Critically, cancer-specific CD8 T cells were identified using MHC-I tetramers containing peptides from the Merkel cell polyomavirus (MCPyV), the etiologic agent of this cancer. Cell lines and tumor specimens from 76 patients were used to evaluate STING expression and signaling in MCC.
Results: High levels of antigen cancer-specific T cells were observed in the tumor prior to ADU-S100 treatment (12% of T cells recognized an MCPyV epitope). These cancer-specific CD8 T cells exhibited characteristics of exhaustion including high TOX and low TCF1. Following STING agonist treatment, MCPyV-specific CD8 T cells expanded 3-fold with minimal phenotypic changes. An increase in antigen presentation on MCC tumor cells was also observed (1.8% of tumor cells MHC-I positive before treatment, 8.2% after treatment). This was not a result of ADU-S100 acting directly on tumor cells as mIHC analysis of 88 tumor samples confirmed little or no STING expression in tumor cells, but high STING expression on infiltrating immune and stromal cells. Further in vitro studies showed MCPyV-positive MCC cell lines are STING deficient, and ADU-S100 did not induce interferon.
Conclusions: Our study suggests that STING agonists act on intratumoral immune cells to release interferons, which upregulate tumor cell antigen presentation and reinvigorate the anti-tumor response from pre-existing cancer-specific T cells. Thus, patients with anti-PD-(L)1-refractory disease showing downregulation of antigen-presentation may benefit from intralesional STING agonists.

overall_design:
We used 10x Genomic's Chromium Single Cell 5' Reagent Kits (v1.1 Chemistry Dual Index) with Feature Barcoding technology for Cell Surface Protein and Immune Receptor to identify antigen specific T cell V(D)J sequences. This was performed on tumor and blood specimen taken from a patient treated with an intralesional STING agonist. Due to patient privacy concerns, only processed and not raw sequencing reads were deposited.
***raw data were not included due to patient privacy regulations***

first: Saumya

last: Jani

first: Paul

last: Nghiem


title: Expression data of peri-tumoral tonsillar tissues in oropharyngeal cancer

status: Public on Apr 02 2023

city: Kanazawa

first: Naohiro

last: Wakisaka

state: Ishikawa

institute: National Hospital Organization Kanazawa Medical Center

summary:
Secondary lymphoid organs (SLOs) serve as an interface between tumor cells and immune system as an initial site of antigen presentation which is critical for the development of an effective anti-tumor immune response.
We used microarray to analyze the gene expression profile of peri-tumoral tonsillar tissues of oropharyngeal cancer.

overall_design: We analyzed the expression profile at peri-tumoral tonsillar tissues, and compares the profile according to the nodal status.


title: Cancer-specific CD8 T cell frequency at baseline in blood correlates with response to PD-1 blockade in Merkel cell carcinoma

summary: Understanding immunotherapy response and resistance is challenging due to difficulty identifying cancer-specific CD8 T cells. Merkel cell carcinoma (MCC) is typically driven by Merkel cell polyomavirus (MCPyV), facilitating identification of cancer-specific T cells across patients. We characterized cancer-specific T cells in 35 MCC patients, including from a neoadjuvant anti-PD-1 trial. Higher MCPyV-specific CD8 T-cell frequency in pre-treatment blood (but not tumors) correlated with response (p=0.0056). Single cell RNAseq revealed MCPyV-specific CD8 T cells in blood with increased stem/memory signatures and decreased exhaustion signatures relative to their intratumoral counterparts. Number of circulating cancer-specific T cells is likely most linked to primary response to immunotherapy as longitudinal samples documented emergence of additional resistance mechanisms, amidst abundant circulating cancer-specific CD8 T cells. These results suggest that blood acts as an important reservoir of cancer-specific CD8 T cells and suggests adoptive cell therapies may be particularly effective in patients without such cells.

overall_design: We used 10x Genomic's Chromium Single Cell 5' Reagent Kits (v2 Chemistry Dual Index) with Feature Barcoding technology for Cell Surface Protein and Immune Receptor to identify antigen specific T cell V(D)J sequences. This was performed on a performed on T cells expaned from patient tumors using HLA-I matched DNA barcoded MHC-I multimers.


title: Phosphorylation of DNA-PKcs at the S2056 cluster ensures efficient and productive lymphocyte development in XLF-deficient mice

city: New York

first: Yimeng

last: Zhu

state: NY

address: 1130 ST NICHOLAS AVE.

department: Institute for Cancer Genetics

institute: Columbia University

summary: The non-homologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of Kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters – PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But mice carrying alanine substitution at all 5 serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. XLF is a non-essential NHEJ factor. Xlf-/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, MRI, etc.) or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf-/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class switch recombination junctions from DNA-PKcsPQR/PQRXlf-/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.

overall_design: To test whether the presence of XLF masks the role of DNA-PKcs PQR phosphorylation on chromosomal NHEJ, we generated DNA-PKcsPQR/PQRXlf-/- mice. In contrast to single deficient mice, DNA-PKcsPQR/PQRXlf-/- mice display severe lymphocytopenia consistent with V(D)J recombination defects. HTGTS of chromosomal V(D)J recombination junctions reveals markedly increased end-resection, large deletions, and severely reduced SJ fidelity to less than 10%. CSR to IgG1 is also further decreased in DNA-PKcsPQR/PQRXlf-/- cells than in the Xlf-/- control.

first: Brian

last: Lee

first: Shan

last: Zha


title: Host genetic variation guides hepacivirus clearance, chronicity, and liver fibrosis in mice

status: Public on Apr 25 2023

city: Chapel Hill

first: Timothy

last: Sheahan

state: North Carolina

address: 3113 MHRC 135 Dauer Drive, Campus Box 7435

department: Epidemiology

institute: University of North Carolina at Chapel Hill

summary: The dataset describes the host transcriptional response in WT and Collaborative Cross lines infected with Norway rat hepacivirus.

overall_design: We infected WT C57BL/6 mice as well as Collaborative Cross strains CC046, CC071 and CC080 with 1E+05 genome equivelents of Norway rat hepacivirus. Bulk RNAseq was performed on total RNA from the liver at 7 days post infection.


title: Effect of deleting PexRAP/ADHAPR in adipose tissue of chow-fed male C57/BL6J mice

status: Public on Mar 28 2023

city: St Louis

first: Irfan

last: Lodhi

state: MO

address: 660 S Euclid Ave Campus box 8127

institute: Washington University School of Medicine in St. Louis

summary: To investigate the role of PexRAP/ADHAPR in adipose tissue, we use adiponectin-driven Cre recombinase to delete Dhrs7b, the gene which encodes PexRAP/ADHAPR, from all adipose depots. This study assays gene expression in subcutaneous adipose tissue (iWAT) of 8 week old chow-fed male mice.

overall_design: We performed gene expression profiling analysis using data obtained from RNA-seq of iWAT tissue from either cre-negative (control) or cre-positive (PexRAP-AKO) male chow-fed mice

last: Kleiboeker


title: Transcriptional Profiling of Mouse Tumours

status: Public on Apr 05 2023

city: Glasgow

first: Kathryn

last: Gilroy

address: Switchback Road

institute: Beatson Institute

summary: Murine small intestinal and colonic tumours with mutations in combinations of Kras, P53, Notch1, Apc and Braf.

overall_design: Expression data from murine tumours of the noted genotypes.

first: Raheleh

last: Amirkhah

first: Owen

last: Sansom


title: Rescue of HIF1A protein level by E3-ligase KO

status: Public on Mar 27 2023

city: Durham

first: Christian

last: Cerda-Smith

state: NC

address: 450 Research Drive, LSRC C134, PO Box 3813, Duke University

department: Pharmacology and Cancer Biology

institute: Duke University

summary: The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell-sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx.

overall_design:
FACS-based CRISPR KO screen utilizing a custom E3-ligase targeted sgRNA library in conjunction witells treated with an ABL kinase inhibitor (Nilotinib) and placed into hypoxia for 24 h.
Sample types:
High - top 10% of HIF-1a expressing cells collected on the basis of PE signal (PC9 cells, Nil High biol rep 1/2/3)
Low - bottom 10% of HIF-1a expressing cells collected on the basis of PE signal (PC9 cells, Nil Low biol rep 1/2/3)
Unsorted - ungated control sample not collected on the basis of PE signal (PC9 cells, Nil Unsorted  biol rep 1/2/3)

first: Benjamin

last: Mayro

first: Haley

last: Hutchinson

first: Kris

last: Wood


title: An interactive murine single cell atlas of the lung responses to radiation injury

status: Public on Mar 20 2023

city: Paris

first: Charles

last: Fouillade

address: 25 rue d'Ulm

institute: Institut Curie

summary: Radiation Induced Lung Injury (RILI) is one of the main limiting factors of thorax irradiation, which can induce acute pneumonitis as well as pulmonary fibrosis, the latter being a life-threatening condition. The order of cellular and molecular events in the progression towards fibrosis is key to the physiopathogenesis of the disease, yet their coordination in space and time remains largely unexplored. Here, we present an interactive murine single cell atlas of the lung responses to irradiation. This analysis opens the door for exploration of the spatio-temporal dynamics of the mechanisms that lead to radiation-induced pulmonary fibrosis. It depicts with unprecedented detail cell type-specific radiation-induced responses associated with either lung regeneration or the failure thereof. A better understanding of the mechanisms leading to lung fibrosis will help finding new therapeutic options that could improve patients’ quality of life.

overall_design: Single-cell suspensions were prepared from 10-12 weeks C57BL/6J mice and single cell RNA-seq libraries were generated using 3' V3 or 3'V3 NextGem chemistry kit on Chromium Single cell controller (10x Genomics). This dataset includes 20 samples: 5 non-irradiated control samples; 5 samples from 1 to 5 months 10 Gy post-irradiation; 10 samples from 1 to 5 months 17 Gy post-irradiation (two biological replicates per time point).

first: Sandra

last: Curras-Alonso

first: Juliette

last: Soulier

first: José-Arturo

last: Londono-Vallejo


title: Transcriptomic profiling and electrophysiological analysis from microtransplanted synaptic membranes

city: Irvine

first: Marquis

middle: P.

last: Vawter

address: 837 Health Sciences Rd

department: Psychiatry & Human Behavior

institute: University of California, Irvine

summary: Alterations of synaptic function in individuals with schizophrenia have been found in transcriptomics, proteomics, and genome wide association studies. Impaired synaptic glutamatergic (excitatory) and GABAergic (inhibitory) neurotransmission in affected brain regions (e.g. dorsolateral prefrontal cortex; DLPFC) is thought to be involved in the core symptoms of schizophrenia. However, there are no quantitative measurements of synaptic function in the human DLPFC, therefore concrete and specific functional alterations of voltage gated ion channels of glutamate and GABA receptors are lacking. We have begun to address this problem by directly measuring AMPA- and GABA receptor-mediated synaptic currents in postmortem brains from subjects with schizophrenia and contrasting to controls. We demonstrate in our preliminary work that the function of synaptic receptors is maintained in postmortem brains and is significantly decreased in schizophrenia compared to controls. Our overarching hypothesis is that reductions in both inhibitory and excitatory currents underlie synaptic deficits in schizophrenia. We will test rigor and reproducibility of this hypothesis in independent case-control cohorts. These deficits in synaptic currents can be statistically modeled with transcriptomic data which will be useful in downstream studies that pharmacologically challenge activation of these currents.

overall_design: (1)Test the hypothesis that alterations of the excitatory to inhibitory balance are present in schizophrenia (SZ) brains using microtransplantation of synaptic membranes (MSM) in the DLPFC of schizophrenia cases (n = 15) and controls (n = 15). We measured ion currents of microtransplanted neurotransmitter receptors into frog oocytes using two electrode voltage clamp. (2) Test the hypothesis that integrative analysis of transcriptomic data predicts synaptic dysfunction in SZ at the electrophysiological level. The physiological and pharmacological profile of synaptic AMPA and GABA receptors are mostly determined by their subunit composition, posttranslational modifications, and associated proteins. We collected RNA-seq to characterize genes in the synapse that predict electrophysiological responses.

first: Agenor

last: Limon

first: Anton

last: Schulmann


title: Single-cell transcriptional profiling identifies a spectrum of unconventional intraepithelial T lineage cells in human cord blood

status: Public on Mar 07 2023

city: Ghent

first: Laurenz

last: De Cock

address: Corneel Heymanslaan 10

department: Department of Diagnostic Sciences

country: Belgium

institute: Ghent University

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

overall_design: Refer to individual Series


title: Single-cell transcriptional profiling identifies a spectrum of unconventional intraepithelial T lineage cells in human cord blood [bulk RNA-Seq]

summary: Conventional CD4 and CD8 single positive T cell lineages constitute the main differentiation pathway in the thymus. In human thymus, a minor TCRαβ differentiation pathway diverges from the early double positive stage, consisting of CD10+ PD-1+ cells. These cells are phenotypically and functionally similar to murine agonist-selected intraepithelial T lymphocyte precursors (IELps) which home to the small intestine. Here, the progeny of the human agonist-selected IEL lineage was identified in antigen-inexperienced cord blood (CB) with a polyclonal T cell receptor (TCR) repertoire exhibiting a bias towards early TCR alpha chain rearrangements and elevated autoreactive indices. Single-cell RNA sequencing allowed further delineation of this unconventional lineage in CB. Trajectory analysis, along with TCR repertoire analysis and transcriptomics, suggests a precursor-progeny relationship with the thymic IELps. The distinct, heterogeneous CB population can now be defined as CD3+ TCRαβ+ CD4- CCR7- CD26-. Besides recent thymic emigrants, this population also consists of newly identified effector clusters and previously described populations: the suppressive NK receptor expressing CD8+ Treg population, the KIR/NKG2A+ EOMES+  virtual memory population and the CD8αα+ T cell populations. The population shows a discriminating stable Helios expression and is exclusively able to downregulate CD8β expression, resulting in double negative T cells. The functional properties of this population suggest that the cells expand on inflammatory cues and exert cytotoxic and proinflammatory activity.

overall_design: To identify additional marker genes in cord blood for unconventional intraepithelial T cells, 3 T cell populations were sorted from cord blood: I. TCRγδ+, II. CD3+/low PD-1+ (unconventional intraepithelial T cells) and III. CD3+ PD-1- (conventional T cells). For each sort 3 replicates from 3 different donors wereused in a differential expression analysis with DeSeq2 to identify additional markers. The same populations were sorted from post natal thymus (also 3 replicates from 3 different donors per cell population) to use as a comparison.

first: Lore

last: Billiet

first: Filip

last: Van Nieuwerburgh

first: Bart

last: Vandekerckhove


title: The contribution of RNA oxidation to acute kidney injury: evidence from ISG20-mediated oxidized RNA degradation

status: Public on Mar 03 2023

city: Jinan

first: Meng

last: Jia

address: 44#, Wenhua Xi Road

institute: Shandong university

summary: RNA is more vulnerable to oxidation than other cellular components, which is linked to a range of diseases and pathological conditions, such as AKI. To identify the contribution of RNA oxidation to AKI, the mechanisms of how cells handle oxidized RNA should be elucidated. By RNA sequencing analysis in ischemia/reperfusion-induced AKI, we observed the significant changes of genes important in degradingting oxidative RNA.

overall_design: The gene expression of 3 samples from a 24 h renal ischemia/reperfusion-induced AKI  was compared to 3 sham samples by RNA sequencing.

first: Wei

last: Tang


title: Single-cell transcriptional profiling identifies a spectrum of unconventional intraepithelial T lineage cells in human cord blood [scRNA-Seq]

overall_design: Based on bulk RNA-sequencing of unconventional intraepithelial T cells additional flowcytometric definitions for this population were postulated. These new definitions were subsequently applied to cord blood from two different donors to investigate whether the unconventional intraepithelial T cells are a homogenous population and whether the new definitions identified the exact same cell types.

first: Niels

last: Vandamme


title: Single-cell transcriptional profiling identifies a spectrum of unconventional intraepithelial T lineage cells in human cord blood [TCR sequencing]

overall_design: To further characterize the unconventional intraepithelial T cell lineage in cord blood and post-natal thymus, unconventional and conventional T cells were sorted from 6 cord blood and 2 thymus donors and subsequently subject to TCR sequencing. The resulting TCR repertoires were compared between unconventional and conventional cells and between cord blood and thymus.

first: David

last: Vermijlen


title: Altered transcriptional programming in Nur77-GFPhi naïve CD8 T cells

status: Public on Mar 02 2023

city: Atlanta

first: Chris

last: Scharer

address: 1510 Clifton Rd, Suite 3086A

department: Microbiology and Immunology

institute: Emory University

summary: Naive T cells experience accumulation of TCR signaling in response to self-antigens in the steady state. However, how these signals influence the responsiveness of naive CD8+ T cells to subsequent agonist TCR stimulation remains incompletely understood. We investigated how naive CD8+ T cells that experienced relatively low or high levels of TCR signaling in response to self-antigens respond to stimulation with foreign antigens. A transcriptional reporter of Nr4a1 (Nur77-GFP) revealed substantial heterogeneity of the amount of TCR signaling naive CD8+ T cells accumulate in the steady state. To further understand the impact of differential TCR signaling on CD8 T cells we performed RNA-seq on naive Nur77-GFPhi and lo populations.

overall_design: Triplicate samples of naive CD8 T cells that were Nur77-GFPhi or lo were isolated by FACS and RNA-seq preformed.

first: Christopher

first: Byron

last: Au-Yeung


title: Cancer-specific CD8 T cell frequency at baseline in blood correlates with response to PD-1 blockade in Merkel cell carcinoma.

first: Thomas

last: Pulliam

summary: Understanding immunotherapy resistance is challenging due to difficulty identifying cancer-specific T cells. Merkel cell carcinoma (MCC) is typically driven by Merkel cell polyomavirus (MCPyV), facilitating identification of cancer-specific T cells across patients. We characterized cancer-specific T cells in 35 MCC patients, including participants in a neoadjuvant anti-PD-1 trial. Higher MCPyV-specific CD8 T-cell frequency in pre-treatment blood (but not tumors) correlated with response (p=0.005). Single-cell transcriptomics revealed MCPyV-specific CD8 T cells in blood had increased stem/memory signatures and decreased exhaustion signatures relative to their intratumoral counterparts. While frequency of peripheral cancer-specific T cells was associated with response to initial PD-(L)1 blockade, longitudinal study of acquired resistance revealed immune evasion by tumor-cell MHC-I downregulation despite cancer-specific T cell expansion to 0.5% in blood. Blood thus appears to be an important reservoir of cancer-specific CD8 T cells and adoptive cell therapies may be particularly effective in patients without such cells.

overall_design:
We used 10x Genomic's Chromium Single Cell 5' Reagent Kits (v1.1 Chemistry Dual Index) with Feature Barcoding technology for Cell Surface Protein and Immune Receptor to identify antigen specific T cell V(D)J sequences. This was performed on tumor and blood specimen taken from 7 pateints. Due to patient privacy concerns, only processed and not raw sequencing reads were deposited.
***raw data were not included due to patient privacy regulations***

first: Saumya

last: Jani

first: Paul

last: Nghiem


title: Gene expression profile at single cell level of hindlimb bones from postnatal day 7.5 mice that label hypertrophic chondrocytes and their progeny by Col10a1-Cre;ROSA-ZsGreen.

status: Public on Feb 28 2023

city: Beijing

first: Xiao

last: Yang

address: 38# LifeScience Park, Changping District, Beijing 102206, China

institute: Beijing Institute of Lifeomics

summary: Hypertrophic chondrocytes are considered as a key contributor to bone formation. We used single cell RNA sequencing (scRNA-seq) to analyze the diversity of hypertrophic chondrocytes and their progeny.

overall_design: Unsorted and sorted cells were isolated from hindlimb bones of Col10a1-Cre;ROSA-ZsGreen mice and analyzed using scRNAseq.

first: Guan


title: Peripheral immune-derived matrix metalloproteinase promotes stress susceptibility

status: Public on Feb 24 2023

first: Aarthi

last: Ramakrishnan

state: New York

address: 1425 Madison Avenue

department: Neuroscience

institute: Icahn School of Medicine at Mount Sinai

summary: Psychosocial stress has profound effects on the body, including the peripheral immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3,4,5, the underlying mechanisms are not well understood. Here we show that a myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is elevated in serum from MDD subjects as well as in stress susceptible (SUS) mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), thereby altering social behavior. Using a combination of mass cytometry and single-cell RNA-sequencing, we performed high-dimensional phenotyping of immune cells in circulation and brain and demonstrate that peripheral monocytes are strongly affected by stress. Both peripheral and brain infiltrating monocytes of SUS mice show increased expression of Mmp8 following CSDS. We further show that peripheral MMP8 directly infiltrates the NAc parenchyma to control ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behavior and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a novel mechanism by which peripheral immune factors can affect central nervous system function and behavior in the context of stress. Targeting specific peripheral immune cell-derived proteins, such as matrix metalloproteinases, could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.

overall_design: After 10 days of chronic social defeat stress, blood was collected and Ly6Chigh monocytes, Ly6Clow monocytes, T cells and B cells were isolated, RNA extracted and cell-type specific RNA-sequencing was performed.

first: Flurin

last: Cathomas

first: Hsiao-Yun

last: Lin

first: Kenny

middle: L

last: Chan

first: James

middle: W

last: Murrough

first: Scott

middle: J

last: Russo


title: Single-cell RNA sequencing of liver tissues from NASH and control mice

status: Public on Feb 27 2023

city: Shanghai

first: Huiyi

last: Li

address: No. 1800, Yuntai Rd

department: Department of Gastroenterology

institute: Shanghai East Hospital

summary: The role of single-cell subsets in NASH is unclear. We used single cell RNA sequencing to analyze the diversity of different single-cell subpopulations in NASH.

overall_design: Male C57BL/6 mice (8 weeks old) were randomly fed either a standard diet (low fat) as a control or a methionine-choline deficient diet (MCD; 40% carbohydrate, 10% fat, deficient in methionine and choline) as NASH mice for 6 weeks (each group, n=3). Liver tissues were cut into about 1-2mm3 pieces and digested in SoloTM Tumor Dissociation Kit (Sinotech Genomics Co. Ltd., Shanghai, China, JZ-SC-58201) at 37°C for 30-60min. Then we stopped enzymatic digestion with excess RPMI-1640 medium, and filtered cell with 40 μm Cell strainer. Single cell solution were kept on ice before loading to BD Rhapsody cartridge for single cell transcriptome capturation. Samples from NASH and control groups were pooled separately for scRNAseq analysis.

first: Mingyi

last: Xu