title: Exogenous interleukin-1 beta stimulation regulates equine tenocyte function and gene expression which can only be rescued by pharmacological inhibition of interleukin 1 receptor, but not nuclear factor kappa B, signalling

city: Hatfield

first: Ross

middle: Eric

last: Beaumont

state: Hertfordshire

address: Hawkshead Ln

department: Clinical science and services

institute: Royal Veterinary College

summary: We investigated how Interleukin 1 beta impacts equine tenocyte function and global gene expression in 3D culture and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB or interleukin 1 signalling.

overall_design: mRNA profiles of eight independent lines of adult tenocytes were generated by RNA sequencing, using Illumina NovaSeq6000. The sequence reads that passed quality filters were analyzed using the pseudoaligner Salmon and differential expression quantified using DESeq2. Gene ontology analysis was then conducted using Panther GoSlim and pathway analysis with Enrichr.

first: R

first: E

last: Smith

first: L

last: Zhou

first: N

last: Marr

first: C

last: Thorpe

first: D

last: Guest


title: Differential binding of PARP-1 domains

status: Public on May 25 2023

city: Grand Forks

first: Gbolahan

last: Bamgbose

state: ND

address: 501 N Columbia Rd

department: Biomedical Sciences

country: USA

institute: University of North Dakota

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Evolution of immune and stromal cell states and ecotypes during gastric adenocarcinoma progression

status: Public on Jun 10 2023

city: Houston

first: Linghua

last: Wang

address: 1881 East Road,

institute: MD Anderson Cancer Center

type: Expression profiling by high throughput sequencing

summary: Understanding how the tumor microenvironment (TME) is orchestrated along the continuum of gastric adenocarcinoma (GAC) may uncover novel therapeutic strategies. Here, we performed comprehensive single-cell profiling of precancerous lesions, localized and metastatic GACs, identifying alterations in TME cell states and phenotypes during tumorigenesis and progression. IgA+ plasma cells appear to be the most active regulators in the premalignant microenvironment, whereas immunosuppressive myeloid and stromal subsets dominated the late-stage GACs. We discover six TME ecotypes, four of which are unique to the premalignant/progression in the continuum, with two ecotypes, identified in primary GACs, showing strong associations with histopathologic, genomic characteristics and prognosis. Extensive stromal remodeling is evident with GAC progression. High SDC2 expression in cancer-associated fibroblasts (CAFs) correlates with aggressive phenotypes and poor survival and SDC2 overexpression in CAFs contributes to tumor growth. This study illustrates a roadmap of the GAC TME and highlights potential targets for further investigation.

overall_design:
Six patients (Pt1-5, Pt9) were enrolled and diagnosed at Zhejiang Cancer Hospital. Diagnosis was confirmed by experienced gastrointestinal pathologists and radiologists and fresh biopsies (primary tumors, ovarian or liver metastases, n = 17) were obtained from each patient, with adjacent normal tissue or peripheral blood as controls. The primary tumor tissues and adjacent non-neoplastic tissues were collected by gastroscopy. The ascites samples were collected when patients required a therapeutic paracentesis and cells were isolated for scRNA-seq. The liver metastatic tissues were collected when patients required needle aspiration biopsy during the diagnostic procedure and the ovary metastatic tissues were collected when patients required cytoreductive surgery. None of these six patients were treated with chemotherapy or radiotherapy prior to sample collection.
**Raw data not provided due to patient privacy concerns**

first: Jiangjiang

last: Qin

first: Ruiping

first: Xiangdong

last: Cheng


title: Role of PARP-1 in transcriptional regulation during development


title: Differential binding of PARP-1 domains  (ZNI-YFP ChIP-seq)

summary: We interrogated the differential binding of PARP-1 domains (ZnI, ZnII, AD-WGR) and a ZnI mutant in Drosophila via ChIP-seq of third-instar larvae.

overall_design: We performed ChIP-seq using third-instar larvae expressing ZNI-YFP and Control animals (3 biological replicates per group).

first: Sarah

last: Johnson

first: Tulin

last: Alexei


title: Differential binding of PARP-1 domains  (DZNI-YFP ChIP-seq)

overall_design: We performed ChIP-seq using third-instar larvae expressing ZnI mutant (ΔZnI) and Control animals (3 biological replicates per group).


title: Role of PARP-1 in transcriptional regulation during development (ChIP-seq)

summary: We interrogated the role of PARP-1 in developmental gene regulation in Drosophila via ChIP-seq in third-instar larvae.

overall_design: We performed ChIP-seq using third-instar larvae expressing PARP1-YFP and Control animals (3 biological replicates per group).

first: Alexei

last: Tulin


title: Effect of coculture with normal colonic epithelial cells on gene expression of RKO colon cancer cells

status: Public on May 24 2023

city: Shanghai

first: Bingjie

last: Guan

address: 85 Wujin Road

country: China

institute: Shanghai General Hospital

summary: To investigate the effect of extracellular vesicle-treated normal colonic epithelial cells (FHC) on the activities of oncogenic signaling pathways in colon cancer cells (RKO), RKO cells were cocultured with FHC cells prestimulated with extracellular vesicles (EVs) or PBS.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for the treated RKO cells and the control RKO cells.The treated RKO cells:cocultured with FHC cells prestimulated with EVs; The control RKO cells:cocultured with FHC cells prestimulated with PBS. Three samples were included in each group.

first: Youdong

last: Liu

first: Bowen

last: Xie

first: Dongwang

last: Yan


title: CXCL9/10-engineered dendritic cells promote T cell activation and enhances immune checkpoint blockade for lung cancer.

status: Public on May 22 2023

city: Los Angeles

first: Linh

middle: My

last: Tran

state: California

department: Medicine - Pulmonary & Critical Care

institute: University of Los Angeles

summary: Immune checkpoint blockade (ICB), notably Programmed Death-1/Programmed Death-Ligand 1 (PD-1/PD-L1) inhibition, has revolutionized the treatment of non-small cell lung cancer (NSCLC). However, durable responses are only observed in a subpopulation of patients. Defective antigen presentation and an immunosuppressive tumor microenvironment can lead to deficient T-cell recruitment and ICB resistance. We evaluated in situ vaccination with CXCL9 and CXCL10-engineered dendritic cells (CXCL9/10-DC) as a novel strategy to overcome resistance to ICB using Lkb1-null murine NSCLC model. We utilized single-cell RNA-seq to evaluate alterations of immune infiltration associated with the new therapy, which combines intratumoral CXCL9/10-DC administration and PD-1 inhibition.

overall_design: Murine tumors were harvested on day 14 following subcutaneous inoculation with KPL-3M tumor cells and treatment. Single-cell suspension of murine tumors was stained with Zombie-NIR LIVE/DEAD stain and CD45 antibody before being isolated for alive CD45+ cells by Fluorescence-activated cell sorting (FACS) with a 100-mm nozzle.

first: Raymond

middle: J

last: Lim

first: Ramin

last: Salehi-Rad

middle: M

first: Camelia

last: Dumitras

first: Rui

last: Li

first: Tejas

middle: S

last: Patel

first: Samantha

last: Man

first: Cara

last: Yen

first: William

middle: P

last: Crosson

first: Jensen

middle: W

last: Abascal

first: ZiLing

last: Huang

first: Stephanie

middle: L

last: Ong

first: Kostyantyn

last: Krysan

first: Bin

first: Steven

last: Dubinett


title: Characterizing glucokinase variant mechanisms using a multiplexed abundance assay

city: Copenhagen N

first: Sarah

last: Gersing

address: Ole Maaløes vej 5

country: Denmark

institute: University of Copenhagen

summary: Amino acid substitutions can perturb protein activity in multiple ways. Understanding their mechanistic basis may pinpoint how residues contribute to protein function. Here, we characterize the mechanisms of human glucokinase (GCK) variants, building on our previous comprehensive study on GCK variant activity. We assayed the abundance of 95\% GCK missense and nonsense variants, and found that 59\% of hypoactive variants have a decreased cellular abundance. By combining our abundance scores with predictions of protein thermodynamic stability, we identify residues important for GCK metabolic stability and conformational dynamics. These residues could be targeted to modulate GCK activity, and thereby affect glucose homeostasis.

overall_design: Three regional libraries of human glucokinase variants (aa 2-171, 172-337, 338-465) were transformed into yeast (BY4741), and were grown on SC+leucine+methionine+histidine medium with methotrexate to select for glucokinase variant abundance. The variants were sequenced before and after selection using 14 tiles spanning the glucokinase ORF to determine their change in frequency and thereby effect on protein abundance.

first: Rasmus

last: Hartmann-Petersen


title: Transcriptomics profile of polyQ expressing U2OS cells and clofazimine treated polyQ expressing U2OS cells

status: Public on May 11 2023

city: Solna

first: Xuexin

address: Tomtebodavägen 23A

department: Department of Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

summary: To better understand the mechanism by which CFZ impacts polyQ toxicity, we conducted a transcriptomic analysis in polyQ94-EGFP expressing U2OS (5μM, 8 days). Data from a transcriptomics experiment in polyQ94-U2OS cells treated with CFZ (5 μM, 8day) was compared to a DMSO-treated control.

overall_design: Transcriptomic analysis in polyQ94-EGFP expressing U2OS cells exposed or not to CFZ (5μM, 8days) was performed. Total RNA was extracted from cell pellets using the Agilent Absolutely RNA Miniprep Kit following the manufacturer’s instructions. The sequencing library was constructed with the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen), and approximately 10 million reads were obtained per sample by Illumina sequencing. STAR was used for sequence alignment based on the GRCh38 DNA primary assembly reference build, and quantification was done using featureCounts(Liao et al., 2014) with reference build GRCh38.101. Differential expression (DE) analyses between the groups were performed using DESeq2.

first: Fredrik

middle: F

last: Billai

first: Louise

last: Lidemalm


title: Next Generation Sequencing Facilitates Quantitative Analysis of  FLox and SMYD2 KO Aortic Smooth Muscle Cell Transcriptomes

status: Public on May 12 2023

city: Guangzhou

first: Yu

last: Zhou

state: Guangdong

address: No 58, Zhongshan 2nd street

department: Division of Vascular Surgery

institute: The First Affiliated Hospital, Sun Yat-sen University

summary:
Purpose: Methyltransferase SMYD2 has been implicated in cancer cell growth and cardiomyocytes development. However, its role in  vascular smooth muscle cell(VSMC) is still unknown. This study aims to investigate differentially expressed genes in  Flox and SMYD2 knockout VSMC.
Methods:RNA profiles of  Aortic Smooth Muscle Cell isolated from Flox and SMYD2 knockout (SMYD2−/−) mice were generated by deep sequencing, in triplicate, using BGISEQ-500 platform. Firstly, we filter the low quality reads (More than 20% of the bases qualities are lower than 10),reads with adaptors and reads with unknown bases (N bases more than 5%)to get the clean reads. Then we map those clean reads onto reference genome, followed with novel gene prediction, SNP &INDEL calling and gene splicing detection. Finally, we identify DEGs (differentially expressed genes) between samples and do clustering analysis and functional annotations.
Results: In our project, we sequenced 6 samples on BGISEQ-500 Platform in total and generated about 4.69 Gb per sample. The average genome mapping rate is 96.62% and the average gene mapping rate is 80.92%. 18,112 genes were identified in which 16,992 of them are known genes and 1,351 of them are novel genes. 13,680 novel transcipts were identified in which 11,251 of them are previously unknown splicing event for known genes, 1,351 of them are novel coding transcripts without any known features, and the remaining 1,078 are long noncoding RNA.
Conclusions: Our study represents the first detailed analysis of SMYD2 KO VSMC transcriptomes, with biologic replicates, generated by RNA-seq technology.

overall_design: Aortic Smooth Muscle Cell mRNA profiles of  Flox and SMYD2 KO mice were generated by deep sequencing, in triplicate, using BGISEQ-500 platform.

first: Shaligram

last: Sharma

first: Xiaoqing

last: Guan

first: Zuojun

last: Hu

first: Chunying


title: Fun30-Myc ChIP-seq in Spo11 wild type and spo11-yf mutants

status: Public on May 01 2023

city: Emeryville

first: Pei-Ching

state: CA

address: 1545 Park Ave

institute: Metagenomi

summary: Resection, nucleolytic processing of DSB ends is necessary to generate 3' single-stranded DNA tails (ssDNA) for homologous recombination (HR). Meiotic recombination initiated by Spo11 induced double-strand breaks (DSBs) is essential for the accurate segregation of homologous chromosomes. After cleavage, Spo11 stays covalently linked to the DSB ends, which requires MRX/Sae2 incision on broken molecules to allow following Exo1-mediated resection. Exonuclease I activity is inhibited by nucleosome bound DNA in vitro. To ensure resection proceeds, resection machineries must overcome chromatin barrier.  Here we show that DSB dependent enrichment of Fun30 proteins at hotspots and meiotic axes.

overall_design: 16 samples total: two biological replicate ChIP-seq of S.cerevisiae for each genetic background and at 4h after meiosis induction

first: Hajime

last: Murakami

first: Scott

last: Keeney


title: A new tractable method for generating Human Alveolar Macrophage Like cells in vitro to study lung inflammatory processes and diseases

status: Public on May 08 2023

city: San Antonio

first: Larry

last: Schlesinger

state: Texas

address: 8715, W Military Drive, Larry Schlesinger Laboratory, Texas Biomedical Research Institute

department: Host Pathogen Interactions program

institute: Texas Biomedical Research Institute

summary:
Alveolar macrophages (AM) are unique lung resident myeloid cells and often the first cell type to contact airborne pathogens. The contribution of human AMs (HAM) to pulmonary diseases remains poorly understood due to the difficulty in accessing them from human donors and their rapid phenotypic change during in vitro culture. There remains an unmet need for cost effective methods for generating human cells with a HAM phenotype, particularly important for translational studies and clinical benefits to humans, including analyses of host cell responses to vaccine candidates. We developed cell culture conditions that mimic
the lung alveolar environment in humans using lung lipids, that is, Infasurf (calfactant, natural bovine surfactant) and lung-associated cytokines (granulocyte macrophage colony–stimulating factor, transforming growth factor-β, and interleukin 10) that facilitate the conversion of blood-obtained monocytes to an AM-like (AML) phenotype and function in tissue culture. Similar to HAM, AML cells are particularly susceptible to both Mycobacterium tuberculosis and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. This study reveals the importance of alveolar space components in the development and maintenance of HAM phenotype and function and provides a readily accessible model to study HAM in infectious and inflammatory disease processes,
as well as therapies and vaccines.

overall_design:
Freshy isolated human alveolar macrophages (HAM, n=2) and 2h adherent monocyte derived macrophages (MDM, n=3) and alveolar macrophage-like (AML, n=3) cells were washed once with 1% Dulbecco's phosphate-buffered saline (DPBS) (Gibco), lysed in TRIzol (Invitrogen) and RNA was isolated using Direct-zol RNA Miniprep kit, R2052 (Zymo Research) as per the manufacturer’s instructions. Isolated RNA was quantified using the Qubit 4 Fluorimeter (Invitrogen). RNA quality was assessed with the 4200 TapeStation System (Agilent). Samples with an RNA integrity number (RIN) higher than 7 were used for RNA-seq. RNA-seq libraries were prepared from 300ng of total RNA using the mRNA-seq library preparation and 50bp single read sequencing with approximately 25-30M reads per sample in the NEB Next RNA Ultra Kit (Qiagen) with poly (A) enrichment. RNA sequencing was carried out using the HiSeq 3000 platform (Illumina).
Raw sequencing reads were preprocessed using Trim Galore! to trim the adapters and low-quality sequences. Trimmed reads were mapped to the human hg38 reference genome using HISAT2. Read counts for each sample were obtained using feature Counts. Differential gene expression analysis was carried out using  DESeq2. The Benjamini-Hochberg procedure was used to control the false discovery rate (FDR), and the shrunken log2 fold changes (LFCs) were calculated using the adaptive shrinkage (ash) estimator with an Empirical Bayes approach. Genes with FDR-adjusted p-value <0.05 and LFC of more than 1 or less than -1 were considered to be differentially expressed. Gene Set Enrichment Analysis (GSEA) was carried out to identify over-represented gene ontology (GO) terms and biological pathways. Pathway and network analyses were performed using Ingenuity Pathway Analysis (IPA) (Qiagen). Functional protein association networks were analyzed using StringApp and network visualization was generated using Cytoscape version 3.8.2. Heatmaps of specific genes were generated using the pheatmap package in R.

first: Susanta

last: Pahari

first: Eusondia

last: Arnett

first: Jan

last: Simper

first: Abul

last: Azad

first: Israel

last: Guerrero-Arguero

first: Chengjin

last: Ye

first: Hao

last: Zhang

first: Hong

last: Cai

first: Yufeng

last: Wang

first: Zhao

last: Lai

first: Natalie

last: Jarvis

first: Miranda

last: Lumbreras

first: Diego

last: Maselli

first: Jay

last: Peters

first: Jordi

middle: B

last: Torrelles

first: Luis

last: Martinez-Sobrido


title: Spatial single cell analysis of tumor microenvironment remodeling pattern in primary central nervous system lymphoma

city: Jinan

first: Chunyan

last: Ji

address: Wenhua West Road

institute: Qilu Hospital

summary: To determine the overall tumor microenvironment (TME), characteristics, and transition mechanisms in primary central nervous system lymphoma (PCNSL), we performed spatial transcriptomics and matched the corresponding single-cell sequencing data of PCNSL patients. We found that tumor cells may achieve a “TME remodeling pattern” through an “immune pressure-sensing model”, in which they could choose to reshape the TME into a barrier environment or a cold environment according to the immune pressure. A key FKBP5+ tumor subgroup was found to be responsible for pushing tumors into the barrier environment, which provides a possible way to evaluate the stage of PCNSL. The specific mechanism of the TME remodeling pattern and the key molecules of the immune pressure-sensing model were identified through the spatial communication analysis. Finally, we discovered the spatial and temporal distributions and variation characteristics of immune checkpoint molecules and CAR-T target molecules in immunotherapy. These data clarified the TME remodeling pattern of PCNSL, provided a reference for its immunotherapy, and provided suggestions for the TME remodeling mechanism of other cancers.

overall_design: Here, 14964 single-cell transcriptomes of PCNSL were combined with spatial transcriptome analyses of hot, IME, IMS, and cold TMEs. The TMEs were characterized. In brief, the main cell types in the TME of PCNSL patients were identified by single cell sequencing, and were mapped to the corresponding spatial locations in 4 TMEs. Through integrative analysis, we defined and annotated tumor cell subpopulations according to their spatial distribution and functional characteristics and found that various tumor cells work together to reshape the TME into a barrier or cold environment through an "immune pressure-sensing model", thus realizing the "TME remodeling pattern". A key FKBP5+ tumor subgroup was found to be closely associated with TME remodeling, which provides a possible method for assessing brain tumor staging. The spatial communication mode between tumor cells and immune cells in each TME was also identified, thus refining the specific mechanism of the TME remodeling pattern and identifying the key molecules of the immune pressure-sensing model. Corresponding treatment suggestions were proposed according to the spatial characteristics of each TME in current PCNSL immunotherapy. These results reveal the spatial heterogeneity of PCNSL, highlight the localization and status of cell types and potential intercellular signals in the TME, and provide resources for further research on TMEs and updating immunotherapy methods.

first: Yuan

last: Xia

first: Tao

last: Sun

first: Jingjing


title: Gene expression under short-term moderate hypoxia and in the HIF-1 negative regulator mutants in Caenorhabditis elegans

status: Public on Apr 15 2023

city: Ames

first: Jo Anne

last: Powell-Coffman

state: Iowa

address: 2213 Pammel Dr.

department: Department of Genetics, Development and Cell Biology

institute: Iowa State University

summary: Hypoxia inducible transcription factor HIF is the master regulator of hypoxia response in metazoans. The stability and activity of HIF are tightly regulated. In the model organism C. elegans, the homolog of HIF is HIF-1. The stability and activity of HIF-1 are negatively regulated by vhl-1, egl-9, rhy-1 and swan-1 when HIF-1 is stabilized by vhl-1 mutation. Here, we are using Affymetrix microarray to measure the immediate gene expression changes under short-term hypoxia treatment (2 hours of 0.5% oxygen treatment). We also want to understand whether the HIF-1 acvitity negative regulators function in common pathway(s) to regulate HIF-1 activity or not. We find that gene expression patterns in these HIF-1 activity negative regulator mutants are extraordinarily similar, which supports the model that they function in common pathway(s) to regulate HIF-1 activity.

overall_design: Randomized complete block design was followed for the microarray experiments, with three biological replicates treated as three blocks. Each block included eight treatments: N2 wild type, N2 wild type with hypoxia treatment, hif-1(ia04) loss-of-function mutants, hif-1(ia04) loss-of-function mutants with hypoxia treatment, vhl-1(ok161) loss-of-function mutants, rhy-1(ok1402) loss-of-function mutants, egl-9(sa307) loss-of-function mutants, and swan-1(ok267);vhl-1(ok161) loss-of-function double mutants.

first: Jo

middle: A

first: Dingxia

last: Feng

first: Long

last: Qu


title: Role of PARP-1 and PR-Set7 in transcriptional regulation during development (RNA-seq)

first: Gbolahan

last: Bamgbose

summary: We interrogated the role of PARP-1 and PR-Set7 in developmental gene regulation in Drosophila via RNA-seq in third-instar larvae.

overall_design: We performed RNA-seq of WT, Parp mutant and PR-Set7 mutant third-instar larvae (3 biological replicates per group)

first: Sarah

last: Johnson

first: Alexei

last: Tulin


title: Cytokimera GIL-11 rescued IL-6R deficient mice from partial hepatectomy-induced death by signaling via non-natural gp130:LIFR:IL-11R complexes

status: Public on Apr 19 2023

city: Düsseldorf

first: Puyan

last: Rafii

state: NRW

address: Universitätsstraß 1

department: Biochemistry & Molecularbiology

country: Germany

institute: Heinrich-Heine-University

summary: All except one cytokine of the Interleukin (IL-)6 family share glycoprotein (gp) 130 as the common b receptor chain. Whereas Interleukin (IL-)11 signal via the non-signaling IL-11 receptor (IL-11R) and gp130 homodimers, leukemia inhibitory factor (LIF) recruits gp130:LIF receptor (LIFR) heterodimers. Using IL-11 as a framework, we exchange the gp130 binding site III of IL-11 with the LIFR binding site III of LIF. The resulting synthetic cytokimera GIL-11 efficiently recruits the non-natural receptor signaling complex consisting of gp130, IL-11R and LIFR resulting in signal transduction and proliferation of factor-depending Ba/F3 cells. Besides LIF and IL-11, GIL-11 does not activate receptor complexes consisting of gp130:LIFR or gp130:IL-11R, respectively. Human GIL-11 shows cross-reactivity to mouse and rescued IL-6R deficient mice following partial hepatectomy, demonstrating gp130:IL-11R:LIFR signaling efficiently induced liver regeneration. With the development of the cytokimera GIL-11, we devise the functional assembly of the non-natural cytokine receptor complex of gp130:IL-11R:LIFR.

overall_design: Synthetic and control cytokines were applied in four replicates each.

first: Christiane

last: Seibel

first: Hendrik

middle: T

last: Weitz

first: Jürgen

last: Scheller


title: Transcriptomic analysis of flower bud dormancy in the sweet cherry cultivar 'Fertard'

city: Villenave d'Ornon

first: Benedicte

last: Wenden

address: 71 avenue Edouard Bourlaux

country: France

institute: INRA

summary: Temperate perennial plants have to adapt their growth in order to survive over winter conditions and protect their tissues by entering into dormancy.  The dormancy phenomenon is a complex process regulated by many factors, externals (photoperiod and temperature) or internals through the transport towards the buds.  In order to better understand the molecular mechanisms involved in the regulation of dormancy, we conducted a transcriptomic analysis of flower buds from the late flowering sweet cherry cultivar 'Fertard' during dormancy, from October 2017 to March 2018.

overall_design: Flower buds of sweet cherry cultivar 'Fertard' were sampled from October 2017 to March 2018 at 6 dates: October 17th, December 8th, January 3rd, January 15th, February 12th and March 12th, with three biological replicates, corresponding to independent buds  from three different trees.

first: Bénédicte

first: Mathieu

last: Fouché

first: Hélène

last: Bonnet


title: Annelid adult cell type diversity and their pluripotent cellular origins

status: Public on Apr 28 2023

city: Exeter

first: Jordi

last: Solana

address: Northcote House

department: Health and Life Sciences

country: United Kingdom

institute: University of Exeter

summary: We used scRNA-seq to profile 75,218 single cell transcriptomes of Pristina leidyi, a highly regenerative and asexually-reproducing freshwater annelid. We characterized all major adult cell types, their transcription factors and gene networks, and uncovered an abundant population of putative stem cells with a pluripotent signature.

overall_design:
We obtained cell dissociations from adult populations of Pristina leidyi containing intact organisms at mixed stages, including fission stages.
PacBio Iso-Seq sequencing was done on a single mixed sample, and no quantitative analysis is intended from the sample.

first: Patricia

last: Álvarez-Campos

first: Helena

last: García-Castro

first: Elena

last: Emili

first: Alberto

last: Pérez-Posada

first: David

last: Salamanca-Díaz

first: Vincent

last: Mason

first: Bria

last: Metzger

first: Nathan

last: Kenny

first: B

middle: D

last: Özpolat


title: Identification of promoter activity in gene-less cassettes from Vibrionaceae superintegrons

status: Public on Apr 17 2023

city: Madrid

first: José

middle: Antonio

last: Escudero

state: Madrid

address: Av Puerta de Hierro s/n

department: Sanidad Animal

country: Spain

institute: Universidad Complutense de Madrid

summary: Integrons are genetic platforms that acquire new genes encoded in integron cassettes (ICs), building arrays of adaptive functions for bacteria. ICs generally encode promoterless genes, whose expression relies on the PC promoter within the integron platform. Cassette arrays are assumed to be operon-like structures in which expression is dependent on the distance to the Pc. This is especially relevant in large sedentary chromosomal integrons (SCIs,) like the ones in Vibrio species. We have identified 29 gene-less cassettes in 4 Vibrio SCIs, and explored whether their function could be related to regulating the transcription of adjacent ICs. We show that most gene-less cassettes have promoter activity on the sense strand, enhancing the expression of downstream cassettes. Accordingly, we found that most of the superintegron in Vibrio cholerae is not silent. These promoter cassettes can trigger the expression of a silent dfrB9 resistance cassette downstream, increasing trimethoprim resistance >512-fold in V. cholerae and Escherichia coli. Additionally, one cassette had an antisense promoter capable of reducing trimethoprim resistance through transcriptional interference. Our findings highlight the regulatory role of gene-less cassettes in the expression of adjacent cassettes, emphasizing their significance in large SCIs and their clinical importance if captured by mobile integrons.

overall_design: Gene expression analysis of RNA-seq data for Vibrio cholerae N16961 strain at exponential (OD600 0.8) and stationary (OD600 2.8) growth phase at 37˚C in LB medium. Three biological replicates per condition are included.

first: Paula

last: Blanco

last: Hipólito

first: Filipa

last: Trigo da Roza

first: Laura

last: Toribio-Celestino

first: Alba

middle: C

last: Ortega

first: Álvaro

last: San Millán


title: Identifying the direct targets for transcription factor HIF-1a in Caenorhabditis elegans by ChIP-seq.

first: Jo Anne

last: Powell-Coffman

summary: Hypoxia-inducible transcription factor HIF is the key regulator of hypoxia response. It is conserved from human to the model organism C. elegans. The homolog of HIF  in C. elegans is HIF-1. In C. elegans,  there are six alternative splicing isoforms for HIF-1. Isoform a (HIF1a) is the predominant one with important biological functions for stress response and longevity. Here, by performing chromatin immunoprecipitation DNA-sequencing (ChIP-seq), we identified the direct targets for HIF-1a at whole genome level.

overall_design: ChIP-seq with anti-HA

first: Jo

first: Dingxia

last: Feng


title: Gene expression profile at single cell level of normal cells from irradiated and untreated mouse brain.

status: Public on Apr 01 2023

first: Jianxiong

last: Ji

state: Shandong

address: 107 Wenhua Xi Road

department: Department of Neurosurgery

institute: Department of Neurosurgery, Qilu Hospital of Shandong University

summary: The goals of this study are to compare the transcriptomes in all cell types from brain microenvironment between irradiated and untreated mouse

overall_design: Examination of untreated and irradiated mouse brain using single-nucleus RNA sequencing

first: Kaikai

last: Ding

first: Jian

last: Wang

first: Xingang

last: Li


title: Young human glial progenitor cells outcompete and replace older and diseased human cells when transplanted into adult human glial chimeric mice

status: Public on Apr 04 2023

city: Rochester

first: Steven

last: Goldman

state: NY

address: 601 Elmwood Ave

department: Center for Translational Neuromedicine

institute: University of Rochester Medical Center

summary: Human glial progenitor cells (hGPCs) are highly migratory, and glial replacement has the potential to treat those neurological disorders in which astrocytic and oligodendrocytic pathology are contributory. Yet it remains unknown whether allografted human glia can out compete diseased cells to achieve therapeutic replacement in the adult human brain.To that end, we engrafted healthy wild-type (WT) hGPCs into the striata of adult mice that had been earlier chimerized neonatally with mutant HTT-expressing hGPCs generated from Huntington disease (HD)-derived human embryonic stemcells. The WT hGPCs effectively out competed and ultimately eliminated their HD counterparts, repopulating the host striata with healthy humanglia. Single-cell transcriptomics revealed that WT hGPCs actively assumed a dominant competitor phenotype upon interaction with their resident HD counter parts.The outcomes of clonal competition depended primarily upon the age difference between competing clones, in that adult-transplanted WT GPCs effectively out competed their isogenic WT counter parts that had been transplanted neonatally, and which were thus necessarily older.These data suggest that both aged and diseased human glia may be broadly replaced in the adult brain by younger and healthier human glial progenitor cells.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: For HD rescue experiments, striata of Rag1 null mice were transplanted neonatally with HD (G20) hESC-derived EGFP tagged hGPCs neonatally, WT (G19) hESC-derived mCherry tagged hGPCs in adulthood (36 weeks), or co-engrafted with both.  WT rescue experiments consisted of neonatal grafts of EGFP tagged G19 hGPCs, adult transplants of mCherry tagged G19 hGPCs, or co-engraftment of both.  Animals were sacrificed at ~72 weeks, striata dissociated, and single cells isolated via FACS for mCherry, EGFP, or double negative (mouse) cells for scRNA-seq (10X genomics, v3.1 chemistry).

first: Ricardo

last: Vieira

first: John

middle: N

last: Mariani

first: Nguyen

last: Hyunh

first: Hans

last: Stephensen

first: Renee

last: Solly

first: Ashley

last: Tate

last: Schanz

first: Lisa

last: Zou

first: Marzieh

last: Mousaei

first: Jon

last: Sporring

first: Abdellatif

last: Benraiss


title: Human skeletal muscle methylome after low carbohydrate energy balanced exercise

status: Public on Mar 29 2023

city: Oslo

first: Adam

last: Sharples

address: 220 Sognsveien

country: Norway

institute: Norwegian School of Sport Sciences

type: Methylation profiling by genome tiling array

summary: We aimed to investigate the human skeletal muscle (SkM) DNA methylome after exercise in low carbohydrate (CHO) energy balance (with high fat) compared with exercise in low-CHO energy deficit (with low fat) conditions. The objective to identify novel epigenetically regulated genes and pathways associated with ‘train-low sleep-low’ paradigms. The sleep-low conditions included 9 males that cycled to deplete muscle glycogen while reaching a set energy expenditure. Post-exercise, low-CHO meals (protein-matched) completely replaced (using high-fat) or only partially replaced (low-fat) the energy expended. The following morning resting baseline biopsies were taken and the participants then undertook 75 minutes of cycling exercise, with skeletal muscle biopsies collected 30 minutes and 3.5 hours post exercise. Discovery of genome-wide DNA methylation was undertaken using Illumina EPIC arrays and targeted gene expression analysis was conducted by RT-qPCR. At baseline participants under energy balance (high fat) demonstrated a predominantly hypermethylated (60%) profile across the genome compared to energy deficit-low fat conditions. However, post exercise performed in energy balance (with high fat) elicited a more prominent hypomethylation signature 30 minutes post-exercise in gene regulatory regions important for transcription (CpG islands within promoter regions) compared with exercise in energy deficit (with low fat) conditions. Such hypomethylation was enriched within pathways related to: IL6-JAK-STAT signalling, metabolic processes, p53 / cell cycle and oxidative / fatty acid metabolism. Hypomethylation within the promoter regions of genes: HDAC2, MECR, IGF2 and c13orf16 were associated with significant increases in gene expression in the post-exercise period in energy balance compared with energy deficit. Furthermore, histone deacetylase, HDAC11 was oppositely regulated at the gene expression level compared with HDAC2, where HDAC11 was hypomethylated yet increased in energy deficit compared with energy balance conditions. Overall, we identify some novel epigenetically regulated genes associated with train-low sleep-low paradigms.

overall_design:
Methods

Ethics

The study was approved by the Norwegian School of Sport Sciences (NIH) Ethics Committee (Application ID 01-020517) and conformed to the standards of the Declaration of Helsinki. The study was registered in the Norwegian Centre for Research Data (NSD) with reference number 54131/3/ASF. All subjects were informed about the nature of the study and possible risks involved and gave written consent prior to participating in the study.

Participants characteristics

Nine well-trained males (tier 2/3 athletes (1)) completed the study. Participants characteristics were: VO2max: 66 ± 6 ml/kg/min, height: 185 ± 5 cm, body mass: 81 ± 8 kg, body fat: 17 ± 5%.

Experimental protocol

Briefly, in a randomised, counterbalanced, crossover design, participants visited the laboratory on two separate occasions to undergo two different ‘sleep-low’ interventions which were only distinguished based on the dietary intervention. The intervention aimed at depleting SkM glycogen with cycle ergometer-based exercise in the evening of day 1 (~18:00), followed by a low-CHO diet (to avoid muscle glycogen resynthesis). The participants slept at the same premises as the laboratory and performed a second exercise session, completed with low muscle glycogen that took place in the morning of day 2 (~7:00). The glycogen depleting exercise elicited an energy expenditure of 30 kcal/kg fat-free mass (FFM) with alternating exercise of 2 mins at 85% of aerobic peak power output (PPO) and 2 min at 50% PPO (total duration ~2 h). Immediately following each glycogen-depleting session, participants consumed the low-CHO meals which either completely (energy balance, high-fat; EB-HF) or partially (energy deficit, low-fat; ED-LF) replaced the energy expended during glycogen depleting exercise (see ‘dietary interventions’ section below for details on the meals). In the morning of day 2, the structured cycle ergometer exercise session that lasted 75 mins was comprised of mostly low-intensity exercise (50% PPO) but included 4 x 30 seconds and 5 x 1 minute high-intensity intervals. Further details of the experimental protocol have been published elsewhere (2).

Dietary interventions

Diet was controlled and standardised for both interventions for the 24 hrs before visiting the laboratory. Specifically, the diets were pre-packaged and provided 40 kcal/kg FFM/day containing 1.2, 6.0 and 1.35 g/kg FFM/day of fat, CHO, and protein, respectively. Immediately after the exercise in the evening of Day 1, participants consumed one of two low-CHO diets: either a high-fat, energy balance diet (EB-HF), which provided 30 kcal/kg FFM, completely replacing the energy expended during exercise and was composed of 2.5 g/kg FFM (73% energy) fat, 1.2 g/kg FFM (16% energy) CHO and 0.84 g/kg FFM (11% Energy) protein, or a low-fat, energy deficit (ED-LF) diet which provided 9 kcal/kg FFM, partially replacing the energy expended during exercise and was composed of 0.1 g/kg FFM (10% energy) fat, 1.2 g/kg FFM (53% energy) CHO and 0.84 g/kg FFM (37% energy) protein. On day 2, both groups ingested a recovery drink 30 min after the morning exercise containing: 1.2 g/kg FFM CHO and 0.38 g/kg FFM of protein, as this nutrient composition is common practice for athletes to maximise training adaptation (3). Diets were designed to provide the same amount of CHO and protein while providing divergent amounts of energy (deficit and balance), with the energy difference depending solely on the difference in exogenous fat consumption.

Biopsies

Muscle biopsies were taken from the vastus lateralis using a 6 mm Bergström needle modified for manual suction, following local anaesthesia (1% lidocaine, AstraZeneca, Cambridge, UK). Muscle biopsies were taken on day 2 at rest immediately before the start of exercise (baseline), and at 30 mins and 3.5 h after the exercise bout. From the 9 subjects completing the study, we identified a random subpopulation of 4 participants biopsies from each condition and each time point to analyse genome-wide DNA methylation (detailed methods below). Based on the genes identified to possess alterations in DNA methylation, we then validated those changes with gene expression of the same genes across the entire cohort of 9 participants (see ´RNA isolation, primer design & gene expression analysis´ methods section below). This helped to determine whether the identified changes at the genome-wide DNA methylation level in the subpopulation were associated with changes in gene expression of the entire cohort. Baseline characteristics of subpopulation for DNA methylome analysis were: VO2max: 70 ± 5 ml/kg/min, height: 185 ± 6 cm, body mass: 77 ± 6 kg, body fat: 14 ± 4%.

Tissue homogenization and DNA isolation

Muscle samples were homogenized for 45 seconds at 6,000 rpm × 3 (5 min on ice in-between intervals) in lysis buffer (180 µl buffer ATL with 20 µl proteinase K) provided in the DNeasy spin column kit (Qiagen, UK) using a Roche Magnalyser instrument and homogenization tubes containing ceramic beads (Roche, UK). The DNA was then isolated using the DNeasy spin column kit (Qiagen, UK)  bisulfite converted using the EZ DNA Methylation Kit (Zymo Research, CA, United States) as per the manufacturer’s instructions.

DNA methylation analysis

All DNA methylation experiments were performed in accordance with Illumina manufacturer instructions for the Infinium Methylation EPIC BeadChip Array. Methods for the amplification, fragmentation, precipitation and resuspension of amplified DNA, hybridisation to EPIC beadchip, extension and staining of the bisulfite converted DNA (BCD) can be found in detail in our open access methods paper (4, 5). EPIC BeadChips were imaged using the Illumina iScan System (Illumina, United States).

DNA methylome analysis, differentially methylated positions (DMPs), pathway enrichment analysis (KEGG and GO pathways) and differentially methylated region (DMR) analysis

Following MethylationEPIC BeadChip arrays, raw .IDAT files were processed using Partek Genomics Suite V.7 (Partek Inc. Missouri, USA) and annotated using the MethylationEPIC_v-1-0_B4 manifest file. The mean detection p-value for all samples was 0.0002, which was well below the recommended 0.01 (6). The difference between the average median methylated and average median unmethylated signal was 0.08, well below the recommended difference of less than 0.5 (6). Upon import of the data we filtered out probes located in known single nucleotide polymorphisms (SNPs) and any known cross-reactive probes using previously defined SNP and cross-reactive probe lists from EPIC BeadChip 850K validation studies (7). Although the average detection p-value for each sample across all probes was very low (on average 0.0002), we also excluded any individual probes with a detection p-value that was above 0.01 as recommended previously (6). Out of a total of 865,860 probes in the EPIC array, removal of known SNPs, cross-reactive probes, those with a detection p-value above 0.01 resulted in 809,832 probes being taken forward for downstream analysis. Following this, background normalisation was performed via functional normalisation (with noob background correction) as previously described (8). After functional normalisation, we also undertook quality control procedures via principal component analysis (PCA). One sample in the EB-HF trial was removed due to a larger variation than that expected within that condition (variation defined as values above 2.2 standard deviations for that condition). Following normalisation and quality control procedures, we undertook differentially methylated position (DMP) analysis by converting β-values to M-values (M-value = log2(β / (1 - β)), as M-values show distributions that are more statistically valid for the differential analysis of methylation levels (9). We then performed a two-way ANOVA for condition (high-fat, energy balance/EB-HF and low-fat energy deficit/ED-LF) and time (baseline, 30 minutes, 3.5 hrs) with planned contrast/pairwise comparisons of: EB-HF baseline vs. ED-LF baseline, EB-HF 30 min vs. ED-LF 30 min, EB-HF 3.5 hrs vs. ED-LF 3.5 hrs. For initial discovery of CpG sites that were deemed statistically significant, DMPs with an unadjusted P value of ≤ 0.01 were accepted for downstream analysis (Kyoto Encyclopedia of Genes and Genomes/KEGG pathway, Gene Ontology/GO and differentially methylated region/DMR analysis - see below). We then undertook CpG enrichment analysis on these DMPs within GO terms and KEGG pathways (10-12) using Partek Genomics Suite and Partek Pathway software at the significance level of FDR ≤ 0.05. Differentially methylated region (DMR) analysis was performed to identify where several CpGs were differentially methylated within a short chromosomal locations/regions, undertaken using the Bioconductor package DMRcate (DOI: 10.18129/B9.bioc.DMRcate). Finally, to plot and visualise temporal changes in methylation across the post-exercise period (baseline, 30 min and 3.5 hr) within each condition (EB-HF and ED-LF) we implemented Self Organising Map (SOM) profiling of the change in mean methylation within each condition using Partek Genomics Suite.

References

1.	McKay AKA, Stellingwerff T, Smith ES, Martin DT, Mujika I, Goosey-Tolfrey VL, Sheppard J, and Burke LM. Defining Training and Performance Caliber: A Participant Classification Framework. International journal of sports physiology and performance 17: 317-331, 2022.
2.	Areta JL, Iraki J, Owens DJ, Joanisse S, Philp A, Morton JP, and Hallén J. Achieving energy balance with a high-fat meal does not enhance skeletal muscle adaptation and impairs glycaemic response in a sleep-low training model. Experimental physiology 105: 1778-1791, 2020.
3.	Moore DR, Camera DM, Areta JL, and Hawley JA. Beyond muscle hypertrophy: why dietary protein is important for endurance athletes. Appl Physiol Nutr Metab 39: 987-997, 2014.
4.	Seaborne RA, Strauss J, Cocks M, Shepherd S, O'Brien TD, Someren KAV, Bell PG, Murgatroyd C, Morton JP, Stewart CE, Mein CA, and Sharples AP. Methylome of human skeletal muscle after acute & chronic resistance exercise training, detraining & retraining. Scientific Data (Nature) 5: 180213, 2018.
5.	Seaborne RA, Strauss J, Cocks M, Shepherd S, O'Brien TD, van Someren KA, Bell PG, Murgatroyd C, Morton JP, Stewart CE, and Sharples AP. Human Skeletal Muscle Possesses an Epigenetic Memory of Hypertrophy. Scientific Reports (Nature) 8: 1898, 2018.
6.	Maksimovic J, Phipson B, and Oshlack A. A cross-package Bioconductor workflow for analysing methylation array data [version 1; referees: 3 approved, 1 approved with reservations]. F1000Research 5: 2016.
7.	Pidsley R, Zotenko E, Peters TJ, Lawrence MG, Risbridger GP, Molloy P, Van Djik S, Muhlhausler B, Stirzaker C, and Clark SJ. Critical evaluation of the Illumina MethylationEPIC BeadChip microarray for whole-genome DNA methylation profiling. Genome biology 17: 208, 2016.
8.	Maksimovic J, Gordon L, and Oshlack A. SWAN: Subset-quantile within array normalization for illumina infinium HumanMethylation450 BeadChips. Genome Biol 13: R44, 2012.
9.	Du P, Zhang X, Huang C-C, Jafari N, Kibbe WA, Hou L, and Lin SM. Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis. BMC Bioinformatics 11: 587, 2010.
10.	Kanehisa M, and Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic acids research 28: 27-30, 2000.
11.	Kanehisa M, Furumichi M, Tanabe M, Sato Y, and Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res 45: D353-d361, 2017.
12.	Kanehisa M, Sato Y, Kawashima M, Furumichi M, and Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res 44: D457-462, 2016.


title: The effect of exosomes on ovarian cancer cells with regard to gene expression

status: Public on Mar 25 2023

city: Suita

first: MASAKI

last: KOBAYASHI

state: Osaka

address: 2-2 Yamadaoka

department: Department of Obstetrics and Gynecology

country: Japan

institute: Osaka University Graduate School of Medicine

summary: In order to know whether fibroblast-derived exosomes affect cancer cell behavior, we sprinkledtreated those onto CaOV3 and OVCAR3, and analyzed those effects using gene expression microarrays. The gene expression of ovarian cancer cells sprinkled with exosomes were compared with counterparts of those without being exposed to exosomes. In the pathways related to cancer growth which could be analyzed, there were no significant differences between them.

overall_design: The gene expression of cancer cells was measured at 24 hours after exposure to exosomes isolated from the supernatant fluids of fibroblast cultures. It was compared to the the gene expression of cancer cells without exposure to exosomes.

first: Masaki

last: Kobayashi


title: Temporal Coordination of the Transcription Factor Response to H2O2stress

status: Public on Mar 20 2023

city: Tucson

first: Andrew

last: Paek

state: Arizona

address: Life Sciences South, 1007 E. Lowell Stre, PO Box 210106

department: Molecular and Cellular Biology

institute: University of Arizona

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

overall_design: Refer to individual Series


title: Temporal Coordination of the Transcription Factor Response to H2O2stress [scATAC-seq and scRNA-seq]

summary: The p53 and FOXO transcription factors (TFs) share many similarities despite their distinct evolutionary origins. Both TFs are activated by a variety of cellular stresses and upregulate genes in similar pathways including cell-cycle arrest and apoptosis. Oxidative stress from excess H2O2activates both FOXO1 and p53, yet whether they are activated at the same time is unclear. Here we found that cells respond to high H2O2levels in two temporal phases. In the first phase FOXO1 rapidly shuttles to the nucleus while p53 levels remain low. In the second phase FOXO1 exits the nucleus and p53 levels rise. We found that other oxidative stress induced TFs are activated in the first phase with FOXO1 (NF-κB, NFAT1), or the second phase with p53 (NRF2, JUN) but not both following H2O2stress. The two TF phases result in large differences in gene expression patterns. Finally, we provide evidence that 2-Cys peroxiredoxins control the timing of the TF phases in response to H2O2.

overall_design:
MCF7 cells were plated (50,000 cells/well) on plastic 6 well plates and allowed to attach for 48 hours. Cells were then treated with PBS control, 50 µM H2O2, and 75 µM H2O2 for 5 hours. Nuclei were then isolated using the Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression protocol (CG000365). All further steps with the kit were performed by the University of Arizona Genetics Core. Sequencing was carried out using Novogene.  Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (Product code: 1000285) from 10X Genomics was used for transposition, GEM generation and Barcoding, ATAC Library Construction, cDNA Amplification, Gene expression and library construction (CG000338).
Please note that processed data files  were generated from all 16 files (linked to each sample records) together using 10X genomics cellranger -arc program

first: Elizabeth

last: Jose

first: Woody

last: March-Steinman


title: RNA-Seq of Oxaliplatin-treated HCT116 human colon cancer cells

city: Montpellier

first: Alexandre

last: Djiane

address: 208 rue des apothicaires

department: IRCM

institute: Inserm

summary:
Aim: mRNA profile of HCT116 human colon cancer cells to study the early response of cells to oxaliplatin exposure at low doses (0.5µM)
Results: After treatment with oxaliplatin for 24h at 0.5µM, only a very small fraction of genes were mis-regulated. They fall mainly include P53-response genes, genes regulating inflammation, or genes regulating cytoskeleton.

overall_design: Triplicates of HCT116 cells in culture for 24 hours. Treated with oxaliplatin at 0.5µM = OXA). Non-treated control cells = NT


title: Lipid engulfed macrophages interfere with protective T cell immunity and favor tumor progression in human colon carcinogenesis

status: Public on Mar 19 2023

city: Orbassano

first: Giulio

last: Ferrero

state: TO

address: Regione Gonzole, 10

department: Dept. Clinical and Biological Sciences

country: Italy

institute: University of Turin

summary: Obesity and altered lipid metabolism is associated with colorectal cancer (CRC). In this context, the presence of colonic lipid-engulfed macrophages (foam cells, FC) may be involved in hindering protective T cell immunity, thus favoring gut carcinogenesis.

overall_design: By analysing FC-enriched versus FC-deprived tissues in stage I-II CRC, we investigated the function of dyslipidemic myeloid subsets with respect to antitumoral immunosurveillance.

first: Elena

last: Daveri

first: Licia

last: Rivoltini


title: Temporal Coordination of the Transcription Factor Response to H2O2stress [bulk RNA-seq]

overall_design: MCF7 cells (50,000 cells/well) were plated on plastic 6 well plates and allowed to attach for 2 days. There were nine different treatment groups: MCF7 + PBS Control, MCF7 + 50 µM H2O2, MCF7 + 75 µM H2O2,  MCF7 + 20 µM J14 + PBS Control, MCF7 + 20 µM J14 + 50 µM H2O2, MCF7 + 20 µM J14 + 75 µM H2O2,  MCF7 SFRX-OE + PBS Control, MCF7 SFRX-OE + 50 µM H2O2, MCF7 SFRX-OE + 75 µM H2O2. SFRX-OE indicate the cell line harbored a construct that expressed the human Sulfiredoxin gene from the PGK promoter. J14 is a Sulfiredoxin inhibitor. Five hours after treatment, RNA was isolated using RNeasy Mini Kit (Cat. No. 74104) from Qiagen. The isolated RNA was then sent to Novogene for sequencing.


title: Induction of fetal meiotic oocytes from embryonic stem cells in cynomolgus monkeys (RNA-Seq II)

status: Public on Mar 17 2023

city: Kyoto

first: Yukihiro

last: Yabuta

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku

department: Anatomy and Cell Biology

institute: Kyoto University, Graduate school of medicine

summary: Human in vitro oogenesis provides a framework for clarifying the mechanism of human oogenesis. To create its benchmark, it is vital to promote in vitro oogenesis using a model physiologically close to humans. Here, we establish a foundation for in vitro oogenesis in cynomolgus (cy) monkeys (Macaca fascicularis): cy female embryonic stem cells harboring one active and one inactive X chromosome (Xa and Xi, respectively) differentiate robustly into primordial germ cell-like cells, which in xenogeneic reconstituted ovaries develop efficiently into oogonia and, remarkably, further into meiotic oocytes at the zygotene stage. This differentiation entails comprehensive epigenetic reprogramming, including Xi reprogramming, yet Xa and Xi remain epigenetically asymmetric with, as partly observed in vivo, incomplete Xi reactivation. In humans and monkeys, the Xi epigenome in pluripotent stem cells functions as an Xi-reprogramming determinant. We further show that developmental pathway over-activations with suboptimal up-regulation of relevant meiotic genes impede in vitro meiotic progression. Cy in vitro oogenesis exhibits critical homology with the human system, including with respect to bottlenecks, providing a salient model for advancing human in vitro oogenesis.

sample_organism:
Macaca fascicularis
Homo sapiens

platform_organism:
Macaca fascicularis
Homo sapiens

overall_design: RNA-Seq analysis of Cynomolgus xrOvary and human iPSC

first: Sayuri

last: Gyobu-Motani

first: Ken

last: Mizuta

first: Yoshitaka

last: Katou

first: Ikuhiro

last: Okamoto

first: Masanori

last: Kawasaki

first: Ayaka

last: Kitamura

first: Tomoyuki

last: Tsukiyama

first: Chizuru

last: Iwatani

first: Hideaki

last: Tsuchiya

first: Taro

last: Tsujimura

first: Takuya

last: Yamamoto

first: Tomonori

last: Nakamura

first: Mitinori

last: Saitou


title: Induction of fetal meiotic oocytes from embryonic stem cells in cynomolgus monkeys ( RNA-Seq I )


title: Induction of fetal meiotic oocytes from embryonic stem cells in cynomolgus monkeys (EM-Seq)

type: Methylation profiling by high throughput sequencing

overall_design: Whole genome EMseq analysis of Cynomolgus xrOvary and human iPSC


title: Induction of fetal meiotic oocytes from embryonic stem cells in cynomolgus monkeys


title: Induction of fetal meiotic oocytes from embryonic stem cells in cynomolgus monkeys (scRNA-Seq)


title: Cis-regulatory control of transcriptional timing and noise in response to estrogen [ChIP-seq]

status: Public on Mar 15 2023

city: Salt Lake City

first: Jason

last: Gertz

state: UT

address: 1950 Cir of Hope Dr

institute: University of Utah

summary: Cis-Regulatory Elements (CREs) precisely control transcription levels, temporal dynamics, and cell-cell variation - often referred to as transcriptional noise. However, the combination of regulatory proteins and epigenetic features necessary to control different transcription attributes is not fully understood. Here, single-cell RNA-seq (scRNA-seq) is conducted during a time course of estrogen treatment to identify genomic predictors of expression timing and noise. We find that multiple active enhancers predict faster temporal responses. Synthetic modulation of enhancer activity verifies that activating enhancers accelerates expression responses, while inhibiting enhancers results in a delayed response. Noise is controlled by a balance of promoter and enhancer activity. Active promoters are found at genes with low noise levels, whereas active enhancers are associated with high noise. Finally, we observe that co-expression across single cells is an emergent property dependent on chromatin looping, timing, and noise levels. Overall, our results indicate a fundamental tradeoff between a gene’s ability to quickly respond to incoming signals and maintain low variation across cells.

overall_design: ChIP-seq was used to analyze protein-DNA interactions in endometrial cancer and breast cancer cell lines

first: Matthew

last: Ginley-Hidinger


title: Cis-regulatory control of transcriptional timing and noise in response to estrogen [Hi-ChIP]

overall_design: H3K27ac HiChIP is conducted in endometrial cancer (Ishikawa), breast cancer (T-47D), and prostate cancer (LNCaP) cells. Replicates and treatments are combined to call all possible loops


title: Cis-regulatory control of transcriptional timing and noise in response to estrogen [PRO-seq]

overall_design: PRO-seq is used to map nascent transcripts in the context of estrogen treatment in endometrial cancer (Ishikawa) and breast cancer (T-47D) cells.


title: Cis-regulatory control of transcriptional timing and noise in response to estrogen [scRNA-seq]

overall_design: scRNA-seq (10x genomics) was used to measure expression of endometrial and breast cancer cell lines grown in the presence of estrogen for four timepoints. Timepoints are mixed and demultiplexed using MULTIseq


title: Cis-regulatory control of transcriptional timing and noise in response to estrogen


title: Augmenting SARS-CoV-2 vaccine induced spike protein-specific CD4+ T cell responses through IL-1β and its receptors.

status: Public on Mar 01 2023

city: New Haven

first: Hong-Jai

last: Park

state: CT

address: S510, 300 Cedar Street

department: Internal Medicine

institute: Yale University

summary: Immunity to SARS-CoV-2 infection provided by COVID-19 mRNA vaccines decline over time. Thus, there is a need for interventions to augment and sustain such immunity. To address this, we characterized S protein-specific CD4+ T cells in healthy individuals who received COVID-19 mRNA vaccines utilizing high-dimensional single cell RNA-seq and mass cytometry. S protein-specific CD4+ T cells highly expressed IL-1 receptor (R) 1 and its decoy receptor IL-1R2. IL-1b promoted IFN-g expression by S protein-stimulated CD4+ T cells, which was furthered by adding anti-IL-1R2-blocking antibodies, supporting the functional implications of IL-1R1 and IL-1R2. Following the 2nd dose of a COVID-19 mRNA vaccine, IL-1R1 expression increased while IL-1R2 expression decreased in S protein-specific CD4+ T cells. Our findings provide novel insight into augmenting COVID-19 mRNA vaccine-induced CD4+ T cell immunity by modulating IL-1β and its receptor system, which could foster a more effective and prolonged immune response.

overall_design: Multiplexed single-cell RNA-seq of human PBMC samples from SARS-CoV-2 mRNA vaccinated healthy subjects. Sorted OX40-CD137-, OX40+CD137-, OX40-CD137+, and OX40+CD137+ CD4+ T cells from spike-stimulated PBMCs, Flu-stimulated OX40+CD137+ and CMV-stimulated OX40+CD137+ PBMCS were stained with cellhashing antibodies and pooled in one tube to generate a library.

first: Min

last: Shin

first: Junghee

first: Hyoungsu

last: Kim

first: Byunghyun

last: Kang

first: Jennefer

last: Par-Young

first: Serhan

last: Unlu

first: Yuliya

last: Afinogenova

last: Catanzaro

first: Juan

last: Young

first: Minhyung

first: Sang

last: Lee

first: Sungyong

last: You

first: Michael

middle: K

last: Racke

first: Richard

last: Bucala

first: Insoo


title: Integrative single-cell transcriptomics clarifies adult neurogenesis and macroglia evolution

status: Public on Feb 26 2023

city: Paris

first: Laure

last: Bally-Cuif

address: 25 Rue du Docteur Roux

department: Developmental and stem cell biology department

institute: Institut Pasteur

summary: The evolution of nervous systems hinges on cell type diversification and specialization. Neural glia, also called macroglia, make up a large percentage of the total cell numbers in the brain of several species including humans and perform critical functions related to producing, maintaining and assiting neurons. Despite their importance, glial cells have been much less studied than neurons. Here, in order to investigate the heterogeneity of adult radial glial cells in zebrafish and to study the evolution of macroglia and the adult neurogenesis process they support, we generated a single cell RNAseq dataset of the adult zebrafish telencephalon enriched for radial glial cells using a sox2:gfp transgenic line.

overall_design: Cells from the telencephalon of 12wpf Tg(sox2:gfp/+) fish were isolated by Fluorescence-Activated Cell Sorting (FACS). GFP positive cells were enriched to increase the relative proportion of radial glial cells and the resulting samples were processed for scRNA-seq.

first: David

last: Morizet

first: Isabelle

last: Foucher

first: Alessandro

last: Alunni


title: Hikeshi-Mediated Nuclear Translocation of HSP70 Promotes Myelinogenesis by Preventing FBXW7-mediated Degradation of Sox10

status: Public on Feb 19 2023

city: shanghai

first: Ximing

last: Nian

state: Shanghai

address: Chongqing Nanlu 227

institute: Shanghai Jiao Tong University

summary: Dysregulation of nucleocytoplasmic transport is intensively linked to many neurological diseases. Heat shock proteins 70 (HSP70s), which dramatically change its cellular localization upon stress, play important regulatory roles in nervous system. How the localization of HSP70s is regulated and the underlying physiological functions of this translocation are both poorly understood. Mutation in Hikeshi, encoding a nonconventional nuclear import carrier of HSP70s, lead to inherited leukodystrophy; however, the underlying mechanisms remain poorly understood. Here we showed that Hikeshi and HSP70s are essential for proper CNS myelination and remyelination. Loss of Hikeshi resulted in oligodendrocyte maturation arrest through preventing the nuclear translocation of HSP70s upon oligodendrocyte differentiation. HSP70s interacted with Sox10SOX10 in the nucleus and protected it from E3 ubiquitin ligase FBXW7-mediated ubiquitination degradation. Importantly, we discovered that the Hikeshi-dependent hyperthermia therapy, which induces nuclear import of HSP70s, promoted oligodendrocyte differentiation and remyelination following in vivo demyelinating injury. Overall, we demonstrate that Hikeshi-dependent nuclear translocation of HSP70s is essential for oligodendrocyte differentiation and myelinogenesis, and suggest the potential therapeutic value of hyperthermia therapy in myelin repair.

overall_design: RNA-seq was performed on rat siSCR or siHsc70 immature oligodendrocytes (T3 D2).

first: Liang

last: Zhang

first: Li

last: Li


title: The differentially expressed genes regulated by 5-Aza-CdR

status: Public on Feb 23 2023

city: Shiyan

first: Qin

last: Shanshan

address: Renmin Road 30, Maojian District

institute: Institute of Basic Medical Sciences, School of Basic Medical Sciences, Hubei University of Medicine

summary: This study aimed to identify the differentially expressed genes regulated by 5-Aza-CdR

overall_design: To reveal the differentially expressed genes caused by 5-Aza-CdR, a  DNA methyltransferase (DNMT) inhibitor, we treated two human cell lines with 5-Aza-CdR. The transcriptome sequencing studies were performed by deep sequencing, using Illumina HiSeq 4000.

first: Shanshan

last: Qin

first: Dandan


title: Whole blood transcriptomic profiling in men and women with heart failure

city: Stirling

first: James

last: Timmons

address: Bridge of Allan

institute: Augur Precision Medicine LTD

summary: Background. Chronic Heart Failure (CHF) is a systemic syndrome with a poor prognosis and a need for novel therapies. We investigated whether whole-blood transcriptomic profiling can provide new mechanistic insights into CHF. Baseline whole-blood transcriptome profiles were generated from 944 CHF patients from the BIOSTAT-CHF Study.

first: Nilesh

last: Samani


title: Trancsriptional profiling of B16-C10 tumors treated with PBS, oBHV or MMC+oBHV.

status: Public on Feb 17 2023

city: Hamilton

first: Karen

last: Mossman

state: Ontario

address: 1280 Main St W

department: Medicine

country: Canada

institute: McMaster University

summary: To  explore the mechanism of action upon addition of MMC to oBHV therapy, unbiased transcriptional profiling was conducted on B16-C10 tumors treated with PBS, oBHV or MMC+oBHV harvested at day 5.

overall_design: RNA was extracted from tumors and transcriptome data was assessed using a Clariom S assay.

first: Maria

middle: E

last: Davola

first: Olga

last: Cormier


title: Coordinated chemokine expression defines macrophage subsets across tissues [scRNA-seq: hSk]

status: Public on Mar 03 2023

city: Lebanon

first: Claudia

middle: V

last: Jakubzick

state: NH

address: 1 Medical Center Drive, Rm 626 W Borwell, PO#1250960

department: Microbiology and Immunology

institute: Dartmouth College

summary: Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.

overall_design: Three healthy subjects underwent breast reduction and skin pieces measuring approxiamately 15 cm x 5 cm were obtained. All subjects provided written informed consent, and this study was approved by the Institutional Review Board of Dartmouth-Hitchcock Medical Center (protocol #22781).

first: Xin

first: Arlind

last: Mara

first: Shawn

last: Musial

first: Fred

last: Kolling

first: Sophie

last: Gibbings

first: Nikita

last: Gerebtsov


title: Coordinated chemokine expression defines macrophage subsets across tissues [scRNA-seq: mBAL]

overall_design: Nine naive C57BL/6 mice were euthanized using CO2 inhalation. The bronchoalveolar lavage fluid (BALF) was collected by performing three washes of the lungs using 1 mL of PBS. The BALF samples were then pooled together to generate three biological replicates, with each replicate comprising BALF obtained from three mice.


title: Epigenetic dosage identifies two major and functionally distinct β cell subtypes [scRNA-seq]

status: Public on Feb 16 2023

city: Grand Rapids

first: Luca

last: Fagnocchi

state: MI

address: 333 Bostwick AVE NE

institute: VAI

summary: The mechanisms that specify and stabilize cell subtypes remain poorly understood. Here, we identify two major subtypes of pancreatic β cells based on histone mark heterogeneity (βHI and βLO). βHI cells exhibit ∼4-fold higher levels of H3K27me3, distinct chromatin organization and compaction, and a specific transcriptional pattern. βHI and βLO cells also differ in size, morphology, cytosolic and nuclear ultrastructure, epigenomes, cell surface marker expression, and function, and can be FACS separated into CD24+ and CD24− fractions. Functionally, βHI cells have increased mitochondrial mass, activity, and insulin secretion in vivo and ex vivo. Partial loss of function indicates that H3K27me3 dosage regulates βHI/βLO ratio in vivo, suggesting that control of β cell subtype identity and ratio is at least partially uncoupled. Both subtypes are conserved in humans, with βHI cells enriched in humans with type 2 diabetes. Thus, epigenetic dosage is a novel regulator of cell subtype specification and identifies two functionally distinct β cell subtypes.

overall_design: ChIP-seq on pancreatic β-cell

first: Erez

last: Dror

first: J

last: Pospisilik


title: Scalable co-sequencing of RNA and DNA from individual nuclei

status: Public on Feb 09 2023

city: New York

first: Peter

last: Sims

address: 3960 Broadway, Lasker 203AC

institute: Columbia University

summary: We present DEFND-seq (DNA and Expression From Nucleosome Depletion), a scalable method for co-sequencing RNA and DNA from single nuclei. In DEFND-seq we treat nuclei with lithium diiodosalicylate to disrupt chromatin and expose genoimc DNA. The nuclei are then tagmented with Tn5 transposase, which fragments and tags gDNA. Tagmented nuclei are loaded into a microfluidic droplet generator which combines nuclei, beads containing transcriptomic and genomic barcodes, and reverse transcription reagents into single droplets. Ultimately two libraries are created, one for nuclear mRNA and one for genomic DNA, with each library containing barcodes linking it to its nuclei of origin, thus allowing simultaneous analysis of single nuclei transcriptomes and genomes. Once nuclei have been depleted of nucleosomes, all steps can be performed using a 10x Chromium Controller and 10x Multiome Kit without further experimental modification.

overall_design: DEFND-seq (snRNA/DNA-seq), snATAC-seq, snDNA-seq

first: Timothy

last: Olsen


title: Coordinated chemokine expression defines macrophage subsets across tissues [scRNA-seq: mPL]

overall_design: Nine naive C57BL/6 mice were euthanized using CO2 inhalation and injected intraperitoneally with 5 ml PBS. The peritoneum was massaged for 1 min, and the lavage fluid was aspirated. The lavage samples were then pooled together to generate three biological replicates, with each replicate comprising lavage fluid obtained from three mice.

last: Li


title: Epigenetic dosage identifies two major and functionally distinct β cell subtypes [RNA-seq]

overall_design: Bulk RNA-seq of FACS sorted β-cell


title: Epigenetic dosage identifies two major and functionally distinct β cell subtypes [DNAmethArray]

overall_design: DNA methylation profiles by array of FACS sorted β-cell


title: Epigenetic dosage identifies two major and functionally distinct β cell subtypes [ChIP-seq]

overall_design: H3K27me3 ChIP-seq on FACS sorted pancreatic beta-HI/-LO β-cell


title: Cortex-specific transcriptome profiling after deeper cerebral hypoperfusion in mice

status: Public on Mar 09 2023

city: Shanghai

first: Zengyu

last: Zhang

address: Dong'an Road

department: Neurology

institute: Fudan University

summary:
Little is understood about the underlying cellular and molecular mechanisms related to cerebral cortex damage and glial activation after severe cerebral hypoperfusion.Efforts to explore the relationship between neuropathological and gene expression changes support a role for identifying novel molecular pathways by transcriptomic mechanisms.
To investigate cortex-specific transcriptome profiling after severe cerebral hypoperfusion in mice

overall_design: Cortex-specific gene expression profiling analysis was performed using data obtained from RNA-seq of 4 biological replicates in sham and BCAS mice after hypoperfusion

first: Shaungxing

last: Hou


title: The involvement of CdhR in Porphyromonas gingivalis during Nitric Oxide stress [microarray]

status: Public on Feb 03 2023

city: Huntsville

first: Marie-Claire

last: Boutrin

address: 7000 Adventist Blvd NW

institute: Oakwood University

summary: To understand the role of CdhR and its adjacent gene PG1236 in nitric oxide (NO) stress resistance, isogenic mutants P. gingivalis FLL457 (ΔPG1237::ermF), FLL458 (ΔPG1236::ermF) and FLL459 (ΔPG1236-37::ermF) were made by allelic exchange mutagenesis and their gene expression was studied under control and NO stress conditions. DNA microarray analysis of FLL457 showed that approximately 2% of the genes were up regulated and over 1% of the genes down regulated. Transcriptome analysis of FLL458 and FLL459 under NO stress showed similar modulation patterns in both mutants for a few genes. The PG1236-7 gene cluster seemed to be part of the same transcriptional unit that showed increased expression under NO stress. Recombinant CdhR showed binding activity to the predicted promoter regions of PG1459 and PG0495.

overall_design:
Fresh cultures of P. gingivalis strains (mutants and wild type) were grown from overnight cell cultures under anaerobic conditions at 37ºC in BHI broth. P. gingivalis strains were grown to early exponential phase (OD600:~0.3) in BHI broth under anaerobic conditions at 37ºC. At that time point, the bacteria cultures were stressed with Diethylamine (DEA) NONOate (15µl, 24mM stock concentration) for the NO stress studies and culture samples were taken 15min post-treatment with DEA NONOate. P. gingivalis W83 cultures and total RNA extracted from them using a total RNA isolation kit (Promega, WI). Additional DNase treatment was carried out using DNase kit (Ambion, Austin, TX). DNA microarray gene expression was carried out using Roche NimbleGen customer arrays (100910_CW_P_ging_W83_expr_HX12, Roche, IN) according to the standard NimbleGen procedure (NimbleGen Arrays User’s Guide: Gene Expression Analysis v5.1).  cDNA was synthesized from the RNA samples using Transcriptor High Fidelity cDNA Synthesis kit (Roche, IN). Both RNA and cDNA quality were checked using Agilent Bioanalyzer and Agilent RNA 6000 Nano and DNA1000 chips. Differentially expressed genes were determined using fold-change (≥1.25) plus p (≤ 0.05) with FDR = 0.05.
***Please note that [1] raw data files have been lost and thus are not provided, [2] only the list of differentially-expressed genes is available and thus included as processed data*****

first: Hansel

last: Flether

first: Charles

last: Wang

first: Arunima

last: Mishra

first: Yuetan

last: Dou


title: The involvement of CdhR in Porphyromonas gingivalis during Nitric Oxide stress

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

sample_organism:
Porphyromonas gingivalis
Porphyromonas gingivalis W83

platform_organism:
Haemophilus influenzae
Porphyromonas gingivalis

overall_design: Refer to individual Series


title: Tcte1 knockout influence on energy chain transportation, apoptosis and spermatogenesis – implications for male infertility

status: Public on Feb 01 2023

city: Bergen

first: Tomasz

last: Stokowy

address: Nygardsgaten 5

department: IT Division

institute: University of Bergen

summary:
RNA sequencing (RNAseq) in testis tissue has been done to obtain an answer about the influence of Tcte1 null mutation in knockout mice on the expression pattern of other gonadal genes in male animals that may be influential for male fertility status.
Homozygous males (infertile) revealed oligoasthenoteratozoospermia (incl. circular pathway of sperm motility), while heterozygous animals (fertile) manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the mutant testicular tissue showed the influence of Tcte1 mutations on the expression pattern of genes responsible for mitochondrial ATP processing, linked with apoptosis, or spermatogenesis.

overall_design: Gene expression profiling analysis using data obtained from RNA-seq of testicular tissue from wild type, heterozygous and homozygous male mice.

first: Marta

last: Olszewska

first: Agnieszka

last: Malcher

first: Maciej

last: Kurpisz

first: Alexander

last: Yatsenko


title: METTL3-mediated Klf9 mRNA stabilization contributes to white adipose tissue beiging

city: Hangzhou

first: Renxiang

last: Xie

state: Zhejiang

address: 866 Yuhangtang Rd

institute: Zhejiang University

summary: The induction of beige adipocytes in white adipose tissue (WAT), also known as WAT beiging, improves glucose and lipid metabolism. However, the regulation of WAT beiging at the posttranscriptional level remains elusive. Here, we report that METTL3, the methyltransferase of N6-methyladenosine (m6A) mRNA modification, is induced during WAT beiging in mice. Adipose-specific depletion of Mettl3 gene undermines WAT beiging. Transcriptomic analysis of m6A modifications and mRNA expression level reveals global reduction of m6A-modified mRNAs in Mettl3-depleted beige adipose tissue. In particular, METTL3-catalyzed m6A installation on Krüppel-like factor 9 (Klf9) mRNA prevents its degradation. We further demonstrate that the m6A reader protein IGF2BP3 mediates the stabilization of m6A-modified Klf9 mRNA. Overexpressioin of KLF9 protein reverses the impaired WAT beiging elicited by Mettl3 deletion. These findings uncover a novel epitranscriptional mechanism in WAT beiging and identify METTL3-KLF9 axis as a potential therapeutic target for obesity-associated disorders.

overall_design: Investigating the role of METTL3-mediated m6A modification in beiging

first: Sujun

last: Yan

first: Yunyi

last: Gao

first: Yu

last: Qian

first: Zhigang

last: Yang

first: Zhanghui

last: Chen

first: Saisai

last: Wei

first: Xiangwei


title: Coordinated chemokine expression defines macrophage subsets across tissues [scRNA-seq: mSk]

first: Claudia

last: Jakubzick

overall_design: Six naive C57BL/6 mice were euthanized using CO2 inhalation. The skin samples were then pooled together to generate three biological replicates, with each replicate comprising BALF obtained from two mice. To differentiate intravascular and extravascular leukocytes, mice were injected intravenously with APC-Cy7-conjugated anti-CD45 antibody 5 minutes before organ harvest.

first: Xin

last: Li

first: Arlind

last: Mara

first: Shawn

last: Musial

first: Fred

last: Kolling

first: Sophie

last: Gibbings

first: Nikita

last: Gerebtsov


title: FACS purified Nav1.8 lineage sensory neuron whole transcriptome changes following peripheral inflammation (CFA) or sciatic nerve injury (SNI).

status: Public on Jan 30 2023

city: Boston

first: Isaac

middle: Ming-Cheng

last: Chiu

state: MA

address: 77 Avenue Louis Pasteur

department: Immunology

institute: Harvard Medical School

summary:
The goal of this study was to analyze global gene expression in  FACS purified Nav1.8 lineage sensory neurons, which include nociceptor neurons that detect damaging/noxious stimuli, following peripheral inflammation by intraplantar injection of Complete Freund's Adjuvant (CFA) or Sciatic Nerve Injury (SNI) by nerve transection.
Nav1.8 Trangsgenic TdTomato+ neurons were purified from Lumbar L4-L6 dorsal root ganglia (DRG) by flow cytometry from mice on the ipsilateral or contralateral sides, following Complete Freund's Adjuvant injection (day 1) or sciatic nerve transection (day 5). Neurons were then analyzed for transcriptional gene expression by microarray analysis.

overall_design: Nav1.8-Cre transgenic mice (also known as SNS-Cre mice) were bred with Rosa26-TdTomato mice (ai14 mice from jackson Labs) to generate Nav1.8-Cre Tg+ TdTomato+ reporter progeny, where peripheral sensory neurons expresing the sodium channel Nav1.8 are genetically marked with red fluroescence.  In one group of Nav1.8-Cre Tg/TdTomato mice, mice were injected intraplantarly with complete freunds adjuvant. In another group of mice, mice underwent complete sciatic nerve transection. Lumbar region dorsal root ganglia (DRG) were dissected from contralateral or ipsilateral sides of CFA injected mice or sciatic nerve transected mice (3 mice were pooled/sample).  Using flow cytometry, highly red fluorescent neurons were Facs purified, total RNA was extracted, and processed for microarray analysis.  For additional comparisons, Nav1.8-Cre Tg/TdTomato lumbar neurons were previously deposited at GEO database (GSE46546).

first: Clifford

middle: J

last: Woolf


title: Pharmacologic Inhibition of  MALT1 Destabilizes MYC and Halts Survival In Precursor B-Cell Acute Lymphoblastic Leukemia

status: Public on Jan 16 2023

city: New Orleans

first: Nakhle

last: Saba

state: LA

address: 1415 Tulane Ave, Room 118

department: Internal Medicine/Hematology

institute: Tulane University

summary: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a para-caspase required for B-cell receptor (BCR)-mediated NF-κB activation. We sought to examine the role of MALT1 in B-ALL and determine the biological consequences of inhibiting its activity.  First, we tested MALT1 expression by immunoblot in B-cell acute lymphoblastic leukemia (B-ALL) using 16 cell lines representing the disease spectrum and found that MALT1 was expressed in all cell lines at different levels. To determine sensitivity to MALT1 inhibition we used two molecules: Z-VRPR-fmk, a highly selective MALT1 blocking peptide, and MI2, a small molecule MALT1 inhibitor. Z-VRPR-fmk resulted in a dramatic cell growth inhibition in most of our B-ALL cell lines, with appropriate positive (JEKO) and negative (JURKAT) controls. We did not observe a clear correlation between MALT1 level and degree of sensitivity to Z-VRPR-fmk. Interestingly, the two ibrutinib-resistant cell lines RS4;11 and 697, were amongst the top sensitive cell lines to MALT1 inhibition. A similar pattern of cell sensitivity was observed when these cell lines were treated with MI2, with a positive correlation between the IC50 of MI2 and the amount of cell growth inhibition with Z-VRPR-fmk (r=0.74 95%CI (0.47-0.88) p=0.0001). Interestingly, freshly collected PBMCs from patients with B-ALL showed a high sensitivity to MI2 following incubation at serial dilutions for 48h. Furthermore, apoptotic and cell cycle analyses showed a dose-dependent apoptotic induction and cell cycle arrest in the studied ell lines.   Surprisingly, the proteolytic activity of MALT1, studied by measuring its ability to cleave its targets such as CYLD, BCL10, and RelB, was not detected at baseline, nor after proteasomal inhibition with MG-132 or following crosslinking of pre-BCR with anti-IgM in pre-B ALL, the latter successfully increased AKT phosphorylation. Collectively, these data are suggestive of distinct role for MALT1 in B-ALL.       To explore this possibility, we used RNA sequencing to determine the changes in gene expression profiling following an 8h MI2 treatment in 12 sensitive B-ALL cell lines. Out of 39,514 tested genes, there were 160 genes whose expression changed ≥ 2-fold at P < 0.05 (84 down- and 76 up-regulated). Gene Set Enrichment Analysis (GSEA) identified 34 Hallmark and Oncogenic Signature gene sets relevant to B-ALL that were all downregulated by MI2 (FDR < 10%, and normalized enrichment score (NES) ≥ 1.50). Among those gene sets the c-MYC, gene signatures stood out as the most affected, further confirmed with downregulation of the MYC protein by immunoblot analysis in MI2-sensitive cell lines.   In conclusion, MALT1 plays a critical role in B-ALL survival likely through a novel mechanism that involves MYC regulation, independently from pre-BCR/BCR signaling.

overall_design: RNA was extracted from ALL cell line that were treated with either a DMSO placebo or MI-2

first: Firas

last: Safa

first: Terri

last: Rasmussen

first: Ishwarya

last: Satyavarapu

first: Jade

last: Meyers

first: Lorena

last: Fontan

first: Ari

last: Melnick

first: Adrian

last: Wiestner

first: Patricia

last: Lobelle-Rich

first: Ekaterina

last: Kim

first: Jan

last: Burger

first: Yara

last: Mouawad

first: Prescott

last: Deininger

first: Francisco

last: Socola

first: Hana

last: Safah

first: Erik

last: Flemington


title: Host gene expression analysis in oral epithelial cells upon KSHV infection

status: Public on Jan 25 2023

city: Gainesville

first: Bernadett

last: Papp

state: FL

address: 1395 Center Drive D5-18

department: Oral Biology

institute: University of Florida

summary: We performed global host transcriptome analysis in human gingival epithelial cells upon Kaposi's sarcoma-associated herpesvirus (KSHV) infection to identify differentially expressed host genes. KSHV is a human oncogenic virus, which establishes persistent infection of the host. We collected mock as well as KSHV infected human gingival epithelial cells at 8 hours post-infection (hpi) in triplicate, and used the purified RNA for creating RNA-seq libraries for the analysis.

overall_design: RNA-seq libraries were prepared in total, 6 samples, which were corresponding to mock or KSHV infected human gingival epithelial cells at 8 hours post-infection (hpi), both in triplicate.


title: Downregulation of CYR61 promotes hepatocellular carcinoma progression by ameliorating the protumor functions of TGF-β/YAP and impeding the activation of stromal hepatic stellate cells

status: Public on Jan 11 2023

city: Nanchang

first: Xiaohua

last: Yan

address: No. 1299, Xuefu Avenue, Honggutan

institute: Nanchang University

summary: Hepatic stellate cells (HSCs) in the tumor microenvironment play a pivotal role in the development of hepatocellular carcinoma (HCC), which is the major form of primary liver cancers and one of the most lethal cancer types worldwide. TGF-β and Hippo signaling are two critical pathways regulating HSC activation and HCC development through their downstream transcriptional regulators, i.e., Smad proteins and YAP/TEADs, respectively. However, how the two pathways coordinately exert their functions in HCC remains elusive. In this study, we performed integrated analyses of public liver cancer databases and clinical cancer tissues, leading to identification of CYR61 as one of the key candidate genes that may regulate liver cancer progression. TGF-β-activated Smad2/3 and YAP/TEAD4 can form a protein complex to induce CYR61 expression in both cancer cells and stellate cells. As a secreted matricellular protein, CYR61 exerts its tumor suppressor functions via acting on cancer cells directly or regulating the stromal stellate cells. On one hand, CYR61 ameliorates TGF-β- or YAP-mediated liver cancer cell proliferation, colony formation and invasion in vitro, and also tumor growth in nude mice. On the other, CYR61 also impedes cancer cell- or TGF-β-induced stellate cell activation, thereby mitigating the tumor-promoting functions of HSCs as assessed both in vitro and in vivo. In support of its tumor suppressor function, the expression of CYR61 is reduced in HCC tissues and cell lines when compared with their normal counterparts, which is associated with a shorter survival period in cancer patients. Together, these results add new evidence for the interplay between TGF-β and Hippo signaling in regulating gene transcription, and unveil a pivotal role of CYR61 in suppressing liver cancer progression by controlling the activation of hepatic stellate cells in the tumor microenvironment.

overall_design: Three HCC cell lines, MHCC97H, MHCC97L and HCCLM3, were treated with or without TGF-β1 ligand for 4 h

first: Yan

last: Xiaohua


title: Disruption of dNTPs Homeostasis by Ribonucleotide Reductase Hyperactivation Overcomes AML Differentiation Blockade

city: Duarte

first: wei

last: chen

state: CA

address: 1500 E Duarte Rd

department: Cancer Biology-BRI

institute: City of Hope National Medical Center

summary: We assessed global gene expression changes in THP1 cells overexpressing inducible RRM2 non-degradable mutant versus empty vector upon doxycycline induction

overall_design: Expression profiling data in THP1 cells transduced with iRxI/AxA mutant (4 replicates) were compared to MOCK control cells (4 replicates) by RNA sequencing

first: Ling

last: Li

first: Hanying

last: Wang

first: Xin

last: He

first: Wei

last: Chen


title: Epigenetic dosage identifies two major and functionally distinct β cell subtypes

first: Luca

last: Fagnocchi

first: Erez

last: Dror

first: J. Andrew

last: Pospisilik


title: Single-cell RNA sequencing for heart failure heart with the history of hypertension

status: Public on Jan 08 2023

first: Qingbo

last: Xu

address: 79 Qingchun Road

department: Department of Cardiology

institute: First Affiliated Hospital, Zhejiang University School of Medicine

summary: CD34+ stem cells have been used to treat the patients with heart failure, but the outcome is variable. It is of great significance to scrutinize the fate and the mechanism of CD34+ cell differentiation in vivo during heart failure and explore its intervention strategy.

overall_design: scRNA-seq for total non-cardiomyocytes from human heart

first: L

last: Du

first: X

last: Sun

first: Q


title: Cγ and lupanine enhances in vivo the antidiabetic effect and involves modulation of the liver gene expression profile

city: Guadalajara

first: Carmen

middle: Magdalena

last: Gurrola-Díaz

state: Jalisco

address: Sierra Mojada 950

department: Departamento de Biología Molecular y Genómica

country: Mexico

institute: Universidad de Guadalajara

summary:
Previous studies have individually shown the antidiabetic potential of gamma conglutin (Cγ) and lupanine from lupins. Until now, the influence of combines both compounds and the effective dose of the combination is not assessed. Moreover, the resulting gene profile from this novel combination remains to be explored. Therefore, we proposed to study different dose combinations of Cγ and lupanine by the oral glucose tolerance test (OGTT) to identify the greater antidiabetic effect on the T2D rat model. Later, we administered the selected dose combination for a week. Finally, we evaluated the gene expression profile using DNA microarrays and its bioinformatic analysis. We found that the combination of 28 mg/kg BW Cγ + 20 mg/kg BW lupanine positively influences the expression of Pdk4, Ppargc1a, Foxo1, and Foxo3 genes. The reestablishment of Pdk4 gene expression is involved in several biological processes associated with regulation, metabolism, and homeostasis of glucose and fatty acids. For the first time, we reported the beneficial effect of the combination of two functional lupin compounds in a T2D rat model. Nevertheless, further studies are needed to investigate the pharmacokinetics and pharmacodynamics of the Cγ + lupanine combination.
Gene expression analysis of RNA samples from liver tissue of healthy and diabetic-induced rats.

overall_design: Three biological replicates of liver total RNA pools were prepared for each experimental group: healthy rat group, type 2 diabetic control group, and type 2 diabetic rats treated with 28 mg/kg BW Cγ + 20 mg/kg BW lupanine. Each pool consisted of the mixture of RNA isolated from three different animals. A total of three technical replicates (derived from the RNA of nine animals per group) were processed for hybridization into the Affymetrix´s Rat Transcriptome Array 1.0 (Affymetrix, Santa Clara, CA, USA) at the Microarray Unit of the National Institute of Genomic Medicine (INMEGEN), Mexico. The procedures of hybridization and scanning of the microarray were performed according to GeneChipTM Whole Transcript (WT) Expression Arrays User Guide of Affymetrix Corporation. The gene expression files were processed and analyzed with the Applied Biosystem-sTM Transcriptome Analysis Console 4.0.2 (TAC, ThermoFisher, CA, USA). Gene expression data were RMA normalized, and differential expression determined with the following parameters: RMA+DABG (Rattus norvegicus) and expression analysis settings: Fold Change < -2 or > 2, p-value < 0.05, and ANOVA method: ebayes. For functional enrichment analysis, STRING software was used to analyze differentially expressed targets interactions. Gene Ontology (GO), KEGG pathways, and WikiPathways analysis were performed.

first: Paloma

last: Guerra-Ávila

first: Tereso

last: Guzmán

first: José

last: Domínguez-Rosales

first: Pedro

middle: M

last: García-López

first: Michael

last: Wink

first: Alejandra

last: Cervantes-Garduño


title: Immunity to the microbiota promotes sensory neurons regeneration via IL-17A

status: Public on Feb 07 2023

city: Bethesda

first: Verena

last: Link

address: 4 Memorial Drive

department: NIAID

institute: NIH

summary: Tissue physiology and responses to injury can be controlled by the cross-talk between all physiological systems including the nervous and immune system.  How the microbiota influences this dialogue remains unclear. Here, we show that adaptive responses to the microbiota directly promote sensory neuron regeneration. At homeostasis, commensal-specific Th17 co-localize with sensory neurons within the dermis and display a transcriptional profile associated with tissue and nerve repair. Following injury, commensal-specific Th17 cells promote axon growth and local nerve regeneration. Mechanistically, our data support the idea that IL17-A produced by commensal-specific T cells directly signal sensory neurons via IL17RA, the transcription of which is specifically upregulated in injured neurons. Collectively our work reveals that microbiota-specific T cells can bridge biological systems by directly promoting neuronal repair and identify IL17-A as a major determinant of this fundamental process.

overall_design: Examination of transcriptome of Th1 and TH17 T cells during S. aureus infection (ID), association (TA) and recall (TA+ID), as well as ganglion neurons with and without IL17-A treatment

first: Michel

last: Enamorado

first: Yasmine

last: Belkaid


city: Arcadia

first: WEI

last: CHEN

address: 655 e huntington dr

department: Shared Resource

summary: We assessed global gene expression changes in 2'-deoxyguanosine (dG)-treated AML cell lines (U937 and KG1A)

overall_design: Expression profiling data in dG-treated cells (2 replicates) were compared to vehicle (VEH) control cells (2 replicates) by RNA sequencing

first: Ling

last: Li

first: Hanying

last: Wang

first: Xin

last: He

first: Wei

last: Chen


title: RNA-seq and DIP-seq analysis of DNMTs mutant mESCs

status: Public on Jan 01 2023

city: Toyonaka

first: Takamasa

last: Ito

address: 1-1 Machikaneyama

department: Biological Science

institute: Osaka University

summary: DNMT1 plays a major role in embryonic development as a maintenance methyltransferase. Although recent studies have shown that DNMT1 has de novo methylation activity, the detailed role of its function during embryonic development remains unclear. In this study, to further understand the role of DNMT1 de novo methylation, we performed RNA-seq and DIP-seq on DNMTs mutatnt mESCs.

overall_design:
RNA-seq was performed on TKO, TKO+FL and TKO+602 mESCs. DIP-seq was performed on
 TKO_FL and DKO mESCs.

first: T

first: M

last: Ichimaru

last: Tada


title: Rewiring the Mitochondrial Electron Transport Chain Enhances Tumor Antigen Presentation and Immunogenicity [bulk RNA-seq]

status: Public on Dec 31 2022

city: San Diego

first: Yuqing

last: Hang

address: 10010 N Torrey Pines Rd

department: IGC Core

institute: Salk Institute for biological studies

summary: Loss of antigen presentation by MHCI is a common mechanism of tumor immune evasion. Mitochondrial oxidative phosphorylation (OXPHOS) capacity influences MHCI expression, but the underlying molecular mechanisms remain unclear. Here, we demonstrate that the relative flow of electrons through complex I or II of the mitochondrial electron transport chain (ETC) regulates MHCI expression and antigen presentation in cancer cells. Specifically, reducing electron flow from complex II increases mitochondrial succinate which activates transcription of MHCI and antigen processing and presentation (APP) genes. These phenotypes are independent of the interferon (IFN) signaling pathway and driven by succinate-mediated enzymatic inhibition of lysine-specific demethylases, KDM5A/B and destabilization of polycomb repressor complex 2 (PRC2). Finally, knockout of the mitochondrial Complex I inhibitor protein, MCJ, preferentially reduces electron flow through Complex II and increases succinate which drives an enhanced antigen-dependent CD8+ T cell response to mouse melanoma tumors in vivo. These findings suggest that the mitochondrial ETC can be manipulated therapeutically to enhance antitumor immune responses independently of IFNγ.

overall_design: Yale University Mouse Melanoma (YUMM) 1.7 cells were treated with ETC CI inhibitor (Rotenone), CII inhibitor (3-NPA) and DMSO (vehicle control) for 48 hours. RNA samples from two biological replicate of each was seauenced using Illumina novaseq.

first: Kailash

last: Chandra Mangalhara

first: Siva

last: Karthik Varanasi

first: Susan

last: Kaech

first: Gerald

middle: S

last: Shadel


title: Rewiring the Mitochondrial Electron Transport Chain Enhances Tumor Antigen Presentation and Immunogenicity [scRNA-seq and scTCR-seq]

summary: The aim of the study is to analyze and compare the transcriptional profiles and clonotypes at a single cell level of tumor-infiltrating lymphocytes from subcutaneous mouse YUMM melanoma model.

overall_design: Single-cell RNA-seq and single-cell TCR-seq of CD8+ and CD4+ T cells from YUMM control (sgSCR) and MCJ-knockout (MCJ-KO) tumors (n=1) were performed at Day 21 post implantation. Each sample is a pool of 3 tumor samples.

last: Mangalhara