title: ACLY and ACSS2 link nutrient-dependent chromatin accessibility to regulation of CD8 T cell effector responses

summary: Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme Acly (ATP citrate lyase). However, ablation of Acly triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by Acss2 (acyl-CoA synthetase short chain family member 2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When Acly is functional, Acss2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, deletion of Acly renders CD8 T cells dependent on acetate (via Acss2) to maintain acetyl-CoA production and effector function.  Thus, together Acly and Acss2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.

overall_design: C57BL/6 mice (3 biological replicates) were infected with LCMV-Armstrong. At 8 dpi, mice were sacrificed, and single cell suspensions generated

first: Kaymak

last: Irem

first: Oswald

last: Brandon

first: Ma

last: Shixin

first: Johnson

last: Benjamin

first: DeCamp

last: Lisa

first: Kaech

last: Susan

first: Roy

last: Dominic

first: Jone

last: Russell


title: Single-cell RNA and ATAC sequencing revealed distinct molecular characteristics of B cells in systemic lupus erythematosus and primary Sjogren's syndrome

status: Public on Jul 05 2024

city: HeFei

first: zhang

middle: xiao

last: jing

state: AnHui

address: 81 Meishan Road

department: dermatology

country: China

institute: Anhui Medical University

type: Genome binding/occupancy profiling by high throughput sequencing

type: Expression profiling by high throughput sequencing

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

sample_organism:
Homo
Homo sapiens

platform_organism:
Homo
Homo sapiens

overall_design: Refer to individual Series


title: Microglia aging progresses through functional intermediate steps.

status: Public on Jul 04 2024

city: San Francisco

first: Jeremy

middle: Michael

last: Shea

state: CA

address: 513 Parnassus Avenue

department: Anatomy

country: USA

institute: University of California, San Francisco

summary: Aged microglia contribute to maladaptive inflammation, but little is known about their progression from homeostasis to dysfunction during aging. Here, we analyze the spatiotemporal kinetics of microglial aging in the hippocampus. Spatially, the dynamics of age-related inflammatory changes in microglia vastly differ across adjoining regions. Using single cell RNA-Sequencing and in vitro approaches, we find that microglia aging proceeds progressively through functional intermediate states that are necessary for inflammatory activation.

overall_design: RNA-Seq of either Cx3cr1-CreERT::Tgfb1 fl/fl or Cx3cr1-CreERT::Tgfb1 fl/wt adult hippocampal microglia

first: Saul

last: Villeda


title: Microglia aging progresses through functional intermediate steps II

overall_design: RNA-Seq of primary mouse microglia that are LPS or control treated and treated with either DMSO, CCCP, CS-5461, or TGFB1


title: Microglia aging progresses through functional intermediate steps III

overall_design: RNA-Seq of either young (3 months) or old (24 months) microglia or mature (6 months) and aged (18 months) bulk hippocampus.


title: Subtle transcriptomic response of Eurasian perch (Perca fluvatilis) associated with Triaenophorus nodulosus plerocercoid infection

city: Stockholm

first: Anti

last: Vasemägi

address: Stångholmsvägen 2

country: Sweden

institute: Swedish University of Agricultural Sciences

summary: Determining the physiological effects of parasites and characterizing genes involved in host responses to infections are essential to improving our understanding of host-parasite interactions and their ecological and evolutionary consequences. This task, however, is complicated by high diversity and complex life histories of many parasite species. The use of transcriptomics in the context of wild-caught specimens can help ameliorate this by providing both qualitative and quantitative information on gene expression patterns in response to parasites in specific host organs and tissues. Here, we evaluated the physiological impact of the widespread parasite, the pike tapeworm (Triaenophorus nodulosus), on its second intermediate host, the Eurasian perch (Perca fluviatilis).

overall_design: We used an RNAseq approach to analyse gene expression in the liver, the target organ of T. nodulosus plerocercoids, and spleen which is one of the main immune organs in teleost fishes. We compared perch collected from multiple lakes with individuals with (n = 8) and without (n = 6) T. nodulosus plerocercoids in the liver.

first: Konrad

last: Taube

first: Kristina

last: Noreikiene

first: Siim

last: Kahar

first: Riho

last: Gross

first: Mikhail

last: Ozerov


title: Microglia aging progresses through functional intermediate steps

overall_design: Single cell profiling of Cd11b+ cells in the hippocampus at 6-, 12-, 18-, and 24-months


title: METTL3-Mediated N6-methyladenosine mRNA Modification and cGAS-STING Pathway Activity in Kidney Fibrosis

status: Public on Jul 03 2024

city: Taipei

first: Szu-Yuan

last: Li

address: No. 201, Sec. 2, Shih-Pai Road

country: Taiwan

institute: Taipei Veterans General Hospital

summary: Background Chemical modifications on RNA profoundly impact RNA function and regulation. m6A, the most abundant RNA modification in eukaryotes, plays a pivotal role in diverse cellular processes and disease mechanisms. However, its importance is understudied in human chronic kidney disease (CKD) samples regarding its influence on pathological mechanisms. Methods LC-MS/MS and Methylated RNA Immunoprecipitation (MeRIP) sequencing were utilized to examine alterations in m6A levels and patterns in CKD samples. Overexpression of the m6A writer METTL3 in cultured kidney tubular cells was performed to confirm the impact of m6A in tubular cells and explore the biological functions of m6A modification on target genes. Additionally, tubule-specific deletion of Mettl3 (Ksp-Cre Mettl3f/f) mice and the use of anti-sense oligonucleotides inhibiting Mettl3 expression were utilized to reduce m6A modification in an animal kidney disease model. Results By examining 127 human CKD samples, we observed a significant increase in m6A modification and METTL3 expression in diseased kidneys. Epitranscriptomic analysis unveiled an enrichment of m6A modifications in transcripts associated with the activation of inflammatory signaling pathways, particularly the cGAS-STING pathway. m6A hypermethylation increased mRNA stability in cGAS and STING1, as well as elevated the expression of key proteins within the cGAS-STING pathway. Both the tubule-specific deletion of Mettl3 and the use of anti-sense oligonucleotides to inhibit Mettl3 expression protected mice from inflammation, reduced cytokine expression, decreased immune cell recruitment, and attenuated kidney fibrosis. Conclusions Our research revealed heightened METTL3-mediated m6A modification in fibrotic kidneys, particularly enriching the cGAS-STING pathway. This hypermethylation increased mRNA stability for cGAS and STING1, leading to sterile inflammation and fibrosis.

overall_design: kidny biopsy cohort

first: Li

last: Szu-Yuan


title: Monomethylation of Lysine 27 at Histone 3 Confers Lifelong Susceptibility to Stress

city: New York

first: Molly

middle: S

last: Estill

state: NY

address: 1 Gustave L. Levy Place

department: Neuroscience

institute: Mount Sinai School of Medicine

summary: Histone post-translational modifications are critical for mediating persistent alterations in gene expression. By combining unbiased proteomics profiling, and genome-wide approaches, we uncovered a role for mono-methylation of lysine 27 at histone H3 (H3K27me1) in the enduring effects of stress. Specifically, mice exposed to early life stress (ELS) or to chronic social defeat stress (CSDS) in adulthood displayed increased enrichment of H3K27me1, and transient decrease in H3K27me2, in the nucleus accumbens (NAc), a key brain-reward region.

overall_design: Cleavage Under Targets & Release Using Nuclease (CUT&RUN) followed by DNA-sequencing for IgG as well as for histone modifications H3K27me1 and H3K27me2 in nucleus accumbens of adult male mice

first: Angélica

last: Torres-Berrío

first: Aarthi

last: Ramakrishnan

first: Hope

last: Kronman

first: Vishwendra

last: Patel

last: Minier-Toribio

first: Orna

last: Issler

first: Caleb

middle: J

last: Browne

first: Eric

middle: M

last: Parise

first: Yentl

last: van der Zee

first: Deena

last: Walker

first: Freddyson

last: Martínez-Rivera

first: Casey

middle: K

last: Lardner

first: Romain

middle: D

last: Cuttoli

first: Scott

last: Russo

last: Shen

first: Simone

last: Sidoli

last: Nestler


title: NAT10-mediated β-hydroxybutyrylation  Rewires Genomic 3D Conformation and Replication Timing [Hi-C]

city: Beijing

first: wenhui

last: Zhao

address: 5 Yiheyuan Road, Haidian District

institute: Peking University

summary: Spatiotemporal regulation of chromatin replication (replication timing, RT） in eukaryotes is critical to maintain the genomic integrity. Here we focused on epigenetic mechanisms in rewiring genomic 3D conformation and replication timing. The results show that the novel lysine β-hydroxybutyrylation (Kbhb) modifications accelerates chromatin replication without inducing replication defects. This effect was mediated by the NAT10, a novel b-hydroxybutyryl-transferase, through regulating the association of NAT10 and CTCF with chromatin. Depletion of NAT10 and NAT10-mediated Kbhb dramatically reduce chromatin-bound NAT10 and CTCF, resulting in reorganization of genomic 3D conformation with enhanced trans- and cis-interaction in Hi-C matrix, with elevated proportion of A compartments, and with reorganized TADs boundaries. Moreover, reorganization of genomic 3D conformation contributes to rewire replication timing. These results support models in which NAT10-mediated β-hydroxybutyrylation coordinates genomic 3D conformation reorganization with replication timing alteration, and emphatically address the concept that epigenetic mechanisms reconcile genomic 3D conformation with replication timing.

overall_design: Hi-C sequencing was performed in two biological replicates on β-hydroxybutyrate (BHB) treated control (siCtrl) or NAT10 knock-down (siNAT10) U2OS cells.

first: Wenhui

first: Kun

last: Liu


title: Neutrophil immune profile guides spinal cord regeneration in zebrafish

city: Lisboa

first: Carmen

last: de Sena-Tomás

address: Av. Prof. Egas Moniz

country: Portugal

institute: Instituto de Medicina Molecular

summary: Spinal cord injury triggers a strong innate inflammatory response in both non-regenerative mammals and regenerative zebrafish. Neutrophils are the first immune population to be recruited to the injury site. Yet, their role in the repair process, particularly in a regenerative context, remains largely unknown. Here, we show that, promoting neutrophil inflammation resolution by inhibiting Cxcr4 boosts cellular and functional regeneration. Neutrophil-specific RNA-seq analysis reveals an enhanced activation state that correlates with a transient increase in tnf-a expression in macrophage/microglia populations. Conversely, blocking neutrophil recruitment through Cxcr1/2 inhibition diminishes the presence of macrophage/microglia at the injury site and impairs spinal cord regeneration. Altogether, these findings provide new insights into the role of neutrophils in spinal cord regeneration, emphasizing the significant impact of their immune profile on the outcome of the repair process.

overall_design:
To understand the biological mechanisms promoted by neutrophils that improve spinal cord regeneration upon Cxcr4 inhibition, we sorted mpx:GFP+ cells from the injury site of lesioned larvae and from the same region of age-matched uninjured controls and performed Bulk RNA sequencing analysis in AMD3100- and DMSO-treated samples.

Grant ID: 2022.02766.PTDC
Grant title: Improving Spinal Cord Regeneration by modulating Neutrophil inflammation: lessons from a Zebrafish perspective
Funding agency: Fundaçåo para a Ciência e Tecnologia (FCT), Portugal

first: Leonor

last: Rebola Lameira

first: Patricia

last: Naique Taborda

first: Sofia

last: de Oliveira

last: Saúde


title: Single-cell RNA and ATAC sequencing revealed distinct molecular characteristics of B cells in systemic lupus erythematosus and primary Sjogren's syndrome (scATAC-Seq)

first: zhang

last: jing

summary: Objective: Autoimmune diseases (ADs), such as systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS), are complex diseases with poorly understood pathogenetic mechanisms. Epigenetic changes are crucial regulatory elements in B-cell differentiation, and disruptions to these mechanisms may contribute to the development of ADs. We hypothesized that shared mechanisms may contribute to the pathogenesis of SLE and pSS by disrupting transcriptional circuits.

overall_design: B-cells were sorted by Flow cytometry, then entered into single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq). ArchR algorithm was used to integrate scRNA-seq and scATAC-seq data to investigate the transcriptome and chromatin accessibility of five B-cell subpopulations and their differentiation trajectories.

first: Zhaobing

last: Pan

first: Jiali

last: Zhang

first: Xiaojing

first: Qiaoshan

last: Yang

first: Ying

last: Yuan

first: Zhengwei

last: Zhu

first: Leilei

last: Wen

first: Fusheng

last: Zhou


title: Impact of Chronic Olanzapine Administration on Gene Expression in the Mouse Lateral Septum

city: Shanghai

first: Lixuan

last: Huang

address: 227 South Chongqing Road

institute: Shanghai Jiao Tong University School of Medicine

summary: To explore the effects of long-term olanzapine treatment on gene expression in the lateral septum of female mice, we conducted a study using female C57BL/6 mice fed with olanzapine-supplemented chow for 12 weeks. The treated mice exhibited significant weight gain and lipid alterations compared to the control group. Subsequently, RNA-seq was performed on the lateral septum brain regions of both the treated and control mice, followed by gene expression profiling to analyze the data.

overall_design: In this study, we investigated the impact of prolonged olanzapine administration on the lateral septum of female C57BL/6 mice. The mice were divided into two groups: the experimental group received chow supplemented with olanzapine for 12 weeks, while the control group was fed standard chow. After the treatment period, we observed significant differences in weight and lipid profiles between the two groups. RNA sequencing was then performed on the lateral septum brain regions of both groups to identify changes in gene expression profiles, providing insights into the molecular effects of long-term olanzapine exposure in female mice.

last: Sun

first: Peijun

last: Ju

first: Jianhua

last: Chen


title: Venetoclax Resistant Acute Lymphoblastic Leukemia is characterized by Increased Mitochondrial Activity and Can Be Overcome by Co-targeting Oxidative Phosphorylation

city: Ulm

first: Alexandra

last: Niedermayer

address: Eythstraße 24

country: Germany

institute: Ulm University Medical Center

summary: Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Adccordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation, strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL.

overall_design: To model VEN-resistance in BCP-ALL, we used the highly VEN-sensitive RS4;11 cell line and generated five lines by parallel exposure to increasing concentrations of VEN over time with five control lines exposed to corresponding concentrations of solvent . To elucidate mechanisms of VEN-resistance, transcriptome profiles of VEN-sensitive and insensitive lines were analyzed and compared (VEN-sensitive, N=5 and VEN-insensitive N=5). Analyses of the identified differentially regulated genes was done using gene set enrichment analysis (GSEA) .

first: Stefanie

last: Enzenmüller

first: Felix

last: Seyfried

first: Vera

last: Münch

first: Daniel

last: Tews

first: Pamela

last: Fischer-Posovsky

first: Ulrich

last: Rupp

first: Paul

last: Walther

first: Alexander

last: Groß

first: Hans

middle: A

last: Kestler

first: Klaus-Michael

last: Debatin

first: Lüder

middle: H

last: Meyer


title: Luteoloside inhibits proliferation, induces apoptosis and differentiation via β-catenin/c-Myc axis in acute myeloid leukemia

city: Wenzhou, CHINA

first: Xiaofang

last: Wang

state: Zhejiang

address: Shangcai Village, Nanbaixiang, Ouhai District

department: Department of Hematology

institute: The First Affiliated Hospital of Wenzhou Medical University

summary:
Background: Acute myeloid leukemia (AML) is the most common leukemia in adults, that has a poor prognosis due in part to low response to treatment and high recurrence. And drugs commonly used in conventional treatment strategies often cause serious side-effects.
Purpose: To investigate the high efficacy and low toxicity of luteoloside on AML and explore its mechanism of action.
Methods:The proliferation of AML cells treated with different concentrations of luteoloside was assessed by the CCK-8 assay and colony formation assay, and   apoptosis and differentiation were detected by flow cytometry. RNA-Seq analysis and western blotting were performed to explore the mechanism by which luteoloside exerted anti-AML effects. The patient-derived xenografts (PDX) model was established to assess anti-AML effect of luteoloside in vivo, and construction of an orthotopic AML mouse model explored the combination therapy of luteoloside and cytarabine.
Results: Herein, we found that luteoloside could dramatically impair the proliferation and clonogenic capacity of AML cells and induce S phase arrest associated with decrease of Cyclin D1 and increase of Cyclin A2. Furthermore, luteoloside induced apoptosis and promoted differentiation of AML cells. Activation of Wnt/β-catenin signaling by GSK3β inhibitor LiCl reversed the inhibitory effect of luteoloside on AML cell proliferation, apoptosis, and differentiation, due to β-catenin entering into nucleus and reducing the expression of c-Myc. Luteoloside significantly reduced the transplantation rate of primary AML cells in PDX model, and the combination of luteoloside and cytarabine showed a synergistic anti-AML effect in orthotopic AML model.
Conclusion: Our findings demonstrate that luteoloside inhibits proliferation, induces apoptosis and differentiation of AML cells via suppression of β-catenin/c-Myc axis, suggesting that luteoloside can be developed as a potential anti-AML drug.

overall_design: U937 cells were treated with luteoloside or DMSO for 72h and harvested for RNA-seq to investigate the mechanism of luteoloside on AML.

first: Yangya

last: Pan

first: Fanfan

last: Li

first: Shenghui

last: Zhang


title: Comparative Analysis of Gene Expression in AgN2a-4P Expanded B6 NK Cells Treated with Anti-TIM-3 Antibody

city: Madison

first: Aicha

last: Quamine

state: WI

address: 1111 highland ave

institute: University of Wisconsin Madison

summary: Gene expression patterns in IL-15 or IL-15/AgN2a-4P expanded B6 NK cells treated with anti-TIM-3 antibody or IgG2a isotype control were analyzed using the nCounter Mouse PanCancer Immune Profiling Panel. Prior to RNA processing, NK cells were purified using the AutoMACS pro-separator (Miltenyi Biotec, San Jose, CA). The treatment involved a 4-hour exposure to antibodies before gene expression analysis, aiming to elucidate differential expression profiles in response to anti-TIM-3 treatment compared to the control condition.

overall_design: Expanded B6 NK cells were cultured with IL-15/AgN2a-4P and treated with anti-TIM-3 antibody or IgG2a isotype control. NK cells were isolated using AutoMACS Pro, and RNA was extracted from purified cells. Gene expression was analyzed using qRT-PCR with specific primers targeting human and mouse genes.

first: Nicholas

middle: R

last: Mohrdieck

first: Chloe

last: King

first: Anastasia

last: Griggs

first: Jillian

last: Kline

first: Monica

last: Cho

first: Sean

middle: P

last: Rinella

first: Katharine

middle: E

last: Tippins

last: Bates

first: Lei

last: Shi

first: Longzhen

last: Song

last: Hess

first: Tyce

last: Kearl

first: Bryon

last: Johnson

first: Christian

last: Capitini


title: Soybean molasses increases subcutaneous fat deposition while reducing lipid oxidation in the meat of castrated lambs

city: São Carlos

first: Sergio

middle: A. G.

last: Pereira-Junior

state: Sao Paulo

address: Rod. Washington Luís km 235

department: Genetic and Evolution

country: Brazil

institute: Federal University of São Carlos

summary: This study aimed to evaluate the effect of including soybean molasses (SM) on performance, blood parameters, carcass traits, meat quality, fatty acid and muscle (longissimus thoracis) transcriptomic profiles of castrated lambs. Twenty Dorper × Santa Inês lambs (20.06 ± 0.76 kg BW) were assigned to a randomized block design, stratified by BW, with the following treatments: CON - 0 g/kg of SM and SM20 - 200 g/kg of SM on DM basis, allocated in individual pens. The diet consisted of 840 g/kg concentrate and 160 g/kg corn silage for 76 days, with the first 12 days as an adaptation period and the remaining 64 days on the finishing diet. The SM20 diet increased blood urea concentration (P = 0.03) while reduced glucose concentration (P = 0.04). Lambs fed SM showed higher subcutaneous fat deposition (P = 0.04) and higher subcutaneous adipocyte diameter (P < 0.01), in addition to reduced meat lipid oxidation (P < 0.01). Soybean molasses reduced the quantity of branched-chain fatty acids in longissimus thoracis (P = 0.05) and increased the quantity of saturated fatty acids (P = 0.01). In the transcriptomic analysis, 294 genes were identified as differentially expressed, which belong to pathways such as oxidative phosphorylation, citric acid cycle, and monosaccharide metabolic process. In conclusion, diet with SM increased carcass fat deposition, reduced lipid oxidation, and changed the energy metabolism, supporting its use in ruminant nutrition.

overall_design: Crossbreed (Dorper X Santa Inês) castrated lambs (n=20) were randomly divided into two experimental groups and subjected to two experimental diets: control (CON, n=10) or 20% inclusion of soybean molasses (SM20, n=10). Briefly, the samples of longissimus thoracis was collected to physcial and chemical meat analysis, as shear force, intramsucular fat, lipid oxidation and others. Upon slaughter the samples of fresh longissimus thoracis was collected and storage in liquid N2 and further -80°C. For the RNA-Seq analysis, four lambs from each CON and SM20 diet were randomly selected.

first: Sérgio

last: Pereira Jr

first: Ricardo

last: Perecin Nociti

first: Marcos

last: Chiaratti

first: Marco

middle: T

last: Almeida


title: Single cell RNA-seq of BHK-21 cells carrying alphaviral RNA replication system

status: Public on Jul 07 2024

first: Yihan

last: Lin

address: No.5 Yiheyuan Road, Haidian

summary: Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. Toinvestigatetheexpressionheterogeneityofself-replicatingRNAsinrepRNA-v4cells,weperformedsingle-cellRNA-seqanalysisusingthe10xGenomicssequencingmethod.Ouranalysisofthesingle-cellRNA-seqprofilingdatarevealedarelativelyuniformexpressionpatternofself-replicatingRNAswithinrepRNA-v4cells.

overall_design: In this study, 10 μg of repRNA-v4 RNA was electroporated into 4 million BHK-21 cells expressing nsP4 in trans. The cells were cultured for ~3 weeks in the presence of 10 μg/ml of puromycin. ~20000 cells were used for the preparation of 10x Genomics 5' gene expression libraries. Subsequently, RNA-seq was performed on an Illumina's high-throughput sequencing platform NovaSeq 6000. Cleaned reads were demultiplexed, barcode processed, gene counted using the Cell Ranger software v4.0.0. against the Mesocricetus auratus genome (GCF_017639785.1_BCM_Maur_2.0_genomic.fna) and the genome of alphaviral RNA replication system. The genome annotation file (GCF_017639785.1_BCM_Maur_2.0_genomic.gff) was downloaded from NCBI and modified to include annotation about the genome of the alphaviral RNA replication system. Subsequent analyses were performed using the Seurat package with default or recommended parameters.

first: Liang

last: Ma


title: Microglia aging advances through intermediate states that drive inflammatory activation and cognitive decline

first: Jeremy

last: Shea

summary: TGFB-signaling is important for the establishment of microglia homeostasis; however, it's role in aging has remained vague. We found that Tgfb1 and TGFB signaling is altered during hippocampal microglia aging. To investigate the effects that microglia-derived Tgfb1 has on microglia aging, we performed scRNA-Seq on Cd11b+ cells from the hippocampus of mature mice where Tgfb1 had been ablated from Cx3cr1 cells post-maturity.

overall_design: scRNA-Seq of Cx3cr1-CreERT Tgfb1 cKO Cd11b+ cells from mature mouse hippocampi

first: Saul

last: Villeda


title: The role of the gene Mest during mouse digit tip regeneration

city: Boston

first: Jessica

last: Lehoczky

state: MA

address: 60 Fenwood Rd.

department: Orthopedic Surgery

institute: Brigham and Women's Hospital

summary: The distal mouse digit tip undergoes complex tissue regeneration following amputation, a process facilitated by the formation of a cellular structure called the blastema. Through single-cell RNA sequencing of the blastema, we identified the gene Mest (mesoderm specific transcript) highly expressed in a subset of blastemal fibroblasts. To determine if Mest expression is necessary for mouse digit tip regeneration, we analyzed Mest wildtype (WT) versus homozygous knockout (KO) post amputation digits. Through single-cell sequencing and FACS analysis of WT and KO regenerating tissues, we determined that this phenomenon was due to a prolonged immune response in the KO digits.

overall_design: Cohorts of adult Mest genetic knock-out (KO) mice and Mest wild-type (WT) mice underwent digit tip amputation; 12 days post-amputation, digit tip blastemas were collected and used for single cell RNA sequencing and analysis.


title: WI-38 cell line as model for myofibroblast and lipofibroblast switch

city: Giessen

first: Marek

last: Bartkuhn

state: Hessen

address: Aulweg 132

department: Biomedical Informatics and Systems Medicine

institute: Justus-Liebig-University Giessen

summary: Background: Myofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methods: using WI-38 cells, MYF differentiation was triggered using TGF-β1 treatment and LIF differentiation using Metformin treatment. We analyzed the LIF to MYF and MYF to LIF differentiation by pre-treating the WI-38 cells with TGF-β1 or Metformin first, followed by treatment with Metformin and TGF-β1, respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IFP patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. Results: WI-38 and LF-IPF display similar phenotypical and gene expression responses to TGF-β1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 and LF-IPF treated with TGF-β1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-β1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the LIF to MYF reversible switch using WI-38 cells. Conclusions: WI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research

overall_design: Comparative gene expression profiling of RNA-seq data for  the fibroblast cell line WI-38 (no-treated, TGFB1-treated, MET-treated and the reversion treatment TGFB1- and TGFb1 followed by MET treatment).

first: Saverio

last: Bellusci

first: Stefano

last: Rivetti

first: Manuela

last: Marega

first: Esmeralda

last: Vásquez-Pacheco


title: Endocrine and transcriptome changes associated with growth and differentiation of Atlantic (Salmo salar L.) salmon testes and immune activity of adipose tissue

city: Aas

first: Aleksei

last: Krasnov

address: Osloveien 1

department: Fish Health

country: Norway

institute: Nofima AS

summary: The study compared salmon reared at different light regiments (groups A, B and D) and fed with feeds with high and low fat (groups D and E). Analyses in pituitaries and testes addressed problem of early sexual maturation in aquaculture. It is marked by dramatic endocrine changes and rapid growth of the testes, resulting in an increase in the gonad somatic index.  The study examined the association of plasma sex steroids, pituitary and testicular gene expression with gonadal growth. The key characteristic is developmental stage of testis, from I to IV. In addition, the effect of feeds with high and low fat levels on adipose tissue was compared. The key finding is markedly different immune activity in groups D (high fat) and E (low fat).

overall_design:
Salmon post smolts were kept in tanks with brackish water within a flow-through system, with an average salinity of 20 ± 0.4 ppt and mean temperature of 12.1 ± 0.2°C. Different light treatments (constant, increasing and decreasing) were tested during a period of 18 weeks. Sampling was condcuted at the end of this period to asses early sexual maturation traits. This study focused on changes related do different levels of maturity.
Various light treatments were explored, including continuous exposure (Group A), increasing exposure (Group B), and decreasing exposure (Groups C, D and E) were investigated on growth and sexual maturation, sampling was performed after 18 weeks to assess gonadal growth and blood steroid hormone levels. All groups were provided with the same feed, except for Group E, which received a low-fat diet.

first: Skjold

last: Vetle

first: Dessen

last: Jens-Erik

first: Krasnov

last: Aleksei


title: Inactivation of Pseudomonas putida KT2440 pyruvate dehydrogenase relieves catabolite repression and improves the usefulness of this strain as a tool in biotechnology

city: Cantoblanco

first: Juan

middle: Carlos

last: Oliveros

state: Madrid

address: Darwin 3

country: Spain

institute: CNB, CSIC

summary: Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acids cycle. We analysed how the lack of PDH affects Pseudomonas putida metabolism. Inactivating PDH generated a strain that can no longer use compounds whose assimilation converges into pyruvate, including sugars and several amino acids. Compounds generating acetyl-CoA supported growth. Interestingly, inactivation of PDH led to loss of the carbon catabolite repression (CCR) effect that inhibits the assimilation of non-preferred compounds when other preferred ones are also present. P. putida can degrade many aromatic compounds, most of which generate acetyl-CoA, and is a useful bacterium for biotransformation and bioremediation processes. However, the genes involved in these metabolic pathways are frequently inhibited by CCR when substrates such as glucose or mixtures of amino acids are also present. Our results show that the PDH-null strain could efficiently degrade aromatic compounds in the presence of other preferred substrates, a condition in which the wild type strain could not mineralise them. Since lack of PDH limits the assimilation of many sugars and amino acids, and relieved CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that imply growth with mixtures of preferred substrates and aromatic compounds.

overall_design: We compared the transcriptome of the bacteriaum Pseudomonas putida KT2440 with that of a mutant derivative in which the aceE gene, coding for a component of the pyruvate dehydrogenase, had been inactivated.

first: Renata

last: Moreno

first: Luis

last: Yuste

first: Gracia

last: Morales

first: Fernando

last: Rojo


title: DNA methylation demonstrates bronchoalveolar cell senescence in people living with HIV: An observational cohort study

city: Calgary

first: Ana

middle: Isabel

last: Hernandez Cordero

state: Alberta

address: 303 13th Avenue SW, unit 1004

country: Canada

institute: Heart Lung Innovation Centre

type: Methylation profiling by genome tiling array

summary: Background: Age-related comorbidities remain a burden in People living with HIV (PLWH). DNA methylation may be a link between HIV, aging, and the increased risk of lung comorbidities. Here, we investigate whether bronchoalveolar lavage (BAL) cells of PLWH demonstrate epigenome wide disruptions and advanced epigenetic aging.  Methods: BAL cell DNA methylation profiles from 25 PLWH and 16 uninfected controls were tested for differential methylation of transposable elements Alu and LINE-1, a marker of aging. We used a weighted gene correlation network analysis to identify co-methylation networks associated with HIV and age. We tested the effect of HIV on DNA methylation using a robust linear model. An enrichment analysis was conducted to identify differentially methylated pathways (false discovery rate <0.10).  Results: The BAL cells of PLWH were marked by global hypomethylation in both Alu and LINE-1 elements. Six co-methylated CpG networks were identified that were significantly associated with age; of these, the red module was significantly differentially methylated in PLWH and enriched pathways associated with HIV, Ras signaling, T cell receptors, and bacterial invasion of epithelial cells. We identified 6,428 differentially methylated sites associated with HIV.  Conclusions: We have shown here for the first time that alterations in the DNA methylation of BAL cells (mainly macrophages) in the lung HIV show in a pattern of advanced aging. In conjunction with our findings in blood cells and airway cells, this study strongly support that HIV may contribute to increase the risk of lung comorbidities through the epigenetic regulation of aging.

overall_design: 41  samples profiles for Bronchoalveolar lavage DNA methyaltion

first: Janice

last: Leung

first: Don

last: Sin


title: Menin deficiency induces autism-like behaviors by regulating Foxg1 transcription and participates in Foxg1-related encephalopathy

city: XiaMen

first: Honghua

last: zheng

state: FuJian

address: XiaMen University xiang’an campus

institute: Institute of Neuroscience xiamen university

summary: FOXG1 syndrome is a developmental encephalopathy with a high phenotypic variability, which results from FOXG1 mutations. However, the upstream transcriptional regulation of Foxg1 expression remains unclear. Here we report that both deficiency and overexpression of Men1 (protein: menin, a pathogenic gene of MEN1 syndrome) result in autism-like behaviors, including social defects, increased repetitive behaviors and cognition impairments. We employed multifaceted transcriptome analyses and found that Foxg1 signaling is mostly altered in Men1 deficiency mice, through its regulation over Alpha Thalassemia/Mental Retardation Syndrome X-Linked (Atrx) factor. Atrx recruits menin to bind to the transcriptional start region of Foxg1 and mediates the regulation of Foxg1 expression by H3K4me3 modification. Notably, the described changes in menin deficient mice were rescued by over-expression of Foxg1, leading to normalized spine growth and hippocampal synaptic plasticity. Collectively, these results indicate a putative role of menin in maintaining Foxg1 expression, and menin signaling may serve as Foxg1-related encephalopathy therapeutic targets.

overall_design: CUT&Tag was performed with Hyperactive In-Situ ChIp Library Prep Kit forlllumina (pG-Tn5) (TD901, Vazyme Biotech) according to the manufacturer'sinstructions. In brief, primary neurons were collected and bounded to Concanavalin A-coated beads. Subsequently, cells were resuspended in antibodybuffer and incubated with primary antibodies against menin antibody (A300-105A; Bethyl) and secondary antibodies in order. The samples were incubated with pA-Tn5 transposase. Aftertransposon activation and tagmentation, DNA was isolated, amplified, and purified to construct library, The library for sequencing was constructed and VAHTS DNAClean Beads (N411, Vazyme Biotech) were used for purification steps. The librarywas quantified with VAHTS Library Quantification Kit for lllumina (VazymeBiotech) and sequenced on an lllumina novaseq 150PE. The sequencing was carried out by Novogene (Novogene, Beijing, China).

first: Jie

last: Zhang

first: Shuzhong

last: Wang

first: Kai

last: zhuang


title: Downregulation of circular RNA_102928 contributes to lung inflammation and epithelial apoptosis in smoke inhalation injury

first: yong

last: Jiang

address: No.168, Changhai Road, Yangpu District

department: Department of Burn Surgery

institute: Changhai Hospital, The People's Liberation Army Naval Medical University

summary:
Smoke inhalation injury is the leading cause of mortality in patients with burns. Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs implicated in various diseases. However, their specific involvement in smoke inhalation injury remains poorly understood.
High-throughput microarray analysis was used to identify differentially expressed circRNAs in plasma samples from patients with smoke inhalation injury compared to healthy controls. Five circRNAs were initially identified, and three were validated by quantitative real-time PCR (qRT-PCR). Bioinformatic analysis was used to establish circRNA–miRNA–mRNA networks. The predicted circRNA-miRNA-mRNA axes were further investigated in a rat inhalation injury model and in A549 cells. Microarray analysis revealed 65 differentially expressed circRNAs, of which three (hsa_circRNA_102928, hsa_circRNA_102059, and hsa_circRNA_101115) were confirmed by qRT-PCR, with one downregulated and two upregulated circRNAs. Bioinformatic analysis predicted 11 potential circRNA-microRNA-mRNA axes associated with smoke inhalation injury development, including the circRNA_102928/let-7f-2-3p/MDM1 axis. In a smoke inhalation injury rat model, the downregulation of circRNA_102928 and MDM1 mRNA, as well as the upregulation of let-7f-2-3p expression, coincided with prominent inflammation in the lungs. Overexpression of circRNA_102928 dramatically reduced let-7f-2-3p and significantly increased MDM1 mRNA levels in A549 cells. Furthermore, circRNA_102928 overexpression notably mitigated tumor necrosis factor-alpha and interleukin 1 beta expression and attenuated apoptosis in A549 cells. This study highlighted the role of the circRNA_102928/let-7f-2-3p/MDM1 axis in mediating lung inflammation and epithelial apoptosis in wood smoke inhalation injuries.

overall_design: Plasma samples were collected from five patients with smoke inhalation injuries on the third day who were admitted to the hospital within 24 hours after injury. Five plasma samples were collected from sex- and age-matched healthy volunteers as controls.

first: yu

last: Sun


title: A MOZ-TIF2 leukemic mouse model displays KAT6 inhibitor-sensitive H3K23 propionylation and dysregulation of a set of highly active developmental genes [ChIP-seq]

first: Mitzi

middle: I

last: Kuroda

address: 77 Avenue Louis Pasteur

department: Genetics

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

summary: Aberrant regulation of chromatin modifiers is a common occurrence across many cancer types, and a key priority is to determine how specific alteration of these proteins, often enzymes, can be targeted therapeutically. MOZ, a histone acyltransferase, is recurrently fused to coactivators CBP, p300, and TIF2 in cases of Acute Myeloid Leukemia (AML). Using either pharmacological inhibition or targeted protein degradation in a mouse model for MOZ-TIF2-driven leukemia, we show that KAT6 (MOZ/MORF) enzymatic activity and the MOZ-TIF2 protein are necessary for indefinite proliferation in cell culture. Interestingly, MOZ enzymatic activity is selectively required in MOZ-TIF2 cells, as murine MLL-AF9 leukemic cells show no sensitivity to the WM-1119 MOZ inhibitor. MOZ-TIF2 directly regulates a small subset of genes encoding developmental transcription factors, augmenting their high expression. Transcription levels of these genes positively correlate with enrichment of histone H3 propionylation at lysine 23 (H3K23pr), a recently appreciated histone acylation associated with gene activation. Unexpectedly, we also show that MOZ-TIF2 and MLL-AF9 regulate transcription of largely non-overlapping gene sets, and their cellular models exhibit distinct sensitivities to multiple small molecule inhibitors directed against AML pathways. This is despite the shared genetic pathways of wild-type MOZ and MLL. Overall, our data provide insight into how aberrant regulation of MOZ contributes to leukemogenesis. We anticipate these experiments will inform future work identifying targeted therapies in the treatment of AML and other diseases involving MOZ-induced transcriptional dysregulation.

overall_design:
Chromatin immunoprecipitation followed by DNA sequencing of H3K23pr and H3K23ac in mouse LSK cells carrying a human MOZ-TIF2 cDNA transgene treated with either DMSO or 1 uM WM-1119 for 1 hour
Chromatin immunoprecipitation followed by DNA sequencing of H3K23ac in mouse LSK cells carrying a human MOZ-TIF2 cDNA transgene

first: Anne

last: Smolko


title: Binding specificities of transcription factors of the oomycete Phytophthorainfestans reflect conserved and divergent evolutionary patterns and function

city: Riverside

first: Howard

last: Judelson

address: Genomics Building

department: Microbiology and Plant Pathology

institute: University of California-Riverside

summary: Identifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a group of microbes that includes many important plant and animal pathogens. Here we describe the use of protein-binding oligonucleotide microarrays (PBMs) to define the DNA-binding preferences of 14 families of TFs from the oomycete Phytophthora infestans, which causes late blight of potato and tomato. DNA motifs obtained from the PBMs for representatives of each major TF family were validated by electrophoretic mobility shift assays (EMSA) or ChIP-seq. Consistent with the large evolutionary distance of oomycetes from traditional models, only some of the P. infestans DNA-binding preferences resembled those of TFs from human and plants. Some families from P. infestans included clusters with canonical targets and others with novel targets. Paralogs having similar binding preferences often displayed distinct patterns of expression, suggesting functional divergence. Many TFs were predicted to either drive stage-specific expression or serve as general activators based on the representation of their binding sites within developmentally-regulated promoters. One such prediction was confirmed using a reporter gene assay. Our data thus provide a basis for understanding transcriptional regulation in P. infestans by linking TFs with their targets on a genome-wide level.

overall_design: Protein binding microarray (PBM) experiments were performed for Phytophthora infestans proteins. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006.

first: Nguyen

middle: N

last: Vo

first: Ally

last: Yang

first: Wiphawee

last: Leesutthiphonchai

first: Yulong

last: Liu

first: Timothy

last: Hughes


title: Oxytocin and Vasopressin Gene Expression in the Brain as Potential Biomarkers for Cannabidiol Exposure and Anti-inflammatory Efficacy

city: Mississippi State

first: Barbara

last: Kaplan

state: MS

address: 240 Wise Center Drive

department: Comparative Biomedical Sciences

institute: Mississippi State University

summary: Over the past few years there has been an increased interest in cannabidiol (CBD) for general use as well as alternative therapeutic for various diseases. Although CBD has only been FDA approved for seizures under the oral formulation, Epidiolex®, the public has used various unregulated forms of CBD to treat a variety of ailments from pain to anxiety and insomnia. Moreover, an increasing subset of CBD advocates take CBD daily to prevent inflammation. This expanded use of CBD strongly suggests that more research is necessary to elucidate the safety and efficacy of CBD and determine the mechanisms by which it acts. Thus, we conducted two separate studies. In the first, RNA sequencing (RNA-Seq) analysis of brains of female mice undergoing experimental autoimmune encephalomyelitis (EAE) in the presence and absence of CBD was conducted in order to identify potential genes that mediated CBD’s neuroprotective effects. In the second, we assessed some of the same genes in male and female mice treated with CBD in the absence of an immune stimulus. Together these data showed that CBD modestly increased oxytocin (Oxt) and vasopressin (Avp) gene expression in the brains of mice, regardless of whether there was active inflammation, suggesting they might serve as biomarkers for CBD exposure, and perhaps, anti-inflammatory efficacy.

overall_design: Mice were anesthetized using 3% isoflurane and EAE was induced by subcutaneously injecting the MOG35-55 peptide emulsion. The emulsion was prepared with vigorous mixing for 30 min of a solution containing 100 µg MOG35–55 peptide, 500 µg HKMT, and CFA. Each mouse received 100 µL emulsion delivered in 4 sites (2 at the hips, 2 at the shoulders; 25 µL per site). Control mice received 15 µL saline (SAL) at each site. Twenty-four hours after EAE induction, mice were dosed orally with 75 mg/kg CBD in 100µL of corn oil (CO) or 100µL CO for five days. Mice were observed over an 18-day period, and scores were given based on the following scale: 0-Asymptomatic; 0.5-Flaccid tail; 1-Hindlimb paresis/waddling; 1.5-Waddling gait; 2-Unable to prevent being placed in dorsal recumbency; 2.5-Hindlimb dragging; 3-Single hindlimb paralysis; 3.5-Single hindlimb paralysis with other hindlimb dragging; and 4-Complete hindlimb paralysis. Any score above 0 was considered symptomatic. Mice were never allowed to progress past a score of 4 for animal welfare purposes. Scores and samples analyzed were from two, independent cohorts.  Mice were divided into three treatment groups: SAL/CO (control),  EAE/CO, and EAE/CBD. SAL/CO mice did not show any clinical signs and were not included in any downstream analyses. For all results and discussion of our findings, “EAE” will refer to active immunization with MOG in CFA supplemented with HKMT (and no administration of PTx).

first: Frodella

last: Christa M.

first: Pruett

last: Stephen B.

first: Ross

last: Matthew K.

first: Kaplan

last: Barbara L F.


title: A MOZ-TIF2 leukemic mouse model displays KAT6 inhibitor-sensitive H3K23 propionylation and dysregulation of a set of highly active developmental genes [RNA-seq]

overall_design:
To investigate how MOZ-TIF2 alters hematopoietic transcription, we altered MOZ/MORF and MOZ-TIF2 function through MOZ inhibition via WM-1119 treatment or MOZ-TIF2 degradation through dTAG13 treatment. We extracted nascent RNA or mRNA from mouse LSK cells containing either MOZ-TIF2 or MLL-AF9 and performed differential expression analysis comparing treated vs DMSO (control) cells
mRNA was extracted from mouse LSK cells carrying either an MLL-AF9 or MOZ-TIF2 transgene. Cells were treated with either DMSO or 1 uM WM-1119 for 1 hour.


title: Single cell transcriptome analysis identified a unique neutrophil type associated with Alzheimer’s Disease

first: Xiaolin

last: Zhang

state: Shanghai

address: Humin Road 3210

institute: Shanghai Mental Health Center

summary: Neutrophils play essential role in Alzheimer’s disease (AD) pathology, however the extent of heterogeneity remains a poorly explored question in light of developing novel therapies targeting this cells. Here, we investigate the population structure of neutrophils purified from AD mice peripheral blood samples. Using single cell RNA sequencing, we comprehensively map neutrophil populations and find some subsets are specially enriched in AD mice. We describe a special cluster closely associated with AD disease and identify marker. Flow cytometry and immunohistochemical staining of mice brain slices confirmed the subcluster. The unique neutrophil-type has the potential to promote AD development, which may have important implications for future diagnosis and treatment.

overall_design: Single-cell RNA sequencing characterization of neutrophils of peripheral blood in Alzheimer disease mice.

first: Guiqin

last: He

first: Jin

last: Li


title: Podocytes from Hypertensive and Obese Mice Acquire an Inflammatory, Senescent and Aged Phenotype

city: Cleveland

first: Oliver

last: Wessely

state: OH

address: 9500 Euclid Avenue/NC10

department: Cardiovascular and Metabolic Sciences

institute: Cleveland Clinic Foundation

summary: Patients with hypertension or obesity can develop glomerular dysfunction characterized by injury and depletion of podocytes. To better understand the molecular processes involved, young mice were treated with either deoxycorticosterone acetate (DOCA) or fed a high-fat diet (HFD) to induce hypertension or obesity, respectively. The transcriptional changes associated with these phenotypes were measured by unbiased bulk mRNA-sequencing of isolated podocytes from experimental models and their respective controls. Key findings were validated by immunostaining. In addition to a decrease in canonical proteins and reduced podocyte number, podocytes from both hypertensive and obese mice exhibited a sterile inflammatory phenotype characterized by increases in NLRP3 inflammasome, protein cell death-1, and Toll-Like Receptor pathways. Finally, although the mice were young, podocytes in both models exhibited increased expression of senescence and aging genes, including genes consistent with a senescence-associated secretory phenotype. However, there were differences between the hypertension- and obesity-associated senescence phenotypes. Both show stress-induced podocyte senescence characterized by increased p21 and p53. Moreover, in hypertensive mice, this is superimposed upon age-associated podocyte senescence characterized by increased p16 and p19. These results suggest that senescence, aging, and inflammation are critical aspects of the podocyte phenotype in experimental hypertension and obesity in mice.

overall_design: C57BL/6J mice were separated into two groups, (i) treatment with Deoxycorticosterone Acetate Salt (DOCA) or (ii) fed a high fat diet to induce obesity (HFD) and each group had its separate set of controls. Kidneys were removed from the mice at 5.5 months in the case of the DOCA treatment and 7 months in the case of HFD feeding. Podocytes we isolated by MAC sorting and injury and senescence were assessed with mRNA sequencing.

first: Sierra

last: McKinzie

first: Natalya

last: Kaverina

first: R

last: Schweickart

first: Christopher

last: Chaney

first: Jeffrey

middle: W

last: Pippin

first: Stuart

last: Shankland


title: DREAM On, DREAM Off: A Review of the Estrogen Paradox

city: Edmonton

first: Judith

last: Hugh

address: 87 ave and 114 st

department: Laboratory Medicine and Pathology

institute: University of Alberta

summary: It is generally assumed that all estrogen receptor positive (ER+) breast cancers proliferate in response to estrogen and therefore examples of estrogen-induced regression of ER+ cancers are paradoxical. This review reexamines the estrogen regression paradox for the Luminal A subtype of ER+ breast cancers. The proliferative response to estrogen is shown to depend on the level of ER. Mechanistically, a window of opportunity study of pre-operative estradiol suggested that with higher levels of ER, estradiol could activate the DREAM-MMB (Dimerization partner, Retinoblastoma-like proteins, E2F4, and MuvB – MYB-MuvB) pathway to decrease proliferation. The response of breast epithelium and the incidence of breast cancers during hormonal variations that occur during the menstrual cycle and at the menopausal transition respectively suggest that a single hormone, either estrogen, progesterone or androgen could activate the DREAM pathway leading to reversible cell cycle arrest. Conversely, the presence of two hormones, could switch the DREAM-MMB complex to a pro-proliferative pathway. Using publicly available data, we examine the gene expression changes after aromatase inhibitors and ICI 182,780 to provide support for the hypothesis. This review suggests that it might be possible to integrate all current hormonal therapies for Luminal A tumors within a single theoretical schema.

overall_design: To investigate estrogen (E2)-induced changes in gene transcription in luminal breast cancer cells with high (MCF7-ER) and low (MCF7-EM) estrogen receptor expression.

middle: C

first: Lacey

middle: S

last: Haddon

first: John

last: Maringa Githaka


title: RNA-Seq experiment of primary hepatic stellate cells (HSCs) and LX-2 cell line treated with TGFb

city: Bethesda

first: Haiming

last: Cao

state: MD

address: 9000 Rockville Pike

department: NHLBI

institute: NIH

summary: Liver fibrosis stands as the most prominent predictor of overall mortality in non-alcoholic steatohepatitis (NASH). The fibrotic liver features excessive deposition of extracellular matrix (ECM), primarily produced from "activated" hepatic stellate cells (HSCs). Whereas targeting HSC in fibrosis therapeutics shows promise, the current identification of human genetic regulators driving HSC activation is far from complete. This knowledge gap largely emanates from the limited understanding of the vast array of long non-coding RNAs (lncRNAs). Analyzing differentially regulated human lncRNAs under various regulatory patterns often provides the most practical means to inform their function. To get human lncRNA regulators in driving HSC activation, we cultured both primary human stellate cells and LX-2 cells. Then, we treated them with TGFb to mimic the activation process in vitro. Differentially expressed lncRNAs can be obtained from RNAseq results, which may guide the characterization of lncRNAs relevant to HSC activation.

overall_design: Human primary HSCa were thawed in Human Stellate Cell Growth Media (Lonza) and seeded at a density of 4,000-5,000 cells/cm2 on a Collagen I coated plate using the growth media above. The medium is changed every other day. Cryopreserved, immortalized LX-2 cells were obtained from Millipore. The standard procedure was followed by the manufacturer's instructions for thawing, splitting, and freezing of the cell line. For TGFβ treatment, 10ng/ml of reconstituted TGFβ was added to the media. Cells were treated for 24 hours before collecting for RNA isolation.

first: Yonghe

last: Ma


title: Influence of Staphylococcus aureus biofilm infection phase on functional composition of host neutrophils

city: Hannover

first: Rodrigo

last: Gutierrez Jauregui

state: Lower saxony

address: Carl-neuberg Str. 1

department: Immunology

institute: Hannover Medical School

summary: Nosocomial infections resulting from growing biofilms on the surface of indwelling medical devices represent a major therapeutic challenge in an aging population. In this context, current models have so far not addressed the synergy between a developing biofilm and the host’s immune response. Here we employed a mouse model for implant-associated infection from Staphylococcus aureus biofilms and, through functional assays, next generation single cell sequencing and spectral flow cytometry, observed a direct influence of the developing biofilms in the phenotype of tissue infiltrating neutrophils over the course of infection. Our results allowed us to differentiate neutrophil subpopulations, identifying some which may be protective of the biofilm and which could be the target for future therapies.

overall_design: skin infiltrates, from peri-implant tissue after biofilm implant-associated infection, were enzymatically isolated and sorted by Fluorescence-activated cell sorting (FACS) to the prescence of CD45 signaland analyzed using scRNA-seq.

first: Siegfreid

last: Weiss

first: Reinhold

last: Förster


title: House Dust-Derived Mixtures of Organophosphate Esters Alter the Phenotype, Function, Transcriptome, and Lipidome of KGN Human Ovarian Granulosa Cells

city: Ottawa

first: Matthew

last: Meier

state: Ontario

address: 251 Sir Frederick Banting Driveway

department: Health Canada

institute: Government of Canada

summary: Background: Organophosphate esters (OPEs), used as flame retardants and plasticizers, are present ubiquitously in the environment. Previous studies suggested that exposure to OPEs may be detrimental to female fertility in humans. However, no experimental information is available on the effects of OPE mixtures on ovarian granulosa cells, which play essential roles in female reproduction.  Objectives: The goal of this study was to elucidate the effects of OPE mixtures representative of those found in Canadian house dust on ovarian granulosa cells. Methods: We used high-content imaging to investigate the effects an environmentally relevant OPE mixtures on KGN human granulosa cell phenotypes. Perturbation to steroidogenesis was assessed using ELISA and qRT-PCR. A high-throughput transcriptomic approach, TempO-Seq, was used to identify transcriptional changes in a targeted panel of genes. Effects on lipid homeostasis were explored using cholesterol assay and global lipidomic profiling. Results: OPE mixtures with distinct structural characteristics altered multiple phenotypic features of KGN cells with different potencies. The mixtures increased basal production of steroid hormones; this was mediated by significant changes in the expression of critical transcripts involved in steroidogenesis. Further, the total-OPE mixture disrupted cholesterol homeostasis and the composition of intracellular lipid droplets. Discussion: Exposure to house dust-derived mixtures of OPEs may adversely affect female reproductive health by altering a multitude of phenotypic and functional endpoints in granulosa cells. This study provides novel insights into the mechanisms of actions underlying the toxicity induced by OPE mixtures and highlights the need to examine the effects of human relevant chemical mixtures.

overall_design: We used a high-throughput transcriptomic approach, TempO-Seq, to examine the expression of 3386 probes covering 2730 genes in the human S1500+ gene set in KGN cells following exposure to a house dust-derived OPE mixture for 48 hrs.

first: Xiaotong

last: Wang

first: Andrea

last: Rowan-Carroll

middle: J

first: Carole

middle: L

last: Yauk

first: Michael

middle: G

last: Wade

first: Barbara

middle: F

last: Hales

first: Bernard

last: Robaire


title: Inhibition of NLRP3 Inflammasome Activation by A20 through Modulation of NEK7

city: 成都

first: Hanwen

last: Li

state: 四川

address: 四川省成都市武侯区人民南路四川大学华西校区

institute: 四川大学华西口腔医学院

summary: The NLRP3 inflammasome, a pivotal component of innate immunity, has been implicated in various inflammatory disorders. The ubiquitin-editing enzyme A20 is well known to regulate inflammation and maintain homeostasis. However, the precise molecular mechanisms by which A20 modulates the NLRP3 inflammasome remain poorly understood. Here our study revealed that macrophages deficient in A20 exhibit increased protein abundance and elevated mRNA level of NIMA-related kinase 7 (NEK7). Importantly, A20 directly binds with NEK7, mediating its K48-linked ubiquitination, thereby targeting NEK7 for proteasomal degradation. Our results demonstrate that A20 enhance the ubiquitination of NEK7 at K189 and K293 ubiquitinated sites, with K189 playing a crucial role in the binding of NEK7 to A20, albeit not significantly influencing the interaction between NEK7 and NLRP3. Furthermore, A20 disrupts the association of NEK7 with the NLRP3 complex, potentially through the OTU domain and/or synergistic effect of ZnF4 and ZnF7 motifs. Significantly, NEK7 deletion markedly attenuates the activation of the NLRP3 inflammasome in A20-deficient conditions, both in vitro and in vivo. This study uncovers a new mechanism by which A20 inhibits the NLRP3 inflammasome.

overall_design: To investigate the role of A20 in macrophage, we established A20ko iBMDM celll ine. WT iBMDM and A20ko iBMDM were performed high-throughput RNA sequencing


title: Innate immune activation modulates contractile and electrical dysfunction in engineered heart tissue models fordesmoplakin(DSP)cardiomyopathy.

city: Chicago,

first: Daniel

last: Selgrade

state: IL

address: 303 E. Superior Street

institute: Northwestern University

summary: Desmosomes are specialized cell-cell junctions critically important in the heart, where their disruption promotes arrhythmogenic cardiomyopathy (ACM). Individuals carrying truncating variants in the desmosomal gene desmoplakin (DSP) often show a prominent inflammatory component in the absence of known antigenic stimuli, termed “hot phase” arrythmia. To assess the role of innate immune activation in DSP-cardiomyopathy, we generated engineered heart tissues (EHTs) from two patients with heterozygous DSP truncation variants (DSPtv, p.E1597X and p.R1951X). We also generated EHTs with homozygous DSP deletion (DSP-/-) for modeling. DSP-/- EHTs revealed impaired force production and reduced conduction velocity compared to isogenic controls, features consistent with ACM. RNA sequencing and protein characterization demonstrated a dramatic increase in innate immune activation in DSP-/- EHTs. Similarly, patient-derived DSPtv EHTs show enhanced sensitivity to inflammatory stimuli seen as reduced contractility and greater cytokine release. Inhibition of NFκB signaling improved baseline force production and mechanical response to strain in DSPtv EHTs, indicating that anti-inflammatory modulation may improve mechanical function in DSP deficient hearts. Furthermore, genomic correction of p.R1951X using adenine base editing reduced inflammatory biomarkers in cultured EHTs. Taken together, these data identify that DSPtv promote innate immune activation, creating an arrhythmogenic substrate in the concealed phase of ACM.

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design:
To investigate the gene-specific disease processes of desomsomal proteins in ACM, we performed bulk RNA-seq on neonatal rat ventricular myocytes (NRVMs) treated with siRNA to knockdown expression of Desmoplakin (Dsp), Plakoglobin (Jup) and Plakophilin-2 (Pkp2). We then performed differential expression and gene ontology analysis.
We then generated engineered heart tissues (EHTs) from human induced pluripotent stem cells (hiPSC) that contained homozygous deletion of DSP (DSP-/-) and performed bulk RNA-Seq alongside isogenic controls (DSP+/+) to identify differential gene expression in an alternate genetic model for severe ACM. We then performed differential expression and gene ontology analysis as well as motif enrochment analysis among differentially expressed genes.

first: Adi

last: Dubash


title: Chromatin Remodeling Drives Immune-Fibroblast Cell Communication in Heart Failure [scRNA-Seq]

city: San Francisco

first: Angelo

last: Pelonero

state: CA

address: 1650 Owens Street

department: Cardiovascular Disease Institute

institute: Gladstone Institutes (UCSF)

summary: Chronic inflammation and tissue fibrosis are common stress responses that worsen organ function, yet the molecular mechanisms governing their crosstalk are poorly understood. In diseased organs, stress-induced changes in gene expression fuel maladaptive cell state transitions and pathological interaction between diverse cellular compartments. Although chronic fibroblast activation worsens dysfunction of lung, liver, kidney, and heart, and exacerbates many cancers, the stress-sensing mechanisms initiating the transcriptional activation of fibroblasts are not well understood. Here, we show that conditional deletion of the transcription co-activator Brd4 in infiltrating Cx3cr1-positive myeloid cells ameliorates heart failure and is associated with a dramatic reduction in fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancy in vivo in Cx3cr1-positive cells identified a large enhancer proximal to Interleukin-1 beta (Il1b), and a series of CRISPR deletions revealed the precise stress-dependent regulatory element that controlled expression of Il1b in disease. Secreted IL1B functioned non-cell autonomously to activate a p65/RELA-dependent enhancer near the transcription factor MEOX1, resulting in a profibrotic response in human cardiac fibroblasts. In vivo, antibody-mediated IL1B neutralization improved cardiac function and tissue fibrosis in heart failure. Mechanistically, systemic IL1B inhibition or targeted Il1b deletion in Cx3cr1-positive cells prevented stress-induced expression of MEOX1 and inhibited fibroblast activation. The elucidation of BRD4-dependent crosstalk between a specific immune cell subset and fibroblasts through IL1B provides new therapeutic strategies for heart disease and other disorders of chronic inflammation and maladaptive tissue remodeling.

overall_design: Single cell or nuclei RNAseq from adult heart in the context of BET bromodomain inhibition, Brd4 and Il1b genetic deletion, and antibody-mediated neutralization of IL1B

last: Alexanian

first: Pawel

last: Przytycki

first: Tomohiro

last: Nishino


title: An environmentally relevant mixture of Perfluoroalkyl Substances (PFAS) impacts proliferation, steroid hormone synthesis, and gene transcription in primary human granulosa cells

city: Omaha

first: Jordan

last: Rowley

state: NE

address: 42nd and Emile street

department: Genetics Cell Biology and Anatomy

institute: University of Nebraska Medical Center

summary: Perfluoroalkyl substances (PFAS) are a group of synthetic chemicals that are resistant to biodegradation and are environmentally persistent. PFAS are found in many consumer products including non-stick cookware, food packaging materials, upholstery, and personal care products. Accordingly, PFAS are a major source of water and soil contamination. Although use of many PFAS have been phased out, they continue to be detected in human and animal fluids, including human follicular fluid. This study investigated the effects of an environmentally relevant PFAS mixture [perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS)] on the transcriptome and function of human granulosa cells (hGCs). PFOA, PFOS, and PFHxS were detected in 100% of follicle fluid samples. Increased cell proliferation was observed in hGCs treated with the PFAS mixture with no impacts on cellular apoptosis. The PFAS mixture also altered steroid hormone synthesis, increasing both FSH-stimulated and basal progesterone secretion and concomitant upregulation of STAR protein. RNA sequencing revealed inherent differences in transcriptomic profiles in hGCs after PFAS exposure. This study demonstrates functional and transcriptomic changes in hGCs after exposure to a PFAS mixture, improving our knowledge about the impacts of PFAS exposures and female reproductive health. These findings suggest that PFAS compounds have the potential to disrupt normal granulosa cell function with possible long-term consequences on overall reproductive health.

overall_design: Primary hGCs were harvested from follicular aspirates of healthy, reproductive-age women who were undergoing oocyte retrieval for in vitro fertilization. LC/MS-MS performed to identify PFAS compounds in pure follicular fluid. Cells were cultured with vehicle control or a PFAS mixture (2 nM PFHxS, 7 nM PFOA, 10 nM PFOS) for 96h. Analyses of cell proliferation/apoptosis, steroidogenesis, and gene expression were measured via MTT assays/immunofluorescence, ELISA/western blotting, and RNA sequencing/bioinformatics, respectively.

first: Kendra

last: Clark

first: Mamta

last: Shukla

first: Jitu

last: George

first: Stephanie

last: Gustin

first: M

first: John

last: Davis


title: PIK3CA as a therapeutic target for patients with proliferative glomerulonephritis

city: Paris

first: Noémie

last: Robil

address: 29, rue du Faubourg Saint-Jacques

department: Paris Biotech Santé

country: France

institute: GenoSplice technology

summary: Proliferative glomerulonephritis is a severe kidney condition often associated with advanced renal failure. Unfortunately, there is a significant lack of effective treatment options for these disorders. Here, following the identification of a somatic PIK3CA gain-of-function mutation in glomerular epithelial cells of a patient, we demonstrate using multiple genetically engineered mouse models, single cell RNA sequencing and spatial transcriptomics the crucial role played by this pathway in proliferative glomerulonephritis development by promoting podocyte proliferation, dedifferentiation and inflammation. In addition, we show that alpelisib, a clinically approved pharmacological PI3Ka inhibitor, improves glomerular lesions and kidney function in different models of collapsing glomerulopathy and lupus nephritis by targeting podocytes. But surprisingly, we uncovered that pharmacological inhibition of PI3Ka affects B and T lymphocyte population in lupus nephritis mouse models with decrease in the production of proinflammatory cytokines, autoantibodies and complement deposition, all of which are characteristic features of PI3Kd inhibition. These findings were further confirmed in human lymphocytes isolated from patients with active lupus nephritis. In conclusion, we demonstrate the major role played by PI3Ka in proliferative glomerular lesion and show for the first time that alpelisib holds promise as a therapeutic approach that acts on both, glomerular epithelial cells and immune system.

overall_design: 8-week-old PIK3CAWT and PIK3CAPod-HET (PIK3CA gain-of-function mutation specifically in podocytes) mice were treated with vehicle or alpelisib for 4 weeks. 4 mice groups, PIK3CAWT + vehicle, PIK3CAWT + alpelisib, PIK3CAPodo-HET + vehicle, and PIK3CAPodo-HET + alpelisib were processed for scRNAseq profiling to explore the transcriptional programs of PIK3CA overactivation in podocytes, as well as the effect of alpelisib in podocytes. Glomerular enrichment and single-cell suspensions were achieved by using M-450 Dynabeads, Multi Tissue Dissociation Kit 1, and size filters.

first: Junna

last: Yamaguchi

first: Pierre

last: de la Grange

first: Guillaume

last: Canaud


title: Involvement of autophagy in mesaconitine-induced neurotoxicity in HT22 cells revealed through integrated transcriptomic, proteomic, and m6A epitranscriptomic profiling

city: Fuzhou

first: Mengting

last: Chen

address: Fujian Medical University, 1 Xuefubei Road, Minhou, Fuzhou, Fujian province, China

institute: Fujian Medical University

summary: Our study provided a comprehensive mechanistic understanding of the neurotoxic effects induced by MA at multi-omics resolution, and offered valuable insights for enhancing the rational application of traditional Chinese medicine containing aconitum in clinical practice.

overall_design: To investigate whether m6A modification is involved in neurotoxicity following MA treatment, we performed MeRIP-seq on both MA-treated and untreated control cells.

first: Xiaohuang

last: Lin

first: Jian

last: Zhang

first: Zekai

last: Wu

first: Yuan

last: Shi


title: Single cell RNA sequencing of adult regenerating mouse digit tips

city: Boston

first: Jessica

middle: A

last: Lehoczky

address: 60 Fenwood Rd.

institute: Brigham and Women's Hospital

summary: Innate regeneration following digit tip amputation is one of the few examples of epimorphic regeneration in mammals. Digit tip regeneration is mediated by the blastema, the same structure invoked during limb regeneration in some lower vertebrates. By genetic lineage analyses in mice, the digit tip blastema has been defined as a population of heterogeneous, lineage restructed progenitor cells. These previous studies, however, do not comprehensively evaluate blastema heterogeneity or address lineage restruction of closely related cell types. Here we report one additional single cell RNA sequencing sample (28 days post-amputation) to add to our already published 38,000 cells from mouse digit tip blastemas and unamputated control digit tips used to generate an atlas of the cell types participating in digit tip regeneration.

overall_design: Adult wildtype FVB/NJ mice used to generate 28 days post-amputation digit tips for single cell RNA sequencing by 10X genomics. GSM4276219, GSM4276220, GSM4276221, GSM4276222, and GSM4276223 from GSE143888 were re-used for the current study


title: Pyruvate metabolism alters the epigenetic and transcriptional profile of bovine oocytes.

city: Québec

first: Ricardo

middle: Perecin

last: Nociti

state: Québec

address: 2425, Rue de l'Agriculture

department: Animal Science

institute: Université Laval

summary: Pyruvate, the end-product of glycolysis in aerobic conditions, is converted into acetyl-CoA inside the mitochondria of oocytes as a master fuel input for the tricarboxylic acid (TCA) cycle. The citrate generated in the TCA cycle can be directed to the cytoplasm and converted back to acetyl-CoA, being driven to lipid synthesis or, alternatively, being used as the substrate for histones acetylation. This work aimed to verify the impact of pyruvate metabolism on the dynamics of lysine 9 histone 3 acetylation (H3K9ac) and RNA transcription in bovine oocytes during in vitro maturation (IVM). Bovine cumulus-oocyte complexes were subjected to IVM for 24 h considering three experimental groups: Control [IVM medium], sodium dichloroacetate [DCA, a stimulator of pyruvate oxidation into acetyl-CoA] or sodium iodoacetate [IA, a glycolysis inhibitor]. Our results show that both treatments change the metabolic profile of oocytes, stimulating the use of lipids for energy metabolism in the gamete. This leads to changes during IVM in the dynamics of H3K9ac, consequently impacting the oocyte transcriptional profile. A total of 148 and 356 differentially expressed genes were identified in DCA and IA oocyte groups, respectively, when compared to the control group. In summary, our results indicate that changes in the pyruvate metabolism induce the activation of metabolic pathways, such as lipid metabolism, and these metabolic alterations alter acetyl-CoA availability and H3K9ac levels at 24 h of IVM, consequently impacting the mRNA content of bovine oocytes.

overall_design: Bovine Oocytes were submitted to IVM in a maturation medium supplemented with dichloroacetate (DCA) or iodoacetate (IA) for 24h.

first: Marcela

last: Milazzotto

first: João

middle: V

last: Alcantara da Silva


title: A MOZ-TIF2 leukemic mouse model displays KAT6 inhibitor-sensitive H3K23 propionylation and dysregulation of a set of highly active developmental genes [Cut&Run]

first: Mitzi

last: Kuroda

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

summary: Aberrant regulation of chromatin modifiers is a common occurrence across many cancer types, and a key priority is to determine how specific alteration of these proteins, often enzymes, can be targeted therapeutically. MOZ, a histone acyltransferase, is recurrently fused to coactivators CBP, p300, and TIF2 in cases of Acute Myeloid Leukemia (AML). Using either pharmacological inhibition or targeted protein degradation in a mouse model for MOZ-TIF2-driven leukemia, we show that KAT6 (MOZ/MORF) enzymatic activity and the MOZ-TIF2 protein are necessary for indefinite proliferation in cell culture. Interestingly, MOZ enzymatic activity is selectively required in MOZ-TIF2 cells, as murine MLL-AF9 leukemic cells show no sensitivity to the WM-1119 MOZ inhibitor. MOZ-TIF2 directly regulates a small subset of genes encoding developmental transcription factors, augmenting their high expression. Transcription levels of these genes positively correlate with enrichment of histone H3 propionylation at lysine 23 (H3K23pr), a recently appreciated histone acylation associated with gene activation. Unexpectedly, we also show that MOZ-TIF2 and MLL-AF9 regulate transcription of largely non-overlapping gene sets, and their cellular models exhibit distinct sensitivities to multiple small molecule inhibitors directed against AML pathways. This is despite the shared genetic pathways of wild-type MOZ and MLL. Overall, our data provide insight into how aberrant regulation of MOZ contributes to leukemogenesis. We anticipate these experiments will inform future work identifying targeted therapies in the treatment of AML and other diseases involving MOZ-induced transcriptional dysregulation.

overall_design: Cut&Run DNA sequencing against HA or IgG in FKBPV12-HA-MOZ-TIF2 LSK from mouse bone marrow treated with DMSO or 500 nM dTAG13 for 2 hours

first: Anne

last: Smolko


title: Genome-wide Mapping of GATA4 in Breast Cancer MCF-7 Cell Chromatin State.

city: Jinan

first: wenqian

last: yu

state: Shandong

address: No.105, jiefang road

department: Research Center of Translational Medicine

institute: Central Hospital Affiliated to Shandong First Medical University

summary: To investigate the functional role of GATA4 in breast cancer cells, we employed ChIP-seq analysis to identify GATA4's genome-wide transcription targets. The ChIP experiment began with the use of anti-GATA4 antibodies in MCF-7 cells. Subsequently, GATA4-related DNA was unbiasedly amplified, labeled, and sequenced. Using Illumina Novaseq 6000, we identified numerous GATA4-specific binding peaks. We observed a strong enrichment of GATA4 on the promoters of specific genes involved in classical pathways. This study has provided us with new insights into the role of GATA4 in chromatin state and gene transcription in the context of breast cancer.

overall_design: Examination of  GATA4 target genes in MCF-7 cell line

first: Wenqian

last: Yu

first: Shuai

last: Leng


title: Epigenomic partitioning of an polygenic risk score for asthma reveals distinct genetically driven disease pathways

city: Rotterdam

first: Bernard

middle: Setiawan

last: Stikker

state: South Holland

address: Dr Molewaterplein 40

department: Pulmonary Medicine

country: Netherlands

institute: Erasmus MC

summary: Background: Asthma is common chronic inflammatory disease of the airways with a heterogenous clinical presentation. Individual differences in asthma susceptibility remain poorly understood, although genetics is thought to play a major role. Aim: To build a polygenic risk score (PRS) for asthma and determine whether predictive genetic variants can be epigenomically linked to specific pathophysiological mechanisms. Methods: PRSs were constructed using data from genome-wide association studies and performance was validated using data generated in the Rotterdam Study, a Dutch prospective cohort of 14,926 individuals. Outcomes used were asthma, childhood-onset asthma, adulthood-onset asthma, eosinophilic asthma and exacerbations. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) data from 14 primary cell types, including lung epithelial cells and T lymphocytes was used for epigenomic PRS partitioning. Results: All PRSs successfully predicted risk to develop asthma and related outcomes, with the strongest predictive power (2.42 odds ratios per PRS standard deviation, area under the curve of 0.736) achieved for childhood-onset asthma. PRSs allowed for stratification of the Rotterdam Study cohort into groups at low or high risk to develop asthma. PRS partitioning using genome-wide epigenomic profiles identified 5 clusters of variants within gene regulatory regions linked to specific asthma-relevant cells, genes and biological pathways. Conclusions: PRSs can predict whether individuals in a Dutch cohort developed asthma and asthma-related phenotypes, which is most effective for childhood-onset asthma. Importantly, we show that PRS partitioning based on epigenomics data dissects a genetic risk score into blocks of regulatory variants with differential predictive power, which likely represent distinct genetically driven disease pathways. These findings have potential implications for personalized risk mitigation and treatment strategies.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) data for histone 3 lysine 4 dimethylation (H3K4me2) modifications for the following cell types: naive CD8+ T cells (CD3+CD8+CD45RA+), memory CD8+ T cells (CD3+CD8+CD45RO+), naive B cells (CD3-CD19+CD27-IgD+), memory B cells (CD3-CD19+CD27+IgD-) resting eosinophils (CD33-CD66b+SIGLEC8+CD16-) resting neutrophils (CD33-CD66b+SIGLEC8-CD16+), lung epithelium (Bronchial brush in air-liquid interface culture), resting mast cells (Folkerts et al. Allergy, 2020).

first: Lianne

last: Trap

first: Bahar

last: Sedaghati-Kayat

first: Marjolein

last: de Bruijn

first: Wilfred

last: van Ijcken

first: Emmely

last: de Roos

first: Arfan

last: Ikram

first: Rudi

last: Hendriks

first: Guy

last: Brusselle

first: Jeroen

last: van Rooij

first: Ralph

last: Stadhouders


title: Differential transcriptional invasion signatures from patient derived organoid models define a functional prognostic tool for head and neck cancer

city: Utrecht

first: Peter

last: Haughton

address: Heidelberglaan 100

department: Pathology

institute: UMCU

summary: Clinical outcome for patients suffering from head and neck squamous cell carcinoma (HNSCC) remains poor. This is mostly due to highly invasive tumors that cause loco-regional relapses after initial therapeutic intervention. The molecular pathways governing the detrimental invasive growth modes in HNSCC remain however understudied. Here, we have established HNSCC patient derived organoid (PDO) models that recapitulate 3-dimensional invasion in vitro. Single cell mRNA sequencing was applied to study the differences between non-invasive and invasive conditions, and in a collective versus single cell invading PDO model. Differential expression analysis under invasive conditions in Collagen gels reveals an overall upregulation of a YAP-centered transcriptional program, irrespective of the invasion mode. However, we find that collectively invading HNSCC PDO cells show elevated levels of YAP transcription targets when compared to single cell invasion. Also, collectively invading cells are characterized by increased nuclear translocation of YAP within the invasive strands, which coincides with Collagen-I matrix alignment at the invasive front. Using gene set enrichment analysis, we identify immune cell-like migratory pathways in the single cell invading HNSCC PDO, while collective invasion is characterized by overt upregulation of adhesion and migratory pathways. Lastly, based on a clinical head and neck cancer cohort, we demonstrate that the identified collective invasion signature provides a candidate prognostic platform for survival in HNSCC. By uncoupling collective and single cell invasive programs, we have established invasion signatures that may guide new therapeutic options.

overall_design: Single-cell RNA sequencing was performed on two HNSCC patient derived organoid models cultured in BME (non-invasive) or collagen type-I (invasive) matrices.

middle: D

first: Stefan

last: Prekovic

first: Wisse

last: Haakma

first: Else

last: Driehuis

first: Hans

last: Clevers

last: Willems

first: Patrick

last: Derksen


title: A Genome Scale Transcriptional Regulatory Model of the Human Placenta

city: Seattle

first: Research

middle: Scientific

last: Computing

state: WASHINGTON

address: 818 Stewart Street

department: Research Scientific Computing

institute: Seattle Children's Research Institute

summary: Gene regulation is essential to placental function and fetal development. We built a genome-scale transcriptional regulatory network (TRN) of the human placenta using digital genomic footprinting and transcriptomic data. We integrated 475 transcriptomes and 12 DNase hypersensitivity datasets from placental samples to globally and quantitatively map transcription factor (TF)-target gene interactions. We performed siRNA knockdowns of 4 TFs and achieved concordance between the predicted gene targets in our TRN.

overall_design: We constructed a human placental-specific genome scale TRN  with RNA sequencing data compiled from 475 placental collected in two independent birth cohorts. This GEO dataset contains the data from the MWH cohort.

first: Alison

last: Paquette

first: Yoel

last: Sadvosky

first: Helen

last: Jones

first: Nathan

last: Price


title: Immune response to intra-ocular injection of particulate β-(1,3)(1,6) glucan

city: Ann Arbor

first: Michigan

middle: CDB

last: Bioinformatics

state: MI

address: 109 Zina Pitcher Pl

department: Cell & Developmental Biology

institute: University of Michigan

summary: Description of cellular and molecular landscape of vitreal immune cells following optic nerve crush injury plus intra-ocular injection of particulate β-glucan after 3days, in WT and Itgam-/- mice

overall_design: Intro-ocular (i.o.) of highly purified particulate β-(1,3)(1,6) glucan, prepared from s. cerevisiae, triggers a strong immune response in the vitreal humor and the retina. When combined with retro-orbital optic nerve crush injury, β-(1,3)(1,6) glucan elicited ocular inflammation enables robust regenerative axon growth of injured retinal ganglion cells. We observed that in mice deficient for the MAC-1 receptor component integrin alphaM (encoded by Itgam), i.o. β-(1,3)(1,6) glucan results in enhanced RGC axon regeneration when compared to parallel processed WT mice (also treated with i.o. β-(1,3)(1,6) glucan). For a comparative analysis of ocular immune infiltrates at 3days post optic nerve crush plus β-(1,3)(1,6) glucan, we used magnetic beads sorting (anti-CD45 MACS) of all leukocytes in the vitreous and retina. Leukocytes from WT and Itgam-/- eyes were prepared for scRNA-sequencing and processed separately.

first: R

last: Giger

first: C

last: Johnson

first: X-F

last: Zhao


title: A novel, druggable casacade links methionine metabolism to epigenomic reprogramming in squamous cancer

city: Los Angeles

first: Qian

last: Yang

address: 8700 Beverly Blvd

department: Department of Surgery

institute: CEDARS SINAI MEDICAL CENTER

summary: DNA and histone methylation represents perhaps the most significant form of epigenetic regulation in mammalian cells, and their deregulation is most notable and pervasive in cancer. Based on our previous study which demonstrated the interplay between cancer metabolism and epigenetic regulation and the public database showing abnormal expression of LAT1 in upper aerodigestive squamous cell carcinoma (UASCC), we aim to investigate the role of LAT1 as a regulator of epigenomic reprogramming in UASCC. To identify the effect of the blockade of LAT1 on epigenetic regulation, we silenced LAT1 in UASCC cell line and performed RNA-Seq. In addition, we confirmed that Knockdown of LAT1 is associated with the downstream genes of EZH2. Therefore, we performed RNA-Seq after silencing EZH2 by transfection of siRNA and Cut&Tag assays using EZH2 and H3K27me3 antibodies

overall_design: KYSE510 and FADU cell lines were cultured and Cut&Tag assays were performed using the EZH2 antibody. KYSE510  cells silenced either LAT1 or EZH2 using two independent siRNA were generated to profile the transcriptome through deep sequencing.

first: Chehyun

last: Nam

first: Li-Yan

last: Li

first: Hua

first: Boyan

last: Hu

last: Xu

first: En-Min

first: Harold

middle: P

last: Koeffler

first: Uttam

last: Sinha

first: De-Chen

last: Lin


title: Molecular signature of metastatic cutaneous squamous cell carcinomas: a case-control study

city: Reggio Emilia

first: Benedetta

last: Donati

address: Viale Risorgimento, 80

country: Italy

institute: Azienda USL- IRCCS di Reggio Emilia

summary: RNA extracted from diagnostic tumor samples of 48 patients affected by SCC was analyzed on the nCounter system using the PanCancer Progression Panel.

overall_design:
Gene expression profiles were linked to tumor dissemination defined by the development of lymphonodal or distant metastasis
Total RNA was hybridized to capture probes and reporter probes using the NanoString nCounter system according to the manufacturer's protocol

first: Caterina

last: Longo

first: Alessia

last: Ciarrocchi


title: Difference of expression profile between tumor tissue and adjacent tissue in patients with pancreatic cancer

first: Guoying

last: Dong

address: Jinan lixia district Wenhua west road no. 44

department: Cheeloo College of Medicine

institute: Shandong University

summary: In order to explore the differences between tumor tissues and adjacent tissues of pancreatic cancer patients, we plan to find the differences in expression profiles between tumor tissues and adjacent tissues of pancreatic cancer patients by sequencing

overall_design: The tumor tissue of pancreatic cancer patients was compared with its adjacent tissue

first: Yunshan

last: Wang

first: Guangwei

last: Wei


title: The influence of DDX18 high expression in PDAC cells Hs766T on the expression of other intracellular molecules

summary: DDX18 promotes the occurrence and development of pancreatic cancer and the maintenance of stem cell characteristics, but the mechanism through which these effects are accomplished is still unclear. The purpose of this sequencing is to screen the differential genes affected by DDX18 expression

overall_design: We constructed Hs766T with high DDX18 expression and its control, treated the cell extracts and sequenced them, and the sequencing was repeated 3 times for each group.


title: Molecular Profiling of NOD mice reveals a novel regulator of insulitin onset

city: BErgen

first: Simona

last: Chera

address: Haukelandsbakken 15

institute: University of Bergen

summary: Non-obese Diabetes (NOD) mice are an established spontaneous model for Type 1 Diabetes (T1D), emulating diabetes development through insulitis. Using next generation sequencing, coupled with pathways analysis, the molecular fingerprint of early insulitis was mapped in a cohort of mice ranging from four to 12 weeks of age. The resulted dynamic timeline revealed an initial decrease in proliferative capacity followed by the emergence of an inflammatory signature between 6- and 8-weeks, that increases to a regulatory plateau between 10- and 12-weeks.  The inflammatory signature is identified by activation of central immunogenic factors such as Infg, Il1b, Tnfa, and activation of canonical inflammatory signalling. Prodding of the regulatory landscape identified the transcription factor Atf3 as a potential novel regulator of inflammatory signalling in the NOD Islet. Furthermore, the Hedgehog (Hh) signaling pathway correlated with Atf3 regulation, hinting at role for the two in regulating the inflamed islet, however, further studies are needed to establish the nature of this connection.

overall_design: Islets were collected from NOD mice at 2 week intervals between 4-12 weeks of age for Bulk RNAseq. Aim: Investigate the gradual development of Insulitis in the NOD mice.

first: Andreas

last: Frøslev Mathisen

first: Andrei

last: Mircea Vacaru

first: Lucas

last: Unger

first: Luiza

last: Ghila

first: Ana-Maria

last: Vacaru


title: Pancreatic cancer

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

overall_design: Refer to individual Series


title: KHSRP has oncogenic functions and regulates the expression and alternative splicing of DNA repair genes in breast cancer MDA-MB-231 cells

city: Wuhan

first: Dong

last: Chen

state: Hubei

address: 888 Gaoxin Avenue

institute: Nissi Biotech

summary: Breast cancer has become the most common type of cancers worldwide. Its high prevalence and malignant features are associated with various environmental factors and molecules. The KH-type splicing regulatory protein (KHSRP) participates in the development of breast cancer, while the underlying mechanisms are largely unknown. In this study, we silenced KHSRP expression in MDA-MB-231 cells by small interfering RNA (siKHSRP), and then assessed its effects on cellular features. Finally, we performed whole transcriptome sequencing (RNA-seq) experiments to explore the downstream targets of KHSRP, and validated their changed pattern using quantitative polymerase chain reaction. We found KHSRP showed higher expression level and was associated with worse prognosis in breast cancer patients. In siKHSRP samples, the proliferation, invasion, and migration abilities were significantly repressed compared with negative control (NC) samples, while the apoptosis level was increased. By investigating the RNA-seq data, we found KHSRP globally regulates the expression and alternative splicing profiles of MDA-MB-231 cells by identifying 1632 differentially expressed genes (DEGs) and 1630 HKSRP-regulated AS events (RASEs). Functional enriched analysis of DEGs demonstrated that cilium assembly and movement and extracellular matrix organization pathways were specifically enriched in up DEGs, consistent with the repressed migration and invasion abilities in siKHSRP cells. Interestingly, the cell cycle and DNA damage and repair associated pathways were enriched in both down DEGs and RASE genes, suggesting that KHSRP may modulate cell proliferation by regulating genes in these pathways. Finally, we validated the changed expression and AS patterns of genes in cell cycle and DNA damage/repair pathways. Expression levels of BIRC5, CCNA2, CDK1, FEN1, FOXM1, PTTG1, and UHRF1 were downregulated in siKHSRP samples. The AS patterns of PARK7, UBE2A, UBE2D3, NPM1, ERCC1, CSNK1E, CENPX, and SWI5 were also dysregulated in siKHSRP samples. In summary, our study comprehensively explored the downstream targets and their functions of KHSRP in breast cancer cells, highlighting the molecular mechanisms of KHSRP on the oncogenic features of breast cancer. The identified molecular targets could be served as potential therapeutic targets for breast cancer in future.

overall_design: In this study, we silenced KHSRP expression in MDA-MB-231 cells by small interfering RNA (siKHSRP), and then we performed whole transcriptome sequencing (RNA-seq) experiments to explore the downstream targets of KHSRP.

first: Xuelaiti

last: Paizula