title: EWS-FLI1 and HOXD13 control tumor cell plasticity in Ewing sarcoma [CITE-seq]

summary: Posterior homeobox D genes, in particular HOXD13, are over-expressed by Ewing sarcoma, a tumor driven by the oncogenic fusion protein EWS-FLI1. Here, we have found that EWS-FLI1 maintains HOXD13 expression through a GGAA microsatellite enhancer in the developmental posterior HOXD regulatory domain. Activation of this enhancer is EWS-FLI1 dependent and epigenomic silencing of this region leads to loss of HOXD13 expression in Ewing sarcoma cells, but not in unrelated cells. To determine the function of HOXD13 activation in Ewing sarcoma we performed nascent RNA sequencing upon HOXD13 knockdown. We identified NT5E as a target induced by HOXD13. NT5E encodes for the cell surface marker CD73, so we used CITE-seq to profile these cells.

overall_design: A673 and CHLA10 cells were labeled with an CD73-ADT antibody and a unique HTO antibody for 30 min at 4C. Cells were washed, counted and pooled at 1000 cells/uL. Libraries were prepared and sequenced by the University of Michigan Advanced Genomics core using the 10X genomics platform. Libraries generated using the 3’ V3 10X Genomics Chromium Controller following the manufacturer’s  protocol (CG000183) and then paired end sequencing was ran on the NovaSeq 6000. Bcl2fastq2  Conversion Software (Illumina) was used to generate de-multiplexed Fastq files and the  CellRanger (3.1) Pipeline (10X Genomics) was used to align reads and generate count  matrices.

first: Scott

last: Furlan

first: Elizabeth


title: Identification of TWIST1-associated genomic regions in human breast cancer cell MCF7

status: Public on Jun 28 2022

city: Houston

first: Xiaobin

last: Yu

state: TX

address: One Baylor Plaza

department: MCB

country: USA

institute: Baylor College of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: In this study, we aimed to characterize the stimulatory role of TWIST1 in breast cancer cell proliferation, EMT and metastasis, uncover the underlying molecular mechanism, and identify potential therapeutic targets for treating BLBC. RNA-Seq analysis were performed to identify differentially expressed genes influenced by TWIST1 overexpression in breast cancer cell MCF7. Pathway enrichment analysis and gene set enrichment analysis were performed to identify TWIST1-regulated signaling pathways, particually the cancer-associated pathways. ChIP-Seq analysis was further performed to identify TWIST1-associated genomic regions in MCF7-TWIST1 cells. The potential direct target genes of TWIST1 were identified by combined analysis of the gene expression profiling data and ChIP-Seq data.

overall_design: TWIST1-associated genomic regions in MCF7-TWIST1 cultured in DMEM complete medium were measured at 24 hours after cell passage. Two samples were analyzed by using FLAG antibody. Samples were used as a control and one overexpression sample.

first: Jianming

last: Xu


title: Clonotypic analysis of protective influenza M2e-specific lung resident T helper memory cells reveals extensive functional plasticity

status: Public on Jun 29 2022

city: Solna

first: Paulo

last: Czarnewski

address: Tomtebodavägen 23b

country: Sweden

institute: KTH

type: Expression profiling by high throughput sequencing

summary: Intranasal influenza immunization with CTA1-3M2e-DD stimulated M2e-specific Th17 tissue resident memory (Trm) cells that conferred strong antibody-independent protection against infection at 6 months after priming. These cells rapidly expanded in the lung upon infection and effectively restricted virus replication. Single-cell RNAseq transcriptomic and TCR VDJ-analysis of M2e- tetramer-sorted Trm cells on day 3 and 8 post infection revealed complete Th17-lineage dominance (no Th1, Tfh or Foxp3+ Tregs) with extensive functional plasticity involving cytotoxicity (ThCTL) as well as regulatory functions. Unexpectedly, the same clonotype hosted cells with different Th17 subcluster functions (IL-17, IL-22), Tr1 cells and ThCTLs, suggesting a tissue and context-dependent differentiation of reactivated Th17 Trm cells. A gene set enrichment analysis demonstrated up- regulation of regulatory genes in the day 8 recovery phase. Contrary to present thinking lung M2e- specific Th17 Trm cells are sufficient for controlling infection and for preventing tissue injury. These findings will have strong implications for vaccine development.

overall_design: Three immunizations with 5μg CTA1-3M2e-DD in sterile PBS were done with 10 days between the doses. Mice were sacrificed 6 months after the final immunization 3 or 8 days after an influenza A virus challenge infection. Influenza A virus challenge experiments were performed using a lethal dose of 4x LD50 of the mouse-adapted X47(H3N2) virus strain (a reassortant between A/Victoria/3/75 (H3N2) and A/Puerto Rico/8/34 (H1N1)) administrated i.n. to mice under light anaesthesia. Lungs were extracted and collected. Single cell suspensions of lung lymphocytes were prepared with the lung dissociation kit from Miltenyi Biotec (ref 130-095-927). Briefly, the harvested lungs were first added into the gentleMACS C tubes containing enzyme mix and broken up into smaller pieces with gentleMAC OctoDissociator (m_lung_01 program). The samples were then incubated at 37°C for 30 minutes before the complete digestion on gentleMAC OctoDissociator (m_lung_02 program), after which, the cell suspensions were washed once with PBS, passed through a mesh and resuspended in appropriate media for further processing. We assessed the CD4+ T cell response in BALB/c mice after immunizations (day 3 and day 8, post infection with influenza A virus) by flow cytometry using the PE-labeled M2e-tetramer, designed and produced by the NIH Tetramer Core Facility. Sorted lung M2e-teramer+ TCRb+ T cells (CD19-, CD8- F4/80-) from 5 mice at each time point were directly loaded onto a Chromium chip (10X Genomics) and run on the Chromium Controller for droplet generation. Reverse transcription was conducted in droplets and cDNA recovered through demulsification and bead purification. Pre-amplified cDNA further went through 10X library preparation for quantification of both mRNA and VDJ sequences {10.1038/nprot.2018.021} using 10X genomics v2 Chemistry kit. Libraries were sequenced on an Illumina Nextseq500 (High Output) targeting a minimum depth of 10,000 reads per cell across all samples.

first: Nils

last: Lycke


title: Identification of ALKBH5 targets in osteosarcoma by methyl RNA immunoprecipitation [MeRIP-seq]

city: San Antonio

first: Yidong

last: Chen

state: Texas

address: 8403 Floyd Curl Drive, MSC 7784

department: Population Health Sciences

institute: UT Health Science Center at San Antonio

summary: To identify potential target mRNAs that are demethylated by ALKBH5, meRIP-seq with a m6A antibody was conducted in 143B osteosarcoma cells transfected with scrambled or ALKBH5-siRNA.

overall_design: 143B cells were grown in 150 mm tissue culture plates and transfected with with either scrambled-siRNA or ALKBH5-siRNAs (Sigma-Aldrich) for 48 hours.Total RNA / mRNA  isolated from the 143B cells was fragmented and subjected to  immunoprecipitation (IP) using rabbit anti-m6A antibody.  After  incubation and washing , samples were eluted using elution buffer containing m6A nucleoside . The resultant final product was used for RNA-seq.

first: Pooja

last: Yadav

first: Manjeet

last: Rao

first: Zhao

last: Lai


title: The DREAM complex antagonizes WDR5a and represses the productive elongation of transcription in Arabidopsis [ChIP-seq]

city: Beijing

first: Yangyang

last: Fan

address: No.5 Yiheyuan Road Haidian District,

country: China

institute: Peking University

summary: The master transcriptional repressor DREAM (dimerization partner, RB-like, E2F and multi-vulval class B) complex regulates cell cycle in eukaryotes, but much remains unknown about how it transmits repressive signals on chromatin to the primary transcriptional machinery (e.g., Pol II). Through a genetic screen, we identified BTE1 (barrier of transcription elongation 1), a key component in the plant DREAM complex. The subsequent characterization demonstrated that the DREAM complex represses Pol II elongation. The core DREAM subunits E2F transcription factors recruit BTE1 to accumulate at the promoter-proximal regions of target genes. DREAM target genes exhibit characteristic enrichment of H2A.Z and H3K4me2 modification on chromatin. The DREAM complex BTE1 subunit interacts with WDR5a, repressing WDR5a chromatin binding and the productive elongation of transcription on BTE1-targeted genomic loci. Thus, we demonstrate a major role of DREAM complex in the control of transition from transcription initiation to productive elongation around TSSs on the open chromatin via inhibiting WDR5a function.

overall_design: Three biological replicates of each genotype were used for high-throughput sequencing analysis. All experiments wer conducted using  9-day-old seedlings grown in continuous white light.

first: Yuqiu

last: Wang

first: De

first: Xing

middle: W

last: Deng

first: Danmeng

last: Zhu


title: Gene regulation by m6A demethylase ALKBH5 in osteosarcoma

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

overall_design: Refer to individual Series


title: Analysis of Wild Type and Siah2 KO time series liver transcriptome from male and female mice [ZT0-ZT21]

city: Atlanta

first: Jason

last: DeBruyne

state: GA

address: 720 Westview Dr, Research Wing 315

department: Pharmacology & Toxicology

institute: Morehouse School of Medicine

summary: Female-specific role of Siah2 in regulating circadian gene expression in the liver

overall_design: Liver mRNA profiles of  WT and Siah2 KO mice of both sexes were generated by sequencing pooled mRNAs from 3 livers collected every 3 hours during ZT0-ZT21

middle: P


title: Circular and fusion RNAs in medulloblastoma development

status: Public on Jun 27 2022

city: Huddinge

first: Ani

last: Azatyan

state: Stockholm

address: Hälsovägen 7C

department: Biosciences and Nutrition

institute: Karolinska Institutet

type: Non-coding RNA profiling by high throughput sequencing

summary: The expression of circular RNAs in cerebellar cancer versus medulloblastoma subgroups SHH, WNT, Group3 and Group4 has been interrogated. Fusion RNAs containing exons from distinct genes joined at canonical splice sites were also identified.

overall_design: Circular and fusion RNA expression in human medulloblastoma and normal cerebellar samples.

first: Peter

middle: G

last: Zaphiropoulos


title: EWS-FLI1 and HOXD13 control tumor cell plasticity in Ewing sarcoma

city: Boston

first: April

last: Apfelbaum

state: MA

address: 450 Brookline Ave

department: Pediatric Oncology

institute: Dana-farber Cancer Institute


title: EWS-FLI1 and HOXD13 control tumor cell plasticity in Ewing sarcoma [RNA-seq]

summary: Posterior homeobox D genes, in particular HOXD13, are over-expressed by Ewing sarcoma, a tumor driven by the oncogenic fusion protein EWS-FLI1. Here, we have found that EWS-FLI1 maintains HOXD13 expression through a GGAA microsatellite enhancer in the developmental posterior HOXD regulatory domain. Activation of this enhancer is EWS-FLI1 dependent and epigenomic silencing of this region leads to loss of HOXD13 expression in Ewing sarcoma cells, but not in unrelated cells. To determine the function of HOXD13 activation in Ewing sarcoma we performed nascent RNA sequencing or RNA sequencing upon HOXD13 knockdown.

overall_design:
HOXD13 was knocked down by doxy-cycline inducible (0.5 ug/uL) shRNAs in two human Ewing sarcoma cell lines, CHLA10 and A673 for 72 hours.  Three replicates each of targeting shRNA (shHOXD13 #1) and non-targeting control (shNS) were subject to nascent RNA sequencing (Bru-seq).
HOXD13 was knocked down by doxy-cycline inducible (0.5 ug/uL) shRNAs in the human Ewing sarcoma cell lines, TC32, for 72 hours. Three replicates each of targeting shRNA (shHOXD13 #1) and non-targeting control (shNS) were subject to RNA sequencing

first: Brian

last: Magnuson

first: Mats

last: Ljungman

first: Elizabeth

last: Lawlor


title: TWIST1

first: Xiaobin

last: Yu


title: A reference single-cell regulomic and transcriptomic map of cynomolgus monkeys [ATAC-seq]

city: Nanjing

first: Dijun

state: Jiangsu

address: Xianlin

department: Life Sciences

institute: Nanjing University

summary: Non-human primates (NHP) are attractive laboratory animal models that accurately reflect both developmental and pathological features of humans. Here we present a compendium of cell types from the cynomolgus monkey Macaca fascicularis (denoted as ‘Monkey Atlas’) using both single-cell chromatin accessibility (scATAC-seq) and RNA sequencing (scRNA-seq) data at the organism-wide level. The integrated cell map enables in-depth dissection and comparison of molecular dynamics, cell-type composition and cellular heterogeneity across multiple tissues and organs. Using single-cell transcriptomic data, we inferred pseudotime cell trajectories and cell-cell communications to uncover key molecular signatures underlying their cellular processes. Furthermore, we identified various cell-specific cis-regulatory elements and constructed organ-specific gene regulatory networks at the single-cell level. Finally, we performed a comparative analysis of single-cell landscapes among mouse, cynomolgus monkey and human, and we showed that cynomolgus monkey has significantly higher degree of cell-type similarity to human than mouse. Taken together, our study provides a valuable resource for NHP cell biology.

overall_design: The cynomolgus monkey project included 8 ATAC samples

first: Yang

last: Sun

first: Jiao

last: Qu

first: Fa

last: Yang

first: Tao


title: Single seeds exhibit transcriptional heterogeneity during secondary dormancy induction

city: Warsaw

first: Michal

last: Krzyszton

address: Pawinskiego 5a

country: Poland

institute: Institute of Biochemistry and Biophysics, Polish Academy of Sciences

summary: Seeds are highly resilient to the external environment, which allow plants to persist in unpredictable and unfavorable conditions. Some plant species have adopted a bet-hedging strategy to germinate a variable fraction of seeds in any given condition, and this could be explained by population-based threshold models. Here, in the model plant Arabidopsis (Arabidopsis thaliana) we induced secondary dormancy to address the transcriptional heterogeneity among seeds that leads to binary germination/non-germination outcomes. We developed a single seed RNA-seq strategy that allowed us to observe a reduction in seed transcriptional heterogeneity as seeds enter stress conditions, followed by an increase during recovery. We identified groups of genes whose expression showed a specific pattern through a time course and used these groups to position the individual seeds along the transcriptional gradient of germination competence. In agreement, transcriptomes of dormancy-deficient seeds (mutant of DELAY OF GERMINATION 1 gene) showed a shift towards higher values of the germination competence index. Interestingly, a significant fraction of genes with variable expression encoded translation-related factors. In summary, interrogating hundreds of single seed transcriptomes during secondary dormancy-inducing treatment revealed variability among the transcriptomes that could result from the distribution of population-based sensitivity thresholds. Our results also showed that single seed RNA-seq is the method of choice for analyzing seed bet-hedging-related phenomena.

overall_design: 1) Three libraries, each consisting of 32 seeds were prepared for each time point of secondary dormancy induction time course for Arabidopsis seeds (1h, 1d, 3d, 5d, 7d, 7d+24h, pool-split control). 2) Three libraries, each consisting of 32 seeds were prepared for two time points of secondary dormancy induction (3d, 7d+24h) and two genotypes (Col-0 and dog1-4 mutant). 3) Comparison of dry seed transcriptome of Col-0 and dog1-4 mutant (three biological replicas).

first: Szymon

last: Swiezewski


title: JMJD6-DGAT1 Signaling Regulates Lipid Metabolism and Tumorigenesis in ccRCC

city: Chapel Hill

first: Austin

middle: J

last: Hepperla

state: NC

address: 7018B Mary Ellen Jones Building

department: Genetics

institute: University of North Carolina at Chapel Hill

summary: Clear cell renal cell carcinoma (ccRCC) represents a lethal disease and classically resistant to cytotoxic chemotherapy, highlighting the importance of identifying new therapeutic vulnerabilities. We performed a functional siRNA screening for all 2-oxoglutarate (2-OG)-dependent enzymes in ccRCC using 3-D soft agar colony formation assay, and combined with TCGA data, we identified Jumonji domain-containing 6 (JMJD6) as an essential gene for ccRCC tumorigenesis. Downregulation of JMJD6 abolished ccRCC colony formation in vitro and inhibited orthotopic tumor growth in vivo. Integrated analyses of ChIP-seq and RNA-seq identified that JMJD6 can transcriptionally regulate the expression of diacylglycerol O-acyltransferase 1 (DGAT1) by co-occupying the target gene promoter with H3K4me3. Downregulation of JMJD6 caused decreased expression of DGAT1 at both mRNA and protein level. Inhibition or depletion of DGAT1 induced decreased cell growth in vitro or inhibited tumor growth in vivo. Mechanistically, decreased DGAT1 caused decreased LD formation in ccRCC, while restoration of JMJD6 expression can restore DGAT1 level, and LD formation as well as cell growth. In summary, JMJD6 is essential for ccRCC lipid storage and tumorigenesis through a DGAT1-dependent signaling. Therefore, the JMJD6-DGAT1 axis serves as a potential therapeutic avenue for ccRCC.

overall_design: RNA-seq of 786-O clear cell renal cell carcinoma cells before and after JMJD6 knockout with two different sgRNAs (sgRNA2 and sgRNA4) versus control sgRNAs. ChIP-seq of JMJD6 and H3K4me3 with respective inputs in 786-O clear cell renal cell carcinoma cells.

first: Jin

last: Zhou

first: Jeremy

middle: M

last: Simon

first: Qing

last: Zhang


title: The DREAM complex antagonizes WDR5a and represses the productive elongation of transcription in Arabidopsis

first: Yangyang

last: Fan


title: Exploring the cytotoxic effects of dBET1 on multiple molecular   subtypes of Acute myeloid leukemia  cells

city: Suzhou

first: Zimu

address: Zhongnan Street

institute: Children’s Hospital of Soochow University

summary: Acute myeloid leukemia (AML) represents an aggressive hematopoietic malignancy with a prognosis inferior to that of other leukemias. Recent targeted therapies offer new opportunities to achieve better treatment outcomes. However, due to the complex heterogeneity of AML, its prognosis  is remain dismal. In this study, we first identified the correlation between high expression of BRD4 and overall survival of AML patients. Targeted degradation of BRD2, BRD3, and BRD4 proteins by dBET1, a PROTAC against BET family members, showed cytotoxic effects on Kasumi (AML1-ETO), NB4 (PML-RARa), THP-1 (MLL-AF9), and MV4-11 (MLL-AF4) AML cell lines representing different molecular subtypes of AML. Furthermore, we determined that dBET1 treatment  arrested cell cycling and enhanced apoptosis and c-MYC as the downstream target. Collectively, our results indicated that dBET1 has broad cytotoxic effects on AML cells with different molecular lesions and provide more benefits to AML patients.

overall_design: mRNA profiles of NB4 cells treated with 8 μM or same volume DMSO for 24 h were generated by RNA sequencing, in double, using Illumina NovaSeq6000.

first: Kunlong

first: Shaoyan

last: Hu


title: H3K27me3 shapes DNA methylome by inhibiting UHRF1-mediated H3 ubiquitination

first: Falong

last: Lu

address: 1 West Beichen Road Chaoyang District

institute: Institute of Genetics and Developmental Biology

type: Methylation profiling by high throughput sequencing

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: H3K27me3 shapes DNA methylome by inhibiting UHRF1-mediated H3 ubiquitination [Bisulfite-seq]

summary: DNA methylation and histone lysine tri-methylation at H3K27 (H3K27me3) are the two primary epigenetic marks for transcriptional silencing essential for cell fate determination and cell lineage commitment during development1, 2. These two marks are mutually exclusive and target distinct sets of genes in the mammalian genome3. However, whether and how H3K27me3 shapes the DNA methylome remains unknown. Here, we report that the loss of H3K27me3 modification leads to increased DNA methylation at previously marked H3K27me3 sites, revealing that H3K27me3 negatively regulates DNA methylation. Genome-wide analysis of H3 ubiquitination, essential for recruitment and activation of DNA methyltransferase DNMT14, reveals the absence of H3 ubiquitination at H3K27me3 marked nucleosomes. Moreover, loss of H3K27me3 modification induces an increase in H3K18 ubiquitination at the corresponding hypermethylated loci. Importantly, we show that H3K27me3 directly inhibits UHRF1-mediated H3 ubiquitination toward nucleosomes in a defined biochemical assay. Furthermore, UHRF1 is required for the increase in DNA methylation at previously marked H3K27me3 sites in cells with abolished H3K27me3 modification. Taken together, our findings reveal a general mechanism for H3K27me3-mediated shaping of the mammalian DNA methylome via modulation of H3 ubiquitination.

overall_design: We performed WGBS (whole genome bisulfite sequencing) in WT HeLa cell, SUZ12 KO HeLa cell (two replications), WT E14 cell, Suz12 KO E14 cell, Uhrf1 KO E14 cell, Suz12/Uhrf1 double KO E14 cell. We mapped H3K27me3 ChIP-seq and H3K27me3 ChIP-BS-seq(chromatin immunoprecipitation bisulfite sequencing) in WT HeLa cell. We did RNA-seq in WT and SUZ12 KO HeLa cell (two replications). H3K18ub ChIP-seq in WT HeLa cell, SUZ12 KO HeLa cell, WT E14 cell and Uhrf1 KO E14 cell were also performed.

first: H

first: Y

last: Liu

last: Zhu

last: Xie

first: J

first: F


title: H3K27me3 shapes DNA methylome by inhibiting UHRF1-mediated H3 ubiquitination [ChIP-seq]


title: Analysis of Wild Type and Siah2 KO time series liver transcriptome from male and female mice [ZT 10]

first: Jason

last: DeBruyne

overall_design: Liver mRNA profiles of  WT and Siah2 KO mice of both sexes from individuals harvested at ZT 10 only


title: Epigenetic Profiling Linked to Multisystem Inflammatory Syndrome in Children (MIS-C): A Multicenter, Retrospective Study

status: Public on Jun 26 2022

city: Badalona

first: Manel

last: Esteller

state: Barcelona

address: Ctra. de Can Ruti, Cami de les Escoles s/n

department: Cancer and Leukemia Epigenetics and Biology Program (PEBCL)

country: Spain

institute: Josep Carreras Leukaemia Research Institute (IJC)

summary:
Background: Most children and adolescents infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop a mild coronavirus disease 2019 (COVID-19) that usually does not require medical intervention. However, a small proportion of pediatric patients develop a severe clinical condition, Multisystem Inflammatory Syndrome in Children (MIS-C). The involvement of epigenetics in the control of the immune response and viral activity prompted us to carry out an epigenomic study to uncover target loci regulated by DNA methylation that could be altered upon the appearance of MIS-C.
Methods: Peripheral blood samples were recruited from 43 confirmed MIS-C patients with a median age of 7.7 years, most of whom were of West-Eurasian origin. Four or more organ systems were involved in the majority of patients. 69 non-COVID-19 pediatric samples and 15 COVID-19 pediatric samples without MIS-C were used as controls. The cases in the two groups were mixed and divided into discovery (MIS-C = 29 and non-MIS-C = 56) and validation (MIS-C = 14 and non-MIS-C = 28) cohorts, and balanced for age, gender and ethnic background. We interrogated 850,000 CpG sites of the human genome for DNA methylation variants.
Findings: The DNA methylation content of 33 CpG loci was linked with the presence of MIS-C. Of these sites, 18 (54.5%) were located in described genes. The top candidate gene was the immune T-cell mediator ZEB2; and others highly ranked candidates included the regulator of natural killer cell functional competence SH2D1B; VWA8, which contains a domain of the Von Willebrand factor A involved in the pediatric hemostasis disease; and human leukocyte antigen complex member HLA-DRB1; in addition to pro-inflammatory genes such as CUL2 and AIM2. The identified loci were used to construct a DNA methylation profile (EPIMISC) that was associated with MIS-C in both cohorts. The EPIMISC signature was also overrepresented in Kawasaki disease patients, a childhood pathology with also a possible viral trigger, that shares many of the clinical features of MIS-C.
Interpretation: We have characterized DNA methylation loci that are associated with the onset of MIS-C. The identified genes are likely contributors to the characteristic exaggerated host inflammatory response observed in these patients. The described epigenetic signature could also provide new targets for more specific therapies for the disorder.

overall_design: The DNA methylation status of whole blood samples from 43 MIS-C patients, 15 pediatric COVID-19 cases and 69 healthy controls was established using the Infinium MethylationEPIC Array following the manufacturer’s instructions for the automated processing of arrays with a liquid handler (Illumina Infinium HD Methylation Assay Experienced User Card, Automated Protocol 15019521 v01).

first: Veronica

last: Davalos

first: Carlos

middle: A

last: Garcia-Prieto


title: Gene regulation by m6A demethylase ALKBH5 in osteosarcoma [RNA-seq]

first: Yidong

last: Chen

summary: To identify target genes regulated by ALKBH5 in osteosarcoma,  we silenced the expression of ALKBH5 in osteosarcoma cell line-143B and tested its effect on 143B transcriptome.

overall_design: 143B cells were were transfected with either scrambled-siRNA or ALKBH5-siRNAs (Sigma-Aldrich) for 48 hours. Transfection was carried out using the RNAiMAX Reagent (Thermofisher) according to the manufacturer’s protocol. After 48 hours, cells were harvested and total RNA extraction was done using RNeasy Mini Kit (Qiagen).For whole-genome transcriptome profiling, four libraries (2 replicates each for knockdown and control) were generated from total RNA from scrambled-siRNA- and ALKBH5-siRNA-transfected 143B cells, using a TruSeq Stranded mRNA Library Preparation kit according to the manufacturer’s protocol (Illumina Inc.). Samples were sequenced on the Illumina HiSeq 3000 platform (Illumina Inc.) using the 50 base-pair single-read (50SR) sequencing module.

first: Pooja

last: Yadav

first: Manjeet

last: Rao

first: Zhao

last: Lai


title: Role of ILC in bladder infection

status: Public on Jun 25 2022

city: Cambridge

first: John

middle: Robert

last: Ferdinand

address: Department of Medicine

country: United Kingdom

institute: University of Cambridge

summary: The effect of ILCs on UTI was compared in Rag mice within the bladder

overall_design: 9 rag KO mice were given a UTI. 4 were pretreated with an isotype control and 5 with an anti thy1.1 to deplete ILCs

middle: R

first: Alex

last: Riding

first: Kevin

last: Loudon

first: Menna

last: Clatworthy


title: Checkpoint Inhibitors Modulate Plasticity of Innate Lymphoid Cells in Peripheral Blood of Patients with Hepatocellular Carcinoma

status: Public on Jun 23 2022

city: Bethesda

first: Tim

last: Greten

state: Maryland

address: 9000 Rockville Pike

department: National Cancer Institute

institute: National Institutes of Health

summary: Introduction: The role of innate lymphoid cells (ILC) in cancer and specifically in the context of hepatocellular carcinoma (HCC) is not well understood. Immune checkpoint inhibitors (ICI) have been approved for treatment of patients with HCC. The effect of ICI on ILCs has not been explored. Methods: We studied ILCs in PBMCs of 51 patients with HCC at baseline and after treatment with ICI. Flow cytometry was used to study ILC composition. Single-cell RNA-sequencing using a targeted panel of 405 immune-related genes was used to characterize the transcriptomic profiles as well as cellular trajectory of ILCs before and after ICI therapy. Intracellular cytokine staining was used to validate ILC function. Data from large patient cohorts were used to predict association with survival. Results: Characterization of the ILC subgroups in PBMCs of HCC patients revealed a significant increase in ILC1 and a decrease in ILC3 frequencies. Single cell RNA-sequencing identified a subgroup of NK-like ILCs which expressed cytotoxicity markers as well as NKp80/KLRF1. This KLRF1high-NK-like population showed low abundance in patients with HCC and was enhanced after combined anti-CTLA-4 and anti-PD-1 immunotherapy. Trajectory analysis placed this population in between ILC1 and ILC3s. The transcriptomic signature of KLRF1high NK-like ILCs was associated with better progression-free survival in HCC cohorts. Conclusions: We present a previously unknown effect of HCC tumors and ICIs on the composition and plasticity of ILCs in PBMCs.  We identified a cytotoxic KLRF1high-NK-like subgroup which was associated with better survival, absent in untreated HCC patients, and enhanced upon ICI immunotherapy. Thus, ILCs from PBMC can be used to study changes in the innate immune system under immunotherapy.

overall_design: PBMC samples from 3 healthy volunteers, 3 patients with HCC before immunotherapy and 3 patients with HCC after therapy with tremelimumab and durvalumab

first: Bernd

last: Heinrich

middle: F

first: Firouzeh

last: Korangy


title: Small molecule activation of OGG1 increases base excision repair by gaining a new enzymatic function

status: Public on Jun 24 2022

first: Julian

last: Albers

address: Tomtebodavägen 23

department: Department of Oncology and Pathology

institute: Karolinska Institutet, Science for Life Laboratory

summary: Oxidative DNA damage is recognised by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1), initiating repair. Here, we describe a small molecule (TH10785) that interacts with the Phe319 and Gly42 amino acids of OGG1, increases the enzyme activity 10-fold and generates a novel β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and ageing.

overall_design: Examination of the transcriptomic effects of TH10587, PNKP1i or a combination treatment on the transcriptome of U2OS cells.

last: Benitez-Buelga


title: Tissue tropism and host response: similarities and differences between enteric and respiratory enteroviruses

city: Geneva

first: Julien

last: Prados

address: Rue Michel Servet 1

department: SCMU

country: Switzerland

institute: University of Geneva

summary: Enteroviruses (EVs) are one of the most prevalent viruses worldwide. They are characterised by a high genetic and phenotypic diversity, being able to cause a plethora of symptoms. EV-D68, a respiratory EV, and EV-D94, an enteric EV, represent an interesting paradigm of EV tropism heterogeneity. These are closely related viruses, belonging to the same species, but with distinct phenotypic features. Here, we used these two viruses as well as relevant tissue culture models mimicking the respiratory and intestinal epithelia, to highlight key distinctive features of enteric and respiratory EVs. We emphasize the critical role of temperature in restricting the tissue tropism of EV-D68 and the limited replication of EV-D94 in small airway tissues. In parallel, using transcriptomic analysis, we uncover fundamental differences between intestinal and respiratory tissues, in their steady-state as well as in response to infection. Intestinal tissues are more immunotolerant than respiratory tissues both in absence and presence of infection and they present higher turnover. Finally, we highlight the different strategies applied by EV-D94 and EV-D68 towards the host antiviral response in intestinal and respiratory tissues. In summary, our study provides an insightful characterization of the differential pathogenesis of EV-D68 and EV-D94 and the interplay with their main target tissues.

overall_design: Two Human-derived tissues (respiratory and Intestinal) are infected with two viruses (ev-d68 and ev-d94) and compared to a mock infected control condition. Each sample is done in duplicate, and transcriptome is analyzed  by RNA-seq (12 libraries in total).

first: Ines

middle: C

last: Filipe

first: Isabelle

last: Piuz

first: Samuel

last: Constant

first: Song

last: Huang

first: Caroline

last: Tapparel


title: IL22RKO bladders following UTI

summary: WT or IL22RKO mice recived a UTI and bladders were collected for RNAseq

overall_design: WT uninfected or infected bladders and IL22RKO infected bladders

last: Loudin


title: Human umbilical cord blood-derived MSCs trans-differentiate into endometrial cells and regulate Th17/Treg balance through NF-B signaling in rabbit intrauterine adhesions endometrium [RNA-seq]

status: Public on Jun 21 2022

city: Zhengzhou

first: Yong

last: Zhang

state: State...

address: Tongbaibei NO.16 Zhongyuan district, Zhengzhou

institute: Zhengzhou University

summary: Purpose: The fundamental cause of intrauterine adhesions (IUAs) is the destruction and reduction of stem cells in endometrial basal layer, resulting in endometrial reconstruction very difficult. The purpose of this study was to investigate the effects and underlying mechanism of human umbilical cord blood derived mesenchymal stem cells (hUCB-MSC) on the endometrial reconstruction after transplantation.  Methods: hUCB-MSCs were isolated and identified successfully. The rabbit IUA models were established and set five groups (control, 14/28th day after surgery, estrogen and hUCB-MSCs treatment).The number of endometrial glands and the fibrosis rate were evaluated using HE and Masson staining, respectively. Endometrial proliferation, angiogenesis and inflammation was evaluated by immunohistochemical staining of ER, Ki-67and TGF-β1, respectively. Single-cell RNA sequencing (scRNA-seq) was applied to explore the cell differentiation trajectory after hUCB-MSCs transplanted into IUA endometrium. Finally, molecular mechanism of hUCB-MSCs repairing damaged endometrium was investigated by RNA sequencing.  Results: After transplantation of the hUCB-MSCs, the increase of endometrial gland number, estrogen receptor(ER) and Ki-67expression,and the decrease of fibrosis rate and TGF-β expression(P＜0.05), suggested the endometrial repair, angiogenesis and inflammatory suppression. The therapeutic effect of hUCB-MSCs was significantly improved compared with 28th day after surgery and estrogen group. ScRNA-seq demonstrated that the transplanted hUCB-MSCs can trans-differentiate into endometrial cells: epithelial, fibroblast, and macrophage. RNA Sequencing of six IUA samples further revealed that hUCB-MSCs may regulate Th17/Treg balance through NF-B signaling, thus inhibiting the immune response of damaged endometrium.  Conclusions: Our study demonstrated that hUCB-MSCs can repair damaged endometrium through trans-differentiation, immunomodulatory capacities, and NF-κB signaling, suggesting the treatment value of hUCB-MSCs in IUA.

overall_design: The rabbit IUA samples with and without treated with hUCB-MSCs were used for transcriptome sequencing (3 vs 3)

first: Qing

last: Hua

first: Lei

last: Sun

first: Yali

last: Wang


title: Synergistic chemical combination for cell cycle restoration in senescent cells via FoxM1 and E2F1 activation

status: Public on Jun 22 2022

city: Daegu

first: Ji Hwan

last: Park

state: Gyeongbuk

address: 333, Techno jungang-daero, Hyeonpung-eup, Dalseong-gun

department: Newbiology

country: South Korea

institute: DGIST

summary: Cellular senescence wreaks defects in the aging organism. In this study, we co-treated two chemicals: rho-associated protein kinase (ROCK) inhibitor (Y-27632) and ataxia telangiectasia mutated (ATM) inhibitor (KU-60019) to overcome senescence more efficaciously, these two chemicals are already revealed to have senomorphic effects, suppressing senescent phenotypes. We find out that cocktail treatment synergistically alleviates diverse senescent phenotypes in senescent human dermal fibroblast cells. Through transcriptomic approach, FOXM1 and E2F1 are identified as key transcription factor of synergism to normalize senescent cells, of which activities are attenuated by senescence both in vitro and in vivo, but revitalized by co-treatment. Activation of FOXM1 precedes activation of E2F1 and mediates transcription of numerous genes totally shut off by cellular senescence, including E2F1. After that, E2F1 participates in transcriptomic change. For that, Cyclin-dependent kinase (CDKs), where are the confluences of both ATM-Chk2-CDKs and ROCK-Akt-CDKs signaling axes, are direct regulators of these transcription factors. Surprisingly, canonical aging-related CDK inhibitors have nothing to do with the onset of senomorphism. As the order of activations of FOXM1 and E2F1 that are in charge of G2 and G1 phase transition respectively, chemical combination releases senescent cell arrest in order of G2 then G1. Altogether, our data provide new insight into the way of amelioration of aging via pivotal cell cycle transcription factors, FOXM1 and E2F1, which are synergistically stimulated by inhibiting senescence-associated kinases, ATM and ROCK.

overall_design: Single drug (Y27632 and KU60019) and mixture of drug induced gene expresson in old human dermal fibroblast was measured at 3,8 and 15 days after treatment (three replicate for each condition).experiment.

first: EunJae

last: Yang

first: Ji

middle: H

first: Young-Sam

last: Lee

first: Daehee

last: Hwang


title: Adaption of oxidative phosphorylation machinery compensates for hepatic lipotoxicity in early stages of MAFLD [hepatocyte]

city: Duesseldorf

first: Birgit

last: Knebel

address: Auf'm Hennekamp 65

department: Cinical Biochemistry and Pathobiochemistry

country: Germany

institute: German Diabetes Center

summary: Mitochondrial function is an important control variable in the progression of metabolic dysfunction associated fatty liver disease (MAFLD). We hypothesize that organization and function of mitochondrial electron transport chain (ETC) in this pathologic condition is a consequence of shifted substrate availability. Paradoxically, in MAFLD increased de novo lipogenesis (DNL) occurs despite hepatic insulin resistance. Therefore, we addressed this question using our animal model alb-SREBP-1c, which exhibits increased DNL by constitutively active SREBP-1c. Using an omics approach, we show that the abundance of ETC complex subunits and metabolic pathways are altered in liver of these animals. Analyses of cellular metabolic status by functional assays revealed that SREBP-1c-forced DNL induces a limitation of substrates for oxidative phosphorylation that is rescued by enhanced complex II activity. Furthermore, energy metabolism associated gene regulation indicates the counteracting to increase expression of mitochondrial genes and features cell communication by miRNA and exosomal RNA transfer. In conclusion, substrate availability fuels mainly complex II electron flows as a consequence of activated DNL with impact on whole body by liver-specific exosomal RNAs in early stages of MAFLD.

overall_design: C57Bl6 (C57Bl6) and C57Bl6-TgN alb-HA-hSREBP-1c-NT (alb-SREBP-1c)[Knebel et al. PLoS One. 2012;7(2):e31812. doi: 10.1371/journal.pone.0031812] mice were bred and maintained under standard conditions to until alb-SREBP-1c mice develop fatty liver and adipositas phenotype (24 weeks of age). Mice were sacrificed and primary hepatocytes were isolated.

first: Pia

last: Fahlbusch

first: Joerg

last: Kotzka


title: Human umbilical cord blood-derived MSCs trans-differentiate into endometrial cells and regulate Th17/Treg balance through NF-B signaling in rabbit intrauterine adhesions endometrium [scRNA-seq]


title: Prenatal maternal vitamin D deficiency sex-dependently programs adipose tissue metabolism and energy homeostasis in offspring

city: Marseille

first: Jean-Francois

last: Landrier

address: 27 Boulevard Jean Moulin

country: France

summary: In utero environment is crucial to ensure normal development of the fetus and to program metabolic health throughout the life. Beside macronutrients, the role of micronutrients, including vitamin D, begins to be explore. The aim of this study was to decipher the impact of maternal vitamin D deficiency (VDD), in normal and high-fat (HF) diet context, on adipose tissue metabolism and energy homeostasis in offspring, considering sex-specific responses. Body weight, energy expenditure, and spontaneous activity was differential impacted in juvenile male and female offspring born from VDD mice. In adulthood, a HF diet combined with maternal VDD disrupted glucose homeostasis and adiposity in male offspring but not in females. Such phenotypes were associated to different transcriptomic profiles in adipose tissue, which could be related to differential modulation of plasma 17b-estradiol concentrations. Thus, maternal VDD sex-dependently modulated metabolic fate of the offspring, especially when associated with HF diet in adulthood.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for CTRL (LF, HF) and VDD (LF, HF) male and female mice

first: Eva

last: Seipelt

first: Nicole

last: Haroun

first: François


title: Bulk RNA-Sequencing of mouse large intestinal lymphatics (capillaries and collectors)

city: New York

first: Marina

last: Schernthanner

state: NY

address: 1230 York Ave

institute: Rockefeller University

summary:
Purpose: Profile global gene expression of intestinal lymphatics to compare with published datasets
Methods: Intestinal tissues were digested, lymphatic endothelial cells were sorted using lymphatic markers including CD31, Podoplanin, Lyve1. Total RNA was isolated from sorted cells using the Direct-Zol RNA MiniPrep kit (Zymo). Samples were submitted for bulk RNA-Sequencing.
Conclusions: Gobal gene expression profile of intestinal lymphatics for further comparison with published datasets

first: Rachel

middle: E

last: Niec

first: Tinyi

last: Chu

first: Shiri

last: Gur-Cohen

first: Dana

last: Pe'er

first: Elaine

last: Fuchs


title: Human umbilical cord blood-derived MSCs trans-differentiate into endometrial cells and regulate Th17/Treg balance through NF-B signaling in rabbit intrauterine adhesions endometrium

first: Yong

last: Zhang

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

overall_design: Refer to individual Series


title: The Slc25a47 locus is a novel determinant of hepatic mitochondrial function implicated in liver fibrosis

city: Lausanne

first: Nadia

last: Bresciani

address: Route Cantonale

department: SV

institute: EPFL

summary: Background & Aims: Transporters of the SLC25 mitochondrial carrier superfamily bridge cytoplasmic and mitochondrial metabolism by channeling metabolites across mitochondrial membranes and are pivotal for metabolic homeostasis. Despite their physiological relevance as gatekeepers of cellular metabolism, most of the SLC25 family members remain uncharacterized. We undertook a comprehensive tissue distribution analysis of all Slc25 family members across metabolic organs and identified SLC25A47 as a liver-specific mitochondrial carrier.  Method: We used a murine loss-of-function (LOF) model to unravel the role of this transporter in mitochondrial and hepatic homeostasis. We performed extensive metabolic phenotyping and molecular characterization of Slc25a47hep-/- mice.   Results: Slc25a47hep-/- mice displayed a wide variety of metabolic abnormalities, as a result of sustained energy deficiency in the liver originating from impaired mitochondrial respiration in this organ. This mitochondrial phenotype was associated with a robust activation of the mitochondrial stress response in the liver, which in turn, induced the secretion of several mitokines, amongst which FGF21 played a preponderant role in the translation of the effects of the MSR on systemic physiology. To dissect the FGF21-dependent and -independent physiological changes induced by the loss of Slc25a47, we generated a double Slc25a47-Fgf21 LOF mouse model, and demonstrated that several aspects of the hypermetabolic state was entirely driven by hepatic secretion of FGF21. On the other hand, the metabolic fuel inflexibility induced by loss of Slc25a47 could not be rescued by genetical removal of Fgf21.   Conclusion: Collectively, our data place SLC25A47 at the center of mitochondrial homeostasis, which upon dysfunction triggers robust liver-specific and systemic adaptive stress responses.

overall_design: Hepatic mRNA profiles of 9 week- old- Slc25a47hep-/- and Slc25a47hep+/+ mice

first: Hadrien

last: Demagny

first: Vera

last: Lemos

first: Xiaoxu

last: Li


title: Modulating mitofusins to control mitochondrial function and signaling

first: Igor

last: Dolgalev

state: New York

address: 550 1st Ave

institute: NYU Grossman School of Medicine

summary: Purpose: Mitofusin regulate the fusion of the outer mitochondrial membrane. MASM7 is an activator of mitofusins, which promotes mitochondrial fusion. Whereas, MFI8 is an inhibitor of mitofusins, which inhibits mitochondrial fusion, and subsequently promotes mitochondrial fission. The goal of this study is to identify set of genes and pathways that are up-regulated  upon inhibition and activation of mitochondrial fusion.

overall_design: Isolated RNA from two independent biological replicates was analyzed using RNA-seq from mouse embryonic fibroblasts (MEFs) that were treated with MASM7 or MFI8.

first: Emmanouil

last: Zacharioudakis

first: Evripidis

last: Gavathiotis


title: Transcriptional effects of YAP/TAZ knockdown in  fibroblasts

status: Public on Jun 20 2022

city: Padova

first: Silvio

last: Bicciato

address: via U. Bassi 59/b

department: Molecular Medicine

country: Italy

institute: University of Padova

summary: Gene expression profiles of the cells composing the dorsal skin of young control and YAP/TAZ knockout mice with the skin of a old mouse.

overall_design: Single cell profiles were derived from the dorsal skin of young control, young YAP/TAZ knockout and old wild-type mice

first: Hanna

last: Sladitschek

first: Giulia

last: Brumana

first: Alberto

last: Guarnieri

first: Francesca

last: Zanconato

first: Michelangelo

last: Cordenonsi

first: Mattia

last: Forcato

first: Oriana

last: Romano

first: Stefano

last: Piccolo


title: The lack of the TetR-like repressor gene BCG_2177c (Rv2160A) may help mycobacteria overcome intracellular redox stress and survive longer inside macrophages when surrounded by a lipid environment

city: Mexico city

first: Lazaro

last: Garcia-Morales

state: Mexico

address: Av. Intituto Politecnico Nacional

department: MOLECULAR BIOMEDICIN

country: Mexico

institute: Center for Research and Advanced Studies of the National Polytechnic Institute

summary: We analyzed the genes expressed, or the transcriptome, of bacilli Mycobacterium bovis BCG Pasteur (wtBCG) and a mutant strain obtained by transposition of the gene BCG_ BCG_2177c (mtBCG), cultured in the presence of a lipid mixture (fatty acids/cholesterol) as main carbon source. Using RNAseq we identified a group of genes that provides novel insight regarding the metabolic pathways and transcriptional regulation, and pathogenic features that were influenced by BCG_2177c gene, a TetR-like repressor, during the lipid metabolism in slow-growing mycobacteria that could be extrapolated to other members of the MTBC.

sample_organism: Mycobacterium tuberculosis variant bovis BCG

overall_design: Comparative Transcriptomics between two strains of M. bovis BCG Pasteur (wild-type and BCG_2177c-muted strain) cultured in two carbon sources (Dextrose or Lipids mixture), at two states of growth (Exponential and Stationary Phase)

first: Patricia

last: Del Portillo

first: Juan M.

last: Anzola

first: Miguel A.

last: Ares

first: Addy C.

last: Helguera Repetto

first: Jorge F.

last: Cerna-Cortes

first: Alfonso

last: Mendez-Tenorio

first: Maria J.

last: Garcia

first: Isabel

last: Otal

first: Carlos

last: Martín

first: Jorge A.

last: González-y-Merchand

first: Sandra

last: Rivera Gutierrez


title: Transcriptional effects of YAP/TAZ knockdown in human fibroblasts

first: Silvio

last: Bicciato

summary: We compared the transcriptomes of control and YAP/TAZ-depleted fibroblasts, in the presence or absence of cGAS or STING depletion

overall_design: Human WI-38 fibroblasts were transfected with Control, YAP, TAZ (WWTR1), cGAS or STING siRNAs

first: Hanna

last: Sladitschek

first: Giulia

last: Brumana

first: Alberto

last: Guarnieri

first: Francesca

last: Zanconato


title: A lymphatic-stem cell interactome regulates intestinal stem cell activity

first: Marina

last: Schernthanner

summary:
Single-cell RNA-sequencing of murine small intestinal organoid cultured with and without lymphatic endothelial cells:
Purpose: To identify differences in cell composition in murine organoids upon coculture with lymphatic endothelial cells
Results: We recovered 818 cells with a median of 37750 UMIs per cell for the organoid alone and 1233 cells with a median of 79354 UMIs per cells for the cocultured organoid condition.
Conclusions: scRNASeq reveals different cell composition in organoids cocultured with lymphatic endothelial cells compared to control

Single-cell RNA-Sequencing of murine small and large intestinal tissue:
Purpose: To generate a reference dataset of all major cell types present in the small and large intestine of the mouse
Results: For the small intestine we recovered 8842 cells with a median of 4088 UMIs per cell compared to 8646 cells with a median of 10009 UMIs per cell for the large intestine.
Conclusions: scRNASeq recovers all of the major epithelial, immune and stromal cell types in the intestine.

Spatial transcriptomic profiling of murine small and large intestine:
Purpose: To spatially map the major cell types in the mouse intestine and infer cell:cell communication from neighboring cells
Conclusions: Spatial transcriptomic profiling of the mouse small and large intestine reveals all major cell types and allows for cell:cell signaling analyses upon integration with our matching scRNA-Seq data

overall_design:
16 wells each of organoids alone and cocultured organoids were pooled to sort 100k epithelial cells from each. Ctrl and coculture sample were pooled and submitted for 10X single-cell RNA-Sequencing
Bulk intestinal tissue  was digested and we sorted 50k alive cells, enriching for LGR5+ intestinal stem cells (50k) and CD31+ PDPN+ LYVE1+ lymphatic endothelial cells (20-25k). Tissue processing and 10X-based single-cell RNA-sequencing was done separately for the small and large intestine. C57BL/6 and Lgr5-eGFP-ires-CreERT2 (C57BL/6 background) mice were used for sorting experiments.
10X-Visium-based spatial transcriptomics was done on mouse small and large intestine (technical duplicates for each). We used the immunofluorescence-based protocol on 10 µm cryosections of small and large intestinal tubes.

first: Rachel

last: Niec

first: Tinyi

last: Chu

first: Shiri

last: Gur-Cohen

first: Dana

last: Pe'er

first: Elaine

last: Fuchs


title: Clinical significance of interferon signal based on mRNA-microRNA integration and plasma protein analyses in the critically ill patients with COVID19

city: Suita

first: Daisuke

last: Okuzaki

state: Osaka

address: Yamadaoka 3-1

department: Immunology Frontier Research Center

country: Japan

institute: Osaka univ.

summary: It is known that about 60% of all human messenger RNAs (mRNAs) regulated by microRNAs, the role of mRNAs and microRNAs in the critically ill patients with Coronavirus Infection 2019 (COVID-19) is unknown. To evaluate mRNA and microRNA in whole blood of the critically ill patients with COVID-19 and to elucidate the pathogenesis of COVID-19 including the subsequent proteins profile following mRNA and microRNA integration analysis. RNA was extracted from the whole blood in 5 healthy controls and 10 critically ill patients with COVID-19 at the time of admission. mRNA and miRNA were measured by RNA sequence, and gene expression variation and pathway analysis were performed. As the IFNs proteins profile cohort, IFN-α2, IFN-β, IFN-γ, IL-27 and IFN-λ1 were measured on the day of admission (day 1, 181 critical and 22 non-critical patients) and day 6-8 (168 critical patients) in COVID19 patients and 19 healthy controls. Compared to healthy controls, 3488 mRNA and 31 miRNA genes were identified in the differentially expressed genes in the critically ill patients with COVID-19 (p-value<0.05, Log 2 fold change> |2|). In the canonical pathway analysis using Ingenuity Pathway Analysis (IPA), interferon signaling pathway was the most activated. In plasma interferon levels, IFN-β was elevated along with the increase of severity compared to healthy controls. IFN-λ1 was elevated in moderate disease compared to healthy controls, and conversely, IFN-λ1 was lower in severe disease than in moderate disease. Integration of mRNA and microRNA analysis showed activated interferon signaling. The plasma interferon proteins profile revealed that IFN-β (type I) and IFN-λ1 (type III) played an important role in the disease progression of COVID-19.

overall_design: We performed the single RNA seq analysis with the whole blood in 16 healthy controls and 31 critically ill patients with COVID-19 at the time of admission.

first: Yuki

last: Togami

first: Hisatake

last: Matsumoto


title: Murine macrophages in mice infected with UTI

first: John

last: Ferdinand

summary: Mice were infected via a catheter with UPEC and the bladder harvested, macrophages purified and RNA seq performed

overall_design: 3 mice per group. Cells were combined from the 3 and sorted to yeild one pool of sampel which was used for RNAextraction and sequencing

first: Alex

last: Riding

first: Kevin

last: Loudon

first: Menna

last: Clatworthy


title: Transcriptional effects of YAP/TAZ knockout in primary mouse dermal fibroblasts

first: Silvio

last: Bicciato

summary: We compared the transcriptomes of control and YAP/TAZ knockout fibroblasts, and the latter in the presence or absence of a STING inhibitor

overall_design: YAP/TAZ knockout was triggered on primary cultures of mouse dermal fibroblasts by activating CreERT2 (expressed by the ROSA26 locus) with 4OH-Tamoxifen

first: Hanna

last: Sladitschek

first: Michelangelo

last: Cordenonsi

first: Mattia

last: Forcato

first: Oriana

last: Romano

first: Stefano

last: Piccolo


title: ChIP-Seq hnRNP E1, Cervical cancer SiHa cells

status: Public on Jun 16 2022

city: Taiyuan City

first: JinTao

last: Wang

state: Shanxi

address: 56 Xin Jian Nan Road

department: Department of Epidemiology, School of Public Health

institute: Shanxi Medical University

summary: Based on the specificity with which the unique KH structural domains of hnRNP E1 bind with RNA/DNA, considering that HPV16 provides RNA/DNA binding sites, we hypothesized that hnRNP E1 may be crucial to HPV16 oncogenes expression and cervical carcinogenesis. Based on the main purpose of this study was to explore the role of hnRNP E1 on the regulation of HPV16 oncogene expression in cervical cancerization, we conducted ChIP-seq in the untreated SiHa cell line.

first: Jintao

first: Li

last: Song

first: Rui

last: Mao

first: Ling

last: Ding

first: Zhiqiang

last: Tian

first: Mingxuan

last: Zhang

first: Jiahao

first: Ming

first: Yuanjing

last: Lyu

first: Chunliang

last: Liu

first: Meijuan

last: Feng

first: Haixia

last: Jia


title: Pro-Inflammatory Cytokines Mediate the Epithelial-to-Mesenchymal-Like Transition of Pediatric Posterior Fossa Ependymoma (single-nucleus ATAC-seq)

city: Philadelphia

first: Pablo

last: Camara

state: PA

address: 3700 Hamilton Walk

institute: University of Pennsylvania

summary: Pediatric ependymoma is a devastating brain cancer marked by its relapsing pattern and lack of effective chemotherapies. This shortage of treatments is due to limited knowledge about ependymoma tumorigenic mechanisms. By means of single-nucleus chromatin accessibility and gene expression profiling of posterior fossa primary tumors and distal metastases, we reveal key transcription factors and enhancers associated with the differentiation of ependymoma tumor cells into tumor-derived cell lineages and their transition into a mesenchymal-like state. We identify NFkB, AP-1, and MYC as mediators of this transition, and show that the gene expression profiles of tumor cells and infiltrating microglia are consistent with abundant pro-inflammatory signaling between these populations. In line with these results, both TGF-b1 and TNF-a induce the expression of mesenchymal genes on a patient-derived cell model, and TGF-b1 leads to an invasive phenotype. Altogether, these data suggest that tumor gliosis induced by inflammatory cytokines and oxidative stress underlies the mesenchymal phenotype of posterior fossa ependymoma.

overall_design: Single-nucleus ATAC-seq data was generated from 4 primary and 2 metastatic pediatric posterior fossa ependymal tumors using archived flash-frozen tissue.

first: Rachael

last: Aubin

first: Emma

last: Troisi

first: Javier

last: Montelongo

first: Adam

middle: N

last: Alghalith

first: MacLean

last: Nasrallah

first: Mariarita

last: Santi


title: NIS-lncRNA promotes  neuropathic pain by increasing Ccl2 expression

status: Public on Jun 15 2022

city: Newark

first: Yuan-Xiang

last: Tao

state: NJ

address: 183 S Orange Ave

department: Anesthesiology

institute: New Jersey Medical School, Rutgers, The State University of New Jersey

summary: Maladaptive changes of nerve injury–associated genes in dorsal root ganglia (DRGs) are critical for neuropathic pain genesis. Emerging evidence supports the role of long noncoding RNAs (lncRNAs) in regulating gene transcription. Here we identified a conserved lncRNA, named nerve injury–specific lncRNA (NIS-lncRNA) for its upregulation in injured DRGs exclusively in response to nerve injury. This upregulation was triggered by nerve injury–induced increase in DRG ELF1, a transcription factor that bound to the NIS-lncRNA promoter. Blocking this upregulation attenuated nerve injury–induced CCL2 increase in injured DRGs and nociceptive hypersensitivity during the development and maintenance periods of neuropathic pain. Mimicking NIS-lncRNA upregulation elevated CCL2 expression, increased CCL2-mediated excitability in DRG neurons, and produced neuropathic pain symptoms. Mechanistically, NIS-lncRNA recruited more binding of the RNA-interacting protein FUS to the Ccl2 promoter and augmented Ccl2 transcription in injured DRGs. Thus, NIS-lncRNA participates in neuropathic pain likely by promoting FUS-triggered DRG Ccl2 expression and may be a potential target in neuropathic pain management.

overall_design: To elucidate the mechanism by which DRG NIS-lncRNA upregulation participates in neuropathic pain, we performed high-throughput RNA sequencing  to identify the downstream targets regulated by NIS-lncRNA in DRGs of male mice. The ipsilateral L3/4 DRGs were harvested 5 weeks after microinjection with AAV5-Gfp or a mixture of AAV5-V1 and AAV5-V2 into the unilateral L3/4 DRGs of mice. Comparative gene expression profiling analysis of RNA-seq data revealed differentially expressed genes in microinjected DRGs 4 weeks after microinjection of a mixture of AAV5-V1 and AAV5-V2 compared to AAV5-Gfp control.


title: Construction of the axolotl cell landscape using combinatorial hybridization sequencing at single cell resolution [scRNA-seq]

status: Public on Jun 17 2022

city: Hangzhou

first: Fang

last: Ye

state: Zhejiang

address: 866 Yuhangtang Rd

institute: Zhejiang University

summary: Here, we developed a single-cell sequencing method based on combinatorial hybridization to generate a tissue-based transcriptomic landscape of the neotenic and metamorphosed axolotl. We performed gene expression profiling of over 1 million single cells across 19 tissues to construct the first adult axolotl cell landscape. Comparison of single-cell transcriptomes between the tissues of neotenic and metamorphosed axolotls revealed the heterogeneity of non-immune parenchymal cells in different tissues and established their regulatory network. Furthermore, we described dynamic gene expression patterns during limb development in neotenic axolotls. This system-level single-cell analysis of molecular characteristics in neotenic and metamorphosed axolotls, serves as a resource to explore the molecular identity of the axolotl and facilitates better understanding of metamorphosis.

sample_organism:
Ambystoma mexicanum
Homo sapiens
Mus musculus

overall_design: Bulk RNA sequencing datasets for seventeen adult neotenic axolotl tissues (brain, bladder, cloaca, eye, fore limb, hind limb, gill, gonad, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail) and fourteen adult metamorphosed axolotl tissues (bladder, brain, cloaca, eye, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail). Single cell combinatorial hybridization RNA sequencing datasets for nineteen adult neotenic axolotl tissues (brain, bladder, cloaca, eye, fore limb, hind limb, gill, gonad, pancreas, prostate, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail), sixteen adult metamorphosed axolotl tissues (bladder, brain, cloaca, eye, fore limb, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail, gonad).

first: Guoji

last: Guo

first: Xiaoping

last: Han

first: Weigao

last: Ee

first: Jiaqi

last: Li

first: Yuting

last: Fu


title: A Metabolic Regulatory Network for  the C. elegans Intestine

city: Worcteser

first: SUSHILA

last: BHATTACHARYA

address: Albert Sherman Center, Room 5.1051 386 Plantation St.

department: Department of Systems Biology (DSB)

institute: UMass Chan Medical School

summary: Metabolic perturbations can rewire metabolism under different physiological or pathological conditions, in part by transcriptional mechanisms. While numerous efforts have measured gene expression in response to individual metabolic perturbations, methods that determine all metabolic perturbations that affect the expression for a given gene or set of genes have not been available. Here, we use a gene-centered approach to derive a first-pass metabolic regulatory network for Caenorhabditis elegans by performing RNAi of more than 1,400 metabolic genes with a set of 19 promoter reporter strains that express a fluorescent protein in the intestine. We find that metabolic perturbations generally increase promoter activity, which contrasts with transcription factor TF RNAi, which tends to repress promoter activity. We identify several TFs that modulate promoter activity in response to perturbations of the electron transport chain. This work provides a blueprint for a systems-level understanding of how metabolism affects gene expression, and vice versa.

overall_design: C. elegans RNA-sequencing of three experiments. Animals (L1) were treated with 0.3µM Antimycin A & DMSO control until adult stage in biological triplicate.

first: Sushila

last: Bhattacharya

first: Albertha

last: Walhout


title: sham and MI heart transcriptome after 6-week-long light-phase intermittent prednisone or vehicle administration

status: Public on Jun 19 2022

city: Cincinnati

first: Mattia

last: Quattrocelli

state: OH

address: 240 Sabin Way

institute: Cincinnati Children's Hospital Medical Center

summary: Exogenous glucocorticoids interact with the circadian clock, but little attention is paid to their time of intake. Here we investigated the transcriptional effects of prednisone in  heart when pulsed as intermittent once-weekly 1mg/kg i.p. dose in  WT mice at ZT0 at 2-weeks after sham or myocardial infarction (MI; permanent ligation) surgeries.  Aftter 6 weeks of treatment, we performed RNA-seq analysis in myocardial tissue to quantitate gene expression at isoform level.

overall_design: 3 samples sham + vehicle; 3 samples sham + weekly prednisone; 3 samples MI +vehicle; 3 samples MI + weekly prednisone. Each sample was ~1ug RNA isolated through Trizol and further purifed through RNeasy columns.

first: Michelle

last: Wintzinger


title: Massively targeted evaluation of therapeutic CRISPR off-targets in cells

city: Aarhus

first: Lin

last: Lin

address: Høegh-Guldbergs Gade 10

department: Department of Biomedicine

country: Denmark

institute: Aarhus University

summary: Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.

overall_design: SURRO-seq analysis on 12K off-target library

first: Xiaoguang

last: Pan

first: Kunli

last: Qu

first: Yonglun

last: Luo


title: Tet2-mediated epigenetic programing of T follicular helper cell differentiation (RNA-Seq)

city: Salt Lake City

first: Jeffrey

middle: Scott

last: Hale

state: Utah

address: 15 North Medical Drive

department: Pathology

institute: University of Utah

summary: Tet2-mediated demethylation is a key component of epigenetic programing that promotes lineage specific gene expression and contributes to cellular differentiation and function. While the differentiation of CD4+ T cell subsets has been studied extensively, the epigenetic programs that regulate these processes remain unclear. We report that Tet2 acts to restrict the differentiation of T follicular helper (Tfh) cells in CD4+ T cells responding to viral infection. Tet2-deficient CD4+ T cells preferentially differentiated into highly functional germinal center (GC) Tfh cells that provided enhanced help for B cell responses. Using genome-wide expression and methylation analyses combined with Foxo1 ChIPseq analysis, we found that Tet2 coordinates with multiple transcription factors, including Foxo1, to mediate the demethylation and expression of their target genes following activation.

overall_design: Mouse antigen specific WT and Tet2KO CD4+ T cells (SMARTA TCR Transgenic) were sorted from spleens of non-infected mice (CD4+ T cell naïve samples) and LCMV Armstrong infected mice at 7 days post infection (effector Th1 and effector Tfh samples) for RNAsequencing

first: Andrew

middle: S

last: Baessler

first: J


title: Tet2-mediated epigenetic programing of T follicular helper cell differentiation (ChIP-Seq)

overall_design: Foxo1 ChIP-seq on sorted CD44high effector SMARTA CD4+ T cells 7 days post LCMV infection and on sorted naïve CD4+ T cells.

first: Zuolian

last: Shen

first: Jelena

last: Perovanovic

first: Dean

last: Tantin


title: Effects of triiodothyronine on human osteoblast-like cells: novel insights from a global transcriptome analysis

city: Botucatu

first: Célia

middle: Regina

last: Nogueira

address: Av. Prof. Mário Rubens Guimarães Montenegro, s.n.

country: Brazil

institute: Faculdade de Medicina de Botucatu - Unesp

summary: In order to investigate, at the mRNA level, the signaling pathways through which triiodothyronine (T3) affects osteoblast function, human mesenchymal stem cells derived from adipose tissue were subjected to a pre-established osteoinduction protocol, resulting in osteoblast-like cells, which were cultured with or without T3. RNA-Seq was performed using Illumina platform, and differential gene expression was assessed with DESeq2. Among differentially expressed genes, enrichment analysis was performed for biological processes against the Gene Ontology Consortium database, using both ClusterProfiler R package and STRING.

overall_design: The study consists of three groups with three biological replicates each. Osteoblast-like cells were cultured for 72h with no T3 for the control group, and 1nM or 10nM T3 for the remaining groups.

first: Bruna

last: Rodrigues

first: Lucas

last: Mathias

first: Igor

last: Deprá

first: Sarah

last: Cury

first: Miriane

last: Oliveira

first: Regiane

last: Olímpio

first: Maria

middle: T

last: Síbio

first: Bianca

last: Gonçalves


title: Synergistic function screening of multiple checkpoint perturbation in TIL

first: Lijia

last: Ma

address: Shilongshan Road No.18, Cloud Town, Xihu District

department: School of Life Sciences

institute: Westlake university

summary: A synergistic function screening system was established, in which a multiplexed CRIPSR knockout library targeting different checkpoint combinations was constructed and transduced into CD8+T cells to block multiple checkpoints in single cell. Based on this system, we performed a in vivo synergistic function screening of several checkpoints in tumor infiltrating CD8+T cells and find the best combined checkpoint blockade strategy to boost the CD8+T cell-mediated tumor elimination in vivo.

overall_design: In the library designing, a total of 6 checkpoints PDCD1, CTLA4, TIGIT, BTLA, TIM3 and A2AR were selected to make a total of 56 combinations (Table 1). Meanwhile, two distinct biological control groups were created. One group contained 30 combinations covering 5 co-stimulatory molecules for T cell activation, another one contained 15 combinations covering 4 critical molecules in TCR signaling. 6 groups of sgRNA were designed against individual combination. In each combination that contained less than 4 targeting sgRNAs, the non-targeting sgRNAs were included as the patch. At last, a total of 84 non-targeting 4-sgRNA combinations were included as the negative control.

first: Zhike

last: Lu

first: Ke

last: Ni

first: Yingying

last: Wang


title: Enabling multiplexed CRISPR/Cas9 screening via massively parallel in-library ligation

summary: Here we developed a massively parallel in-library ligation methodology to simultaneously perturb four pre-designed targets in CRISPR/Cas9 screening. Thousands of pairs of sequences precisely ligated with their counterparts in library, which enabled simultaneous expression of four gRNAs from each single vector. We demonstrated this novel method with 6,236 4-gene combinations targeting 1,599 immune response related genes, and generated a plasmid library with 1,400x coverage. The library performance was evaluated in a canonical T cell activation experiment, and combinations involved in TCR signaling pathway or TCR complex were successfully identified as positive regulators. Novel combination that is reflecting a potential pathway crosstalk was also verified. This new methodology expands the capacity of the perturbation in CRISPR screening and provided a powerful tool for researches in broad fields to study the combinatorial outcomes from coordinated gene behaviors.

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design:
We examined the library quality in multiple steps over the course of library preparation, including the plasmid library after the 1st golden gate and the final plasmid library after the 4th golden gate. To perform the multiplexed screening, the 4-gRNA expression vectors were transduced to the Jurkat cells with stable Cas9 expression (referred as Jurkat-Cas9 hereafter). After 10-days cell culture and antibiotic selection, cells were stimulated by T Cell Activator. Twenty-four hours after stimulation, the cell activity was assessed based on the surface CD69 expression. Both the top 25% (CD69+) and the bottom 25% (CD69-) cells were collected by cell sorter. NGS sequencing libraries were generated using genomic DNAs collected from the cell populations before-stimulation (Control), CD69+ (Top 25%) post-stimulation and CD69- (Bottom 25%) post-stimulation. The distributions of the 4-gRNA combinations were examined and compared across libraries.The G12 library and G23 library of each sample were treated as technical replicates. For each 4-gene combination, the log2 fold-change of the normalized read counts were calculated between the CD69+ and CD69- samples, and mean of replicates was used.

Please note that processed data generated from multiple samples is linked as Series supplementary file and is indicated in the corresponding sample description field.


title: Tet2-mediated epigenetic programing of T follicular helper cell differentiation (Methyl-Seq)

first: Jeffrey

last: Hale

overall_design: Mouse antigen specific WT and Tet2KO CD4+ T cells (SMARTA TCR Transgenic) were sorted from spleens of non-infected mice (CD4+ T cell naïve samples) and LCMV Armstrong infected mice at 7 days post infection (effector Th1 and effector Tfh samples) for genome wide enzymatic methylation sequencing.

first: Andrew

last: Baessler

first: Mark

last: Wadsworth

first: Katheryne

last: Varley

first: J


title: Tet2-mediated epigenetic programing of T follicular helper cell differentiation

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

overall_design: Refer to individual Series


title: ChIP-sequencing of PRMT1 proficient and deficient microglia

status: Public on Jun 14 2022

city: Montreal

first: Stephane

last: Richard

state: Quebec

address: Lady Davis Institute, 3755 Cot

country: Canada

institute: Lady Davis Institute- Jewish General Hospital

summary:
ChIP_sequencing analysis defined a novel role for the PRMT1 in microglia during normal and 0.2% cuprizone diet 5weeks
We then performed ChIP analysis using data obtained from ChIP-seq of microglia at two time points

overall_design:
Microglia ChIP profiles of 4-6 weeks-old wild-type (PRMT1Ifl/fl) and microglia specific knockout (PRMT1fl/fl; cx3r1cre-ert2) mice during normal and 0.2% cuprizone diet 5 weeks

Samples 1 to 10 (GSM6210097-GSM6210106) represent anti_H3K27Ac ChIP; while Samples 11 to 19 (GSM6210107-GSM6210115) are the inputs; they consisted in 1% aliquots of the samples taken post-sonication, but before IP.

first: Jeesan

last: Lee

first: David

last: Gosselin


title: Cholinergic Anti-inflammatory Pathway (CAP) Activation in a Model of Acute Lung Infection

status: Public on Jun 13 2022

city: Niskayuna

first: John

last: Graf

address: One Research Circle

institute: GE Global Research

summary: The cholinergic anti-inflammatory pathway (CAP), has been well-studied and shown to control peripheral cytokine levels and inflammation. This nerve pathway has been activated pharmacologically or by nerve stimulators and studied in animal models and clinical studies. However, these CAP investigations have typically been performed in models or study populations of severe systemic or pathological inflammation. This transcriptomic profiling study was conducted in rats to understand CAP in a model of actue lung infeaction. Splenic ultrasound stimulation was used to activate CAP in both healthy animals and animals with an acute lung infection.

overall_design: Transcriptomic profiles of blood of 36 adult male Sprague-Dawley rats 18 hours after recieving an injection of either Streptococcus Pneumoniae Serotype 19F bacteria (N=24) or saline (N=12) followed 16 hours later by ultrasound of the spleen for 5 minutes of stimulation (N=18) or sham treatment (N=18).

first: Umair

last: Ahmed

first: Anthony

last: Liew

first: Annette

first: Stavros

last: Zanos

first: Chris

last: Puleo


title: Construction of the axolotl cell landscape using combinatorial hybridization sequencing at single cell resolution [bulk RNA-seq]

first: Fang

last: Ye

summary: We adopted a combinatorial hybridization based single-cell RNA-seq method to generate tissue based transcriptome atlas of adult axolotl and whole organism transcriptome atlas of larva axolotl. Gene expression profiling of over 1million single cells across 19 organs constructed the first adult axolotl cell atlas. Comparison between neoteny and metamorphosis organs revealed transcriptome heterogeneity of structural cells in different tissues and a sophisticated regulatory network. Furthermore, we described dynamic gene expression pattern during neotenic larva axolotl limb development. These data serve as a rich resource to explore molecular identity of axolotl as well as its metamorphosis.

overall_design: Bulk RNA sequencing datasets for seventeen adult neotenic axolotl tissues (brain, bladder, cloaca, eye, fore limb, hind limb, gill, gonad, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail) and fourteen adult metamorphosed axolotl tissues (bladder, brain, cloaca, eye, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail). Single cell combinatorial hybridization RNA sequencing datasets for nineteen adult neotenic axolotl tissues (brain, bladder, cloaca, eye, fore limb, hind limb, gill, gonad, pancreas, prostate, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail), sixteen adult metamorphosed axolotl tissues ( bladder, brain, cloaca, eye, fore limb, hind limb, heart, intestine, kidney, liver, lung, skin, spleen, stomach, tail, gonad) and neotenic larva stage axolotl whole organism covering 5 stages including Day 30, Day 35, Day 45, Day 50, Day 70 postfertilization.

first: Guoji

last: Guo

first: Xiaoping

last: Han

first: Weigao

last: Ee

first: Jiaqi

last: Li

first: Yuting

last: Fu


title: Qualitative differences in diseases-associated mutants reveal molecular signatures and aberrant signaling-crosstalk

city: Minato-ku

first: Mutsuhiro

last: Takekawa

state: Tokyo

address: 4-6-1, Shirokanedai

institute: Institute of Medical Science, The University of Tokyo

summary: We generated HEK293 cells stably expressing diseases-derived mutants and performed a genome-wide transcriptome analysis using the 60K SurePrint G3 Human gene expression microarray technology. By the analysis of their gene expression profiles, we identified diseases-associated signature genes that may serve as diagnostic markers or therapeutic targets for the diseases.

overall_design: We compared mRNA expression of HEK293 cells stably expressing WT, or a representative disease-associated mutant (F53S or K57N).

first: Yuji

last: Kubota

first: Yuko

last: Fujioka

first: Ashwini

last: Patil

first: Yusuke

last: Takagi

first: Ryosuke

last: Naka

first: Daisuke

last: Matsubara

first: Masatomi

last: Iijima

first: Isao

last: Momose

first: Kenta

last: Nakai

first: Nobuo

last: Noda


title: The novel lysine deacetylase ABHD14B regulates transcription of metabolic genes

status: Public on Jun 10 2022

city: PUNE

first: Girish

last: Ratnaparkhi

state: MH

address: Dr. Homi Bhabha Road Pashan Pune 411008

department: Biology

country: India

institute: Indian Institute of Science Education & Research

summary:
Purpose: To understand the role of ABHD14B in cellular metabolism by regulation of transcription. To address this, we performed a RNA-seq experiment with stable knockdowns of ABHD14B in HEK293T cells generated by lentivirus-based shRNA technology.
Methods: Four biological replicates of HEK293T wild-type control, non-targeting shRNA control (NT), and two knockdowns (KD_2 and KD_3) of ABHD14B cells were collected and total RNA was extracted in QIAzol. The total RNA was used to generate a cDNA library using Quantseq 3' mRNA kit Fwd.
Results: Transcriptional profiling revealed that genes involved in cellular metabolism are differentially regulated in HEK293T ABHD14B knockdown cells.
Conclusions: Our study demonstrates the first extensive transcriptomics analysis of mammalian cells upon knocking down ABHD14B, and finds that loss of ABHD14B results in altered expression of glucose and lipid metabolic genes. This observation was complemented by changes in the levels of key intermediates of glucose metabolic pathways and triglycerides. Together, our findings illuminate an important metabolic role played by the KDAC ABHD14B.

first: Rajendran

last: Abinaya

first: Kamat

last: Siddhesh


title: N6-methyladenosine mRNA Modification Is Essential for Human Pancreatic Lineage Specification

first: Yunkun

last: Lu

address: 3 Qingchun East Rd.

department: Sir Run-Run Shaw Hospital


title: N6-methyladenosine mRNA Modification Is Essential for Human Pancreatic Lineage Specification [RNA-Seq]

summary: Recent advances in pancreatic differentiation from human pluripotent stem cells (hPSCs) hold great potentials for disease modeling and regenerative medicine, and more precise control over the dynamic differentiation process is critical. N6-methyladenosine (m6A) is the most prevalent internal messenger RNA (mRNA) modification, while the roles of m6A mark in pancreatic differentiation and development remain elusive. In addition, ALKBH5 is a major mRNA m6A demethylase and its role in pancreatic differentiation has not been reported. Here, we firstly studied mRNA m6A dynamics during pancreatic differentiation from hPSCs. Next, using the CRISPR-based genome editing tool, we generated ALKBH5 knockout hPSC lines and found that ALKBH5 plays important roles in pancreatic specification. After that, we conducted a series of functional and mechanistic studies, and demonstrated that ALKBH5 modulated many important genes involved in pancreatic differentiation. Collectively, our findings identified ALKBH5 as an essential regulator of human pancreatic differentiation and highlighted that m6A modification presents a new layer of regulation at epitranscriptome levels during cell-fate specification.

overall_design:
RNA-seq of hILO-WT and hILO-A5-KO cells.
RNA-seq of PP-WT and PP-A5-KO cells.

first: Xiaojie

last: Ma

first: Jie

last: Cao

first: Ziyu

last: Zhou

first: Jianzhao

last: Liu

first: Saiyong

last: Zhu


title: Analysis of gene expression patterns in denervated muscle atrophy

status: Public on Jun 12 2022

city: Nantong

first: hualin

last: sun

state: jiangsu

address: suzhou

institute: Nantong University

summary: To further analyze the gene expression patterns in denervated muscle atrophy, we have employed whole genome microarray.

overall_design: The rats were randomly divided into the sham and model group. In the sham group [The sciatic nerve was exposed, and then the skin was sewn and disinfected]. In the model group [The sciatic nerve was exposed and disconnected, and then the skin was sewn and disinfected]. The tibialis anterior muscle was collected from rats in both groups at different time points (0.25 h, 0.5 h, 3 h, 6 h, 12 h, 24 h, 3 d, 7d, 14 d, 21 d, and 28 d) after denervation. Gene expression in the skeletal muscle at the different time points after denervation was analyzed by microarrays,

first: Hualin

last: Sun


title: Single-Cell RNA Sequencing of Pediatric Ependymoma uncovers Subclonal Heterogeneity associated with Patient Survival

city: Beijing

first: Haoda

last: Wu

address: Lincui East Road

department: State Key Laboratory of Molecular Development Biology

institute: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

summary: Ependymoma is a malignant glial tumor occurring throughout central nervous system, with one third of relapse rate. The increasing accessibility of single-cell transcriptome has accelerated our understanding of ependymoma. In this study, we generated a high-resolution single-cell dataset with a particular focus on the comparison of subclonal differences within tumor populations. As a proxy to traditional pseudotime analysis, we developed a novel trajectory scoring method to predict survival outcomes and specific molecular characteristics. Moreover, we identified putative cell-cell communication features between relapsed and primary samples and show upregulation of pathways associated with immune cell crosstalk. Our results reveal both inter- and intratumoral gene expression profiles and tumor differentiation and provide a framework for future studies on transcriptomic signatures of individual subclones in brain tumors at single-cell resolution, by which could complement existing published datasets and provide valuable insights into cell-type-specific properties and lay the foundation for therapeutic treatments of diseases.

overall_design: In this study, we report the generation of curated single-cell transcriptomes from EPN patients across PF-A and ST regions. To address intratumoral subclone heterogeneity, we used a deconvolution approach using inferCNV to compare molecular features between subclones in a single PF-A sample as a proof-of-concept. We then identified a subclone-specific cilia-associated program within an individual PF-A EPN sample which was correlated with a more highly differentiated subpopulation. As a proxy to traditional pseudotime analysis commonly used, we also developed and incorporated a trajectory score analysis to our datasets to predict correlations between survival outcomes in EPN and specific molecular characteristics, as well as primary and recurrent tumor populations. Finally, we identified putative cell-cell communication features between relapsed and primary EPN samples and show upregulation of pathways associated with immune cell crosstalk. Our results reveal both inter- and intratumoral gene expression profiles and cell composition differences associated with subclonal variability and provide a framework for future studies on transcriptomic signatures of individual subclones in brain tumors at single-cell resolution.

first: Qing-Feng

first: Jingkai

last: Xu

first: Wenhui

last: Mu


title: Transcription factor HESX1 enhances mesendodermal commitment of human embryonic stem cells by modulating ERK1/2 signaling

city: Shanghai

first: Ran

last: Tong

address: 227 South Chongqing Road, Huangpu District

institute: Shanghai Jiao Tong University

summary: Transcription factors are key determinants of lineage commitment during mammalian development. However, the function and molecular mechanism for the transcription factors in the formation of three primary germ layers during human embryonic development are not fully elucidated.  Here, we report that homeobox-containing transcription factor HESX1 plays a critical role in mesendodermal (ME) commitment of human embryonic stem cells (hESCs). Our results show that expression of HESX1 in hESCs is regulated by OCT4 and NANOG, and that its expression level changes with hESC differentiation. We find that knockdown of HESX1 does not disrupt the undifferentiated state of hESCs, in terms of cell morphology and expression levels of pluripotency- associated genes. However, HESX1 deficiency in hESCs impairs their ME commitment, whereas forced expression of HESX1 significantly enhances ME marker expression during ME commitment. Interestingly, HESX1 knockdown in hESCs represses ERK1/2 signaling activated by ME induction, while overexpression of HESX1 markedly enhances ERK1/2 activity during ME commitment of hESCs. Of note, MEK inhibitor PD0325901 weakens or even eliminates HESX1 overexpression- mediated promotive effects on ME induction in a dosage- dependent manner. Together, this study identifies a novel role of HESX1 in hESC commitment to ME cells and establishes the functional link between a transcription factor and lineage- associated signaling. These findings would help to better understand early human development and develop more efficient protocols to induce hESC differentiation to desired lineages.

overall_design: To investigate the role of HESX1 in hESC self-renewal and mesendodermal(ME) differentiation, we depleted HESX1 in hESCs by two small interfering RNAs (siRNAs) targeting different coding regions of HESX1. A non-targeting (NT) siRNA was used as a negative control. To determine whether HESX1 deficiency would alter hESC gene expression program, the RNA-seq experiment was performed for siNT(NT_mTe), siHESX1 #1(si5_mTe) and siHESX1 #2(si8_mTe) hESC samples, with each having two biological replicates. To determine whether HESX1 would play a role in ME lineage commitment, we applied a previously published protocol for efficient ME induction from hESCs. There were 4 groups of cells: siNT mTeSR1 cells, siNT ME(NT_ME) cells, siHESX1 #1 ME(si5_ME) and siHESX1 #2 ME(si8_ME) cells. We performed RNA-seq experiments for the 4 groups of cells described above with 2 biological replicates for each group.

first: Han

last: Wang

first: Ying

last: Jin

first: Hui

last: Li


title: Effect of EGR3 overexpression in the cell line SEM

status: Public on Jun 09 2022

city: Frankfurt am Main

first: Marius

last: Külp

address: Theodor-Stern-Kai 7

department: Department of Medicine II, Hematology/Oncology

institute: Goethe University Hospital Frankfurt

summary: FLAG-tagged EGR3 was overexpressed in the cell line SEM to investigate promotor binding of EGR3 in the t(4;11) cellular context.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for EGR3::FLAG using a FLAG-antibody in comparision to input in EGR3::FLAG-overexpressing SEM cells.

first: Patrizia

last: Larghero

first: Claus

last: Meyer

first: Rolf

last: Marschalek


title: Transcriptional changes to cytotoxic stresses are driven by HSF-activated genes and enhancers [PRO-seq]

city: Turku

first: Samu

middle: Valtteri

last: Himanen

state: Varsinais-Suomi

address: Tykistökatu 6A

department: Cell Biology

country: Finland

institute: Åbo Akademi University

summary: We utilized precicion run-on sequencing (PRO-seq) to analyze the roles of HSF1 and HSF2  in the reprogramming of transcription under oxidative stress and heat shock. PRO-seq was performed in wild-type (WT), HSF1 knock-out (HSF1 KO) and HSF2 knock-out (HSF2 KO) mouse embryonic fibroblasts (MEFs) that were treated with heat shock (HS) or oxidative stress induced by menadione (MD).

overall_design:
2 replicates of PRO-seq was performed in WT, HSF1 KO and HSF2 KO MEFs that were exposed to HS (42 °C, 1 h) or MD (30 µM, 2 h).
Please note that each processed data file was generated from both replicates and is linked to the corresponding Rep1 sample records.

middle: V

first: Mikael

last: Puustinen

first: Anniina

last: Vihervaara

first: Lea

last: Sistonen


title: Comparative analysis of murine Th17 cell transcriptomes in the presence of Smo inhibitor cyclopamine or carrier control

city: Cambridge

first: Kamal

last: Kishore

address: Robinson Way

department: Bioinformatics Core

institute: CRUK CI

summary: T helper 17 (Th17) cells play a key role in barrier protection against fungal and bacterial pathogens but are also pathological drivers of many inflammatory diseases. We find that Hh signaling inhibition through Smoothened inhibitor cyclopamine selectively inhibits Th17 lineage differentiation but not the development of Th1, Th2, or iTreg CD4+ Th cells. In these experiments we analysed the transcriptomes of Th17 cells that had been polarized in the presence of cyclopamine or carrier control to profile the global transcriptome of Th17 cells in which the Hh pathway had been blocked. The aim of this analysis was to identify the target genes downstream of Smo that may be important for Th17 polarisation.

overall_design: Naïve CD4+ T cells were purified from spleen and peripheral lymph nodes of C57BL/6 mice and stimulated with plate-bound anti-CD3e/CD28 antibodies in the presence of polarizing cytokines (IL-6, IL-23, IL-1b, TGFb) to generate Th17 cells. Th17 cells were polarized as described above, stimulated in the presence of the indicated doses of cyclopamine  (5uM or 7.5uM) or carrier control for three days and harvested at day 3. Six samples/group. Libraries were generated using the TruSeq stranded mRNA kit (Illumina) following the manufacturer’s instructions and sequenced using single-read sequencing with the HiSeq4000 platform (Illumina). Reads were aligned to the mouse genome version GRCm38 using STAR v2.5.3a. Read counts were obtained using feature Counts function in Subread v1.5.267 and read counts were normalized and tested for differential gene expression using the DESeq2 workflow. Multiple testing correction was applied using the Benjamini–Hochberg method.

first: Maike

last: de la Roche


title: Expression data from mouse conventional dendritic cells (cDCs)

city: Perugia

first: Marco

last: Gargaro

state: Perugia

address: Polo Unico Sant'Andrea delle Fratte, P.le Gambuli, Bldg C, 4th Floor

department: Medicine and Surgery

institute: University of Perugia

summary: We used microarrays to understand  the role of  L-Kynurenine in inducing tolerogenic cDC2

overall_design: AhR Flox (Ahr f/f) and Ahr Flox Vav iCre (Ahr f/f Vav iCre) bone marrow derived cDC2 were sort-purified and treated with LPS alone or in combination with L-Kynurenine (L-Kyn) for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain gene expression signature that are induced by L-Kyn in cDC2.

first: Kenneth

last: Murphy

first: Francesca

last: Fallarino


title: Quantitative Analysis of Phaeodactylum tricornutum transcriptomes

city: Xiamen

first: Yanchun You

last: YOU

state: Fujian

address: No. 422, Siming South Road

institute: Xiamen University

summary:
Purpose: Using RNAseq to profile transcriptomes to compare wildtype and PtTryp2 knockout lines across different nutrient conditions, aim to further explore the molecular function of PtTryp2 in regulating cell growth and responses to N- and P-stress.
Methods: Libraries for RNAseq were created from 1µg of mRNA from each culture. The resulting libraries were loaded into the patterned nanoarray, and single-end 50 bp reads were generated on BGIseq500 platform (BGI-Shenzhen, China), with a data output of about 22 M total clean reads for each library.
Results: Transcriptomic data show that PtTryp2 knockout led to the upregulation of most of the nitrogen assimilation and metabolism genes under both N-depleted and replete conditions. In addition, when comparing N-depleted with N-replete conditions, 646 differentially expressed genes (DEGs) were identified in the blank vector control (KOC) but only 187 in PtTryp2-KO1, considerably fewer in the knockout mutant. Based on RNA-seq, remarkably more DEGs were found for the P-depleted versus nutrient-replete comparison in PtTryp2-KO1 (1501) than that in KOC (277). Besides, in PtTryp2-KO1, the majority of these DEGs (77.25%) exhibited greater fold changes than that in KOC.
Conclusions: Transcriptomic data coupling with physiological experiments, suggesting that PtTryp2 knockout led to amplitude magnification of the NO3- and Pi uptake “seesaw”, whereas PtTryp2 overexpression dampened it. All our data demonstrate in concert that PtTryp2 is a coordinate regulator of N-P stoichiometric homeostasis.

overall_design: RNA-seq profiles of 18 Phaeodactylum tricornutum cell samples.

first: Senjie

last: Lin

first: Yanchun

last: You


title: Transcriptional changes to cytotoxic stresses are driven by HSF-activated genes and enhancers [ChIP-seq]

summary: We utilized chromatin immunoprecipitation sequencing (ChIP-seq) to analyze the binding of HSF1 and HSF2  to chromatin under oxidative stress and heat shock. ChIP-seq was performed in mouse embryonic fibroblasts (MEFs) that were exposed to heat shock (HS) or oxidative stress induced by menadione (MD). Antibodies against HSF1 and HSF2 were used for immunoprecipitation.

overall_design:
2 replicates of ChIP-seq were performed in WT MEFs that were treated with HS (42 °C, 1 h) or MD (30 µM, 2 h). Antibodies against HSF1, HSF2 were used for ChIP-seq. IgG and Input are provided for the normalization of the data.
Please note that each processed data file was generated from both replicates and is linked to the corresponding Rep1 sample records.


title: High-Throughput mRNA Sequencing Reveals Potential Therapeutic Targets of Febuxostat in Secondary Injury After Intracerebral Hemorrhage

city: Chengdu

first: Xueyan

last: Wang

address: 37 Guoxue Alley

institute: Sichuan University

summary: Febuxostat is a urate-lowering medication for the treatment of patients with gout. This study was performed to elucidate the effects and underlying mechanisms of febuxostat on neuronal injury induced by intracerebral hemorrhage (ICH) in mice. The results showed that the administration of febuxostat improved neurological severity scores and blood-brain barrier (BBB) permeability. Moreover, febuxostat attenuated neuronal cell death and cytokine levels compared with the ICH group. Next, we conducted a transcriptome analysis of the neuroprotective effects of febuxostat. The overlapping significant differentially expressed genes (DEGs) were identified. Gene ontology (GO) analysis revealed that the overlapping significant DEGs were most enriched in five items. The intersecting DEGs of the aforementioned five pathways were Wisp1, Wnt7b, Frzb, and Pitx2. In addition, GO terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that DEGs were mainly involved in the wnt signaling pathway. Furthermore, the expression of Wisp1 and Wnt7b in the perihematomal region at 72 h post-ICH was observed. The results showed that both Wisp1 and Wnt7b were increased in the ICH group and were decreased by the administration of febuxostat. Taken together, the study showed that febuxostat protected against secondary brain injury after ICH and the Wnt7b-Wisp1 pathway was closely related to neuroprotective effects.

overall_design: Differentially expressed genes were identified among Sham, ICH and Feb groups by using mRNA-Seq

first: Chenyu

last: Zhang

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last: Li

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last: Xu

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last: Xiang

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last: Bai

first: Ying

first: Qi

first: Tiejun

first: Linchuan

last: Liao