title: Identification and characterization of a SARS-CoV-2 specific CD8 T cell response with immunodominant features

city: Amsterdam

first: Pia

last: Kvistborg

state: FOREIGN

address: Plesmanlaan 121

department: Molecular Oncology & Immunology

country: Netherlands

institute: Netherlands Cancer Institute

summary: The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8 T cells. Here, we analyze samples from 31 COVID-19 patients for CD8 T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8 T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8 T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8 T cell responses are detectable up to 5 months post recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8 T cells during convalescence.

overall_design: Single-cell RNA sequencing (incl. TCRseq) using the 10x Genomics Chromium system (5' kit) was performed on CD8 T cells from 6 COVID-19 patients with acute severe and critical disease. Bulk CD8 T cells were sorted from PBMCs and sequenced in two independent experiments (batch I and II). Patients were hashtagged (BioLegend TotalSeq-C anti-human hashtags) and pMHC monomers with indicated epitopes were conjugated to TotalSeq-C-conjugated-streptavidin. Barcode/hashtag assignments (batch I): Patient COVID-087 (Hashtag #1: GTCAACTCTTTAGCG); Epitopes: RLNEVAKNL-MHC (CGTATGCGCTTCAAG), YLQPRTFLL-MHC (CAGCAACTTCATTGT). Patient COVID-096 (Hashtag #3: TTCCGCCTCTCTTTG); Epitopes: PTDNYITTY-MHC (CAGCAACTTCATTGT), TTDPSFLGRY-MHC (GGTATAAGAGCTACG), TVATSRTLSY-MHC (ATGCGATCAGACCGA). Patient COVID-117 (Hashtag #4: AGTAAGTTCAGCGTA); Epitopes: TTDPSFLGRY-MHC (GGTATAAGAGCTACG). Patient COVID-143 (Hashtag #5: AAGTATCGTTTCGCA); Epitopes: CTDDNALAYY-MHC (ATGCGATCAGACCGA), DTDFVNEFY-MHC (CGTATGCGCTTCAAG), PTDNYITTY-MHC (CAGCAACTTCATTGT), TTDPSFLGRY-MHC (GGTATAAGAGCTACG). Patient COVID-153 (Hashtag #7: TGTCTTTCCTGCCAG); Epitopes: PTDNYITTY-MHC (CAGCAACTTCATTGT), TTDPSFLGRY-MHC (GGTATAAGAGCTACG). Barcode/hashtag assignments (batch II): Patient COVID-131 (Hashtag #1: GTCAACTCTTTAGCG), TTDPSFLGRY-MHC (ATGCGATCAGACCGA), VTEHDTLLY-MHC (CGTATGCGCTTCAAG), PTDNYITTY-MHC (CAGCAACTTCATTGT), FTSDYYQLY-MHC (GGTATAAGAGCTACG)

first: Anastasia

last: Gangaev

first: Steven

last: Ketelaars

first: Olga

middle: I

last: Isaeva

first: Sanne

last: Patiwael

first: Anna

last: Dopler

first: Kelly

last: Hoefakker

first: Sara

last: De Biasi

first: Lara

last: Gibellini

first: Cristina

last: Mussini

first: Giovanni

last: Guaraldi

first: Massimo

last: Girardis

first: Cami

middle: M

last: Talavera Ormeno

first: Paul

last: Hekking

first: Neubury

last: Lardy

first: Mireille

last: Toebes

first: Robert

last: Balderas

first: Ton

last: Schumacher

first: Huib

last: Ovaa

first: Andrea

last: Cossarizza


title: Next Generation Sequencing Analysis of BCR in influenza virus specific B cells

status: Public on Mar 17 2021

city: Yokohama

first: Kosuke

last: Miyauchi

state: Kanagawa

address: 1-7-22 Suehiro-sho

country: Japan

institute: RIKEN Center for Integrative Medical Sciences

type: Expression profiling by high throughput sequencing

summary: It is known that antibody responses to influenza virus antigens differ between vaccine and infection. To elucidate this difference, we performed a repatoire analysis of influenza antigen-recognizing B cells.

overall_design: Influenza virus HA protein specific B cells were sorted from vaccinated or infected mice using HA-probes. Total RNA was isolated from sorted B cells and cDNA was synthesized with Igg specific primers. Sequences of V-D-J region of Ig were determined with MiSeq.

first: Masato

last: Kubo


title: Adipose stem cell-derived hepatocyte-like cells from obese patients reveal hepatic steatosis and mitochondrial oxidative dysfunction

status: Public on Mar 19 2021

city: Beijing

first: yaqiong

last: li

address: youanmenwai

country: China

institute: Capital Medical University

summary:
Background & Aims: Obesity is the most important risk factor and a potential treatment target for fatty liver disease (FLD). The continuous adaptation or ‘remodelling’ of hepatic mitochondrial flexibility plays a key role in the pathogenesis of FLD. Human adipose stem cell (hASC)-derived hepatocyte-like cells (HLCs) offer attractive tools for the research and therapy of liver dysfunction. However, evidence showing the ‘remodelling’ of hepatic mitochondrial flexibility or differentiation capability of hASCs in obese patients is lacking.
Methods: The differentiation capability and mitochondrial structure and function of hepatic cells were evaluated by measuring gene expression, de novo lipogenesis and mitochondrial respiration in HLCs derived from hASCs of obese patients and lean controls. The ability of the GSK3 inhibitor CHIR-99021 to modify the mitochondrial oxidative dysfunction was evaluated in the HLCs derived from the hASCs of obese patients.
Results: hASCs from obese patients showed the potential to differentiate into HLCs. HLCs from the hASCs of obese patients exhibited the characteristics of hepatic steatosis, lower expression of the subunits of mitochondrial complex I and lower oxidative phosphorylation levels. CHIR-99021 promoted the expression of NDUFB8, NDUFB9, the subunits of mitochondrial complex I, and the basal oxygen consumption rate of the HLCs derived from the hASCs of obese patients by upregulating the expression of PGC-1α, the transcription factor involved in mitochondrial biogenesis and ‘remodelling’.
Conclusions: The results demonstrate that obese patient ASC-derived HLCs had mitochondrial oxidative dysfunction, which may represent the consequence of hepatic steatosis.

overall_design: Examination of adipose stem cell-derived hepatocyte-like cells from 3 different donors per group.

first: Haiyan

last: Zhang

first: Yaqiong

last: Li


title: Transcriptome analysis of endothelial cells of lymphatic vessel subtypes

status: Public on Mar 30 2021

city: Uppsala

first: Ying

last: Sun

address: Dag Hammarskjölds väg 20

country: Sweden

institute: Uppsala University

summary: To identify genes regulating the functional specification of collecting lymphatic vessels, we performed transcriptome profiling of dermal ECs isolated from adult mouse ear skin by flow cytometry.

overall_design: Dermal cell suspensions from ear skin of 5-week-old mice carrying a lymphatic endothelial cell (LEC) reporter Prox1-GFP (Tg(Prox1-EGFP)KY221Gsat) were first subjected to enrichment for ECs using PECAM1 antibody-coated magnetic beads, followed by sorting of live BEC and LEC populations based on the expression of the lymphatic markers PDPN and Prox1-GFP. LEC subpopulations were further defined by the level of LYVE1 expression, and assigned as LYVE1-high capillaries (cap LEC), LYVE1-low/- collecting vessels (col LEC) and LYVE1-intermed pre-collecting vessels (pre-col LEC). Blood endothelial cells (BEC) were sorted as PECAM1+ PDPN- Prox1-GFP- LYVE1- population. Three replicates were performed for each group.

first: Magda

middle: N

last: Hernández Vásquez

first: Maria

middle: H

last: Ulvmar

first: Alejandra

last: González-Loyola

first: Ioannis

last: Kritikos

first: Liqun

last: He

first: Cornelia

last: Halin

first: Tatiana

middle: V

last: Petrova

first: Taija

last: Mäkinen


title: Next Generation Sequencing of Papillary Thyroid Cancer tissue sample and adjacent normal tissue

status: Public on Mar 29 2021

city: shenyang

first: lv

last: chengzhou

state: liaoing

address: nanjingbeijie 155

department: thyroid surgery

institute: the first hospital of china medical university

type: Non-coding RNA profiling by high throughput sequencing

summary: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of Papillary Thyroid Cancer tissue sample and adjacent normal tissue. The goals of this study are to analysis the different circRNAs expression between Cancer tissue sample and adjacent normal tissue. Quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. We performed circRNA-seq in Papillary Thyroid Cancer tissue sample and adjacent normal tissue. We found that through the deep sequencing of four pairs PTC and adjacent nontumor tissues, we identified 16569 circRNAs, 720 circRNAs were differentials expressed, among them, 301 upregulated and 419 downregulated.

first: Chengzhou

last: Lv

first: Wei

first: Xiaoyu

last: Ji

first: Hao


title: Base-resolution analysis of deoxyuridine at genome scale based on artificial incorporation modified nucleobase

status: Public on Mar 22 2021

city: wuhan

first: han

middle: shao

last: qing

address: whu

institute: school

summary: Deoxyuridine (dU) in DNA can result from deamination of cytosine and dUMP misincorporation. The easy single-nucleotide resolution assay for dU are crucial to understanding its role in genome. Herein, we present a new concept of “base substitution” for accurate single-nucleotide resolution profiling of dU. The method termed AI-Seq (Artificial Incorporation of a modified nucleobase for sequencing) was developed using artificial cytosine (N3-C) to replace dU. After the artificial base construction, dU can read as cytosine during polymerase chain reaction assay. AI-seq was validated on synthetic DNA and then applied to the genome of HEK293T cell line. Collectively, the “base substitution” provides a novel approach for generating comprehensive information about the distribution of dU and can be potentially adapted to detect other epigenetic modifications.

overall_design: N3-C is an artificially synthesized modified cytosine, which is mainly composed of three parts. Hydroxylamine group can label the UNG-treated dU. Cytosine can be incorporated to replace the dU, leading to U-to-C signals in the sequencing reads. Such base substitution enabled U-to-C transition was used from the sequencing reads to obtain dU at single-base resolution. Moreover, the azide group of N3-C can react with DBCO-biotin reagents through a copper-free click reaction for enriching the dU-containing fragment.

first: Y

last: Wang

first: X

first: S

last: Han

last: Weng

last: Zhou


title: Functional mapping of androgen receptor enhancer activity

status: Public on Mar 28 2021

city: Vancouver

first: Nathan

middle: A

last: Lack

state: BC

address: 2260 Oak St

department: Vancouver Prostate Centre

country: Canada

institute: University of British Columbia

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: RNA-Seq data from U2 small nuclear RNA auxiliary factor 1 (U2AF1)-S34F and WT SKM-1 cells

status: Public on Mar 13 2021

city: Shanghai

first: Yuqian

last: Zhu

address: 600 Yi Shan Road

department: Department of Hematology

institute: Shanghai Jiao Tong University Affiliated Sixth People’s Hospital

summary: Mutations in the U2 small nuclear RNA auxiliary factor 1 (U2AF1) gene are the common feature of a major subset in myelodysplastic syndromes (MDS). However, the genetic landscape and molecular pathogenesis of oncogenic U2AF1 S34F mutation in MDS are not totally understood.   To understand how the cancer-associated U2AF1 S34F mutation promotes MDS, we next compared independent RNA samples of SKM-1 cells expressing wild-type U2AF1 and S34F mutant U2AF1 using RNA-sequencing (RNA-seq).Differentially expressed genes (DEGs) and significantly enriched pathways were identified by RNA-seq.

overall_design: We constructed a recombinant lentivirus with FLAG-tagged wild-type or U2AF1 (S34F) mutant and stably transfected SKM-1 cells. Cells were administered the vehicle control and S34F mutants in triplicate, and total RNA was extracted with TRIzol reagent following the vendor's process. RNA sequencing was performed on the Illumina X10 system of LC Sciences (USA).

first: Lingyun

last: Wu


title: Transcriptional analysis of nucleotide excision repair defects

status: Public on Mar 15 2021

city: Cleveland

first: Daniel

last: McGrail

state: Ohio

address: 9500 Euclid Avenue

department: Center for Immunotherapy and Precision Immuno-Oncology

country: USA

institute: Cleveland Clinic

summary: Human mammary MCF-10A cells with shRNA-mediated depletion of genes involved in NER

overall_design: Three biological replicates per sample

middle: J

first: Shiaw-Yih

last: Lin


title: Epigenomic landscape of Lyme disease spirochetes reveals novel motifs

status: Public on Mar 24 2021

city: Hamilton

first: Jenny

last: Wachter

state: MT

address: 4th St

department: NIAID

institute: RML

summary: Borrelia burgdorferi, the etiological agent of Lyme disease, persists in nature through an enzootic cycle consisting of a vertebrate host and an Ixodes tick vector. The sequence motifs modified by two well-characterized restriction/modification loci of B. burgdorferi type strain B31 were recently described, but the methylation profiles of other Lyme disease Borrelia have not been characterized. Herein, the methylomes of B. burgdorferi type strain B31 and 7 clonal derivatives, along with B. burgdorferi N40, B. burgdorferi 297, B. burgdorferi CA-11, B. afzelii PKo, B. afzelii BO23, and B. garinii PBr, were defined through PacBio SMRT sequencing. This analysis revealed 9 novel sequence motifs methylated by the plasmid-encoded restriction/modification enzymes of these Borrelia strains. Furthermore, while a previous analysis of B. burgdorferi B31 revealed an epigenetic impact of methylation on the global transcriptome, the current data contradict those findings; our analyses of wild-type B. burgdorferi B31 revealed no consistent differences in gene expression among isogenic derivatives lacking one or more restriction/modification enzyme(s).

overall_design: 11 B. burgdorferi type strain B31 derivatives either containing or lacking the plasmid-encoded restriction-modification genes bbe02, bbq67, and bbh09 underwent RNA-sequencing. Total RNA was isolated from three biological replicates of B. burgdorferi strain B31-A3, B31-A3bbe02, B31-A3bbq67, and B31-A3bbe02bbq67, and from two biological replicates of B. burgdorferi B31-A3, B31-A3bbe02, B31-A3-68, B31-A3-68bbe02, B31-A3lp28-3, B31-A34, and B31-A34 Tn::H09 in mid-log phase (5-9 x 107 celEls/mL). RNA was quantified and subjected to Agilent Bionalyzer 2200 Tape Station quality assessment. RNAs possessing RIN values > 7.4 were used for downstream analysis. cDNA libraries were synthesized using Ribo-Zero and ScriptSeq complete bacteria kit with indexing primers for synthesis of directional libraries per the manufacturer’s instructions. The quality and quantity of the resulting cDNA libraries were assessed with an Agilent DNA 1000 assay on an Agilent 2100 Bioanalyzer and with a KAPA Library Quantification Kit prior to RNA-seq. RNA-seq libraries were sequenced on an Illumina MiSeq using v3 chemistry at 2 x 75 bp reads.RNA-seq reads were compiled and filtered to remove any reads with PHRED scores less than 10 and aligned to the B. burgdorferi B31 genome (RefSeq AE000783-AE000794 and AE001575-AE001584) using bowtie2. Reads for annotated genes were determined using featureCounts.

first: Patti

last: Rosa


title: 3D Genome Analysis Identifies Enhancer Hijacking Mechanism for High-Risk Factors in Human T-Lineage Acute Lymphoblastic Leukemial (T-ALL)

status: Public on Mar 20 2021

first: Hong

address: Yiheyuan Road 5#

institute: Peking University

summary: The goals of this study are to determine whether alterations in the 3D genome organization are associated with the malignant transformation of T-ALL. We report integrated analyses of 3D genome alterations and differentially expressed genes (DEGs) in 18 newly diagnosed T-ALL patients and 4 healthy T cell controls. We found that 3D genome reorganization at the compartment, topologically associated domains (TAD), and loop levels in different subtypes of T-ALL. Alterations in the 3D genome were associated with nearly 45% of the upregulated genes in T-ALL. We also identified 34 novel translocations in the noncoding regions of the genome and 44 new loops formed between translocated chromosomes.  These translocations can activate the expression of oncogenic transcription factors including HOXA11-A13 by “cis” or “trans” reorganization of chromatin structure. Our analysis demonstrated that T-ALLs with HOXA cluster overexpression were heterogeneous clinical entities, and ectopic expressions of the HOXA11-A13 genes, but not other genes in the HOXA cluster, were associated with immature phenotypes and poor outcomes. Our findings highlight the potentially important roles of 3D genome alterations in the etiology and prognosis of T-ALL.

overall_design: We conducted BL-Hi-C and RNA-seq using purified primary leukemic blasts from 18 newly diagnosed T-ALL patients, including 8 early T-cell precursors ALL (ETP ALL) and 10 non-ETP ALL, two clinical subtypes of T-ALL, as well as normal T cells from 4 healthy volunteers. Raw data and all the processed data were deposited on this GEO Series.All Raw sequence data were also provided at China Genomic Sequence Archive (GSA) (under accession HRA000113 https://bigd.big.ac.cn/gsa-human/browse/HRA000113). UCSC sessions: https://genome.ucsc.edu/s/ChenFengling/TALL

first: Fengling

last: Chen

first: Lu

last: Yang


title: Next Generation Sequencing Facilitates Quantitative Analysis of plasma Exosomal miRNAs of small cell lung cancer

status: Public on Mar 09 2021

city: Hefei

first: Wulin

last: Shan

state: Anhui

address: Huanhudong Road 107

institute: The First Affiliated Hospital of University of Science and Technology of China

summary: Total RNA was isolated from plasma exosomes of two small cell lung cancer patients at the pre- and post-chemoresistant stages. MiRNA sequencing was performed to assess differential expression using next-generation sequencing. The data showed that most miRNAs levels were different in plasma exosomal. Importantly, miR-92b-3p were much higher in the post-chemoresistant stage than in the pre-chemoresistant stage

overall_design: MiRNA sequencing was performed to assess differential expression of miRNAs in  plasma exosomes of two small cell lung cancer patients at the pre- and post-chemoresistant stages using next-generation sequencing.

first: Ming

last: Li


title: Hypermethylation of hepatic mitochondrial ND6 provokes systemic insulin resistance

status: Public on Mar 27 2021

city: Xi'an

first: Ke

last: Cao

state: Shannxi

address: 28# West Xianning Road

institute: Xi'an Jiaotong University

type: Methylation profiling by high throughput sequencing

summary:
Purpose: To reveal the mechanism of mitochondrial DNA methylation in the progression of fatty liver and insulin resistance.

Methods: Liver mitochondrial DNA bisulfite-sequencing of high-fat diet (HFD) and db/db diabetic mice were using Illumina 4000. Western blot, real-time PCR and confocal microscopy were used for further biochemical validation.

Results: In the present study, we found increased mitochondrial localization of DNA methyltransferase 1 (DNMT1) in the liver of high-fat diet (HFD) and db/db diabetic mice. Whole genome bisulfite sequencing of mouse liver mtDNA revealed significant increase of cytosine methylation frequencies including CG, CHG and CHH on both L and H-strand in the diabetic mice comparing with normal control, and ND6 showed the most dramatic increase on the L-strand.

Conclusions: Our present study suggests an epigenetic regulatory of mitochondrial homeostasis and insulin sensitivity by DNMT1, providing novel therapeutic targets for the prevention and treatment of fatty liver and type 2 diabetes.

overall_design: Liver mitochondrial DNA bisulfite-sequencing of male C57BL/6J mice fed a normal diet (NFD, n=8), male C57BL/6J mice fed a high-fat diet (HFD, n=4), and male db/db mice fed a normal diet (NFD, n=4), using Illumina 4000.

first: Zhi

last: Feng

first: Jian

middle: K

last: Liu


title: The role of APOE genotype in modulating the transcriptomics profile of brain endothelial cells in vitro

city: Chicago

first: Leon

middle: Maing

last: Tai

state: IL

address: 808 S.Wood Street, MC512, Room 578

department: Anatomy and Cell Biology

institute: University of Illinois at Chicago

summary: Human APOE genotype is intricately involved in the homeostasis of the central nervous system, which is particularly evident from its association with neurodegenerative disease. Compared to APOE3, APOE4 is associated with greater cognitive decline in aging, poorer outcomes following stroke and traumatic brain injury, and is a major genetic risk factor for Alzheimer’s disease. Human APOE genotypes can differentially modulate neuronal function via both direct and indirect pathways. Of the indirect pathways, increasing evidence supports that APOE4-associated neurovascular dysfunction plays a significant role in neurodegeneration. For example, deficits in cerebral blood flow, neuronal-vascular coupling and blood-brain barrier integrity are observed with APOE4 both during aging and in Alzheimer’s disease patients; data that has been recapitulated in mouse models. Therefore, the identification of cellular mechanistic pathways that contribute to APOE-modulated vascular function is important for fundamental and disease-focused research.  Highly specialized brain endothelial cells (BECs) are essential for virtually all neurovascular functions and, therefore, the influence of apoE on the cerebrovasculature likely involves alterations in brain endothelial cells function, directly or via supporting cells. ApoE synthesis has been demonstrated in multiple cell types in both the brain (e.g. astrocytes, pericytes and neurons) and periphery (e.g. hepatocytes and macrophages). Current research has largely focused on the effects of apoE produced by supporting cell types, particularly astrocytes and pericytes, on BEC function. However, less explored is whether BECs produce apoE to modulate their function. Data from limited studies on apoE production by BECs and APOE genotype-specific functional differences are conflicting. Evaluation of whether the APOE genotype of BECs modulates their function is important, as this could represent a novel mechanisms that contributes to altered cerebrovascular function in aging and AD. The goal of this study was to evaluate the role of human APOE genotype in modulating the phenotype and/or function of brain endothelial cells in vitro. To this end, we utilized primary brain endothelial cells isolated from APOE-targeted replacement mice that express human APOE3 or APOE4 under the endogenous mouse APOE promoter. BECs are highly specialized and functionally complex to maintain homeostasis of the central nervous system and APOE genotype could theoretically alter levels of any number of molecules and associated functions. Thus, without a priori knowledge we initially evaluated the extent that APOE modulated the transcriptomic profile of brain endothelial cells.

overall_design: Two genotypes of brain endothelial cells (APOE3, APOE4) with 8 biological replicates. A biological replicate correspnds to an isoaltion from a different animal.

first: Felecia

last: Marotolli

first: Pinal

last: Kanabar

first: Mark

last: Maienscein-Cline

first: Zarema

last: Arbieva


title: Albendazole induces monocytic differentiation in AML cells through modulating KLF4-DPYSL2A axis

city: Kyoto

first: Ken

last: Morita

address: 53 Kawahara-cho, Syogoin, Sakyo-ku

department: Human Health Sciences

institute: Kyoto University

summary:
To compare the global gene expressions between KLF4-expressed AML cells and albendazole-treated AML cells, we conducted gene expression arrays in THP-1 cells lentivirally-transduced with doxycycline-inducible KLF4 or in THP-1 cells treated with albendazole in different concentrations.
We identified that albendazole induces prominent differentiation in THP-1 cells through up-regulating KLF4 and DPYSL2A expressions. Considering the guaranteed safety and tolerability of albendazole in humans, this drug could easily be repositioned to AML patients once its anti-tumor efficacy is clarified.

overall_design: THP-1 cells were transduced with lentivirus encoding KLF4 under doxycycline responsive promoters. Cells were treated with 3 μM doxycycline for 48 hours to induce gene expressions. In albendazole treatment, THP-1 cells were exposed to it at 0.2 μM or 0.4 μM for 24 hours. Control cells were treated with equivalent amount of DMSO for 24 hours before lysed for RNA extraction.


title: Pulldown of RNA using PrgU-FLAG from Enterococcus faecalis OG1RF pCF10

status: Public on Mar 16 2021

city: Minneapolis

first: Julia

last: Willett

state: MN

address: 689 23rd Avenue SE, 4-149 Micro Research Facility

institute: University of Minnesota

summary: Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the expression of its Type 4 Secretion System (T4SS) genes, controlled by the PQ promoter. The PQ promoter is strictly regulated, partially to limit cell toxicity caused by overproduction of PrgB, a T4SS adhesin. PrgU plays an important role in regulating this toxicity by decreasing PrgB levels. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. We used a combination of in vivo methods to quantify PrgU effects on prgQ transcripts at both single cell and population levels.

overall_design: Comparison of transcript levels in RNA pulldowns using PrgU-FLAG

first: Gary

last: Dunny

middle: E


title: Global landscape mapping of chromatin accessibility in DNA damage-induced senescent human stromal cells

status: Public on Mar 23 2021

first: Yu

last: Sun

address: 320 Yueyang Rd, Life Sciences Building A #1508

department: Shanghai Institute of Nutrition and Health

institute: Chinese Academy of Sciences

summary: We report the application of an improved assay for transposase-accessible chromatin with high-throughput sequencing in profiling changes of chromatin openning state in senescent human stromal cells. By obtaining over four billion bases of sequence from Tn5 transposase-processed chromosome DNAs, we generated genome-wide chromatin-state maps of human primary stromal cells (with a starting cell number of 50,000). This study provides a framework for the application of ATAC-Seq technique towards spatiotemporal chromatin configuration of human stromal cells upon DNA damage-induced senescence.

overall_design: Examination of 3 different cellular states in a primary normal human prosate stromal cell strain.

first: Boyi

last: Zhang


title: Shape of promoter antisense RNAs regulates ligand-induced transcription activation [Shape-Map]

city: La Jolla

first: MICHAEL

middle: G

last: ROSENFELD

state: CA

address: 9500 Gilman Drive

institute: HHMI/UCSD

summary: Demonstration of a massive lncRNA RNA transcriptional program in the mammalian genome has engendered lively discussions about their biological roles, particularly the promoter antisense (PAS) transcripts of coding gene transcription units and abundant species localized to specific subnuclear structures with phase separation properties. Here, we report development of an alternative assay to quantitatively detect RNA distribution in the genome, referred to as ChIRC-seq (Chromatin Isolation by RNA-Cas13a Complex Capture by Cas13a), which has revealed that promoter antisense RNAs (PAS-RNAs) serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK snRNA-dependent inhibitory P-TEFb complex. Induction of PAS-RNA by liganded estrogen receptor ERα (ESR1) releases the basal recruitment of HP1α and KAP1 on target gene promoters, without the spreading characteristic of extended heterochromatic regions, based on PAS-dependent recruitment of H3K9me3 demethylases. Unexpectedly, ERα-bound MegaTrans enhancer activation with features of phase separation events proved to require recruitment of phosphorylated KAP1 to be activated, with its transfer to the cognate promoters licensing estrogen-induced pause release and target gene activation. The ChIRC13a-seq method has proved effective for identification of the cognate promoter for activated enhancers and has revealed that the lncRNAs NEAT1/MALAT1 in phase-separated subnuclear structures interact with robust estrogen-activated MegaTrans TFF1e1 enhancer and other active enhancers, linking acute activation of a subset of enhancers to interactions with specific phase-separated subnuclear architectural structures.

overall_design:
The changes in the gene expression profiles were investigated by ChIP-seq, PRO-seq, and ChIRC-seq.
The secondary structure of pasRNA has been mapped by SHAPE-MaP.

first: Fan

first: Bogdan

last: Tanasa

first: Rudi

last: Micheletti

first: Kenneth

last: Ohgi

first: Aneel

last: Aggarwal

first: Michael

last: Rosenfeld


title: DNA methylation profiling of gastric cancer and precursor lesions

status: Public on Mar 04 2021

city: Badalona

first: Manel

last: Esteller

state: Barcelona

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

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

country: Spain

institute: Josep Carreras Leukaemia Research Institute (IJC)

type: Methylation profiling by genome tiling array

summary: Genome-scale DNA methylation profiling using the Infinium DNA methylation 450K BeadChip platform and samples from gastric cancer (intestinal and diffuse), precursor lesions (multifocal chronic atrophic gastritis and inestina metaplasia), non-atrophic gastritis and normal gastric mucosa.

overall_design: DNA methylation analysis of gastric biopsies from patient suffering gastric cancer (intestinal and diffuse subtypes) and paierd normal adyacent tissues, precursor lesions and normal mucosa using the Infinium DNA 450 methylation BeadChip platform

first: María

last: Berdasco


title: Epigenetic reprogramming of host and viral genes by Human Cytomegalovirus infection in Kasumi-3 myeloid progenitor cells at early times post-infection

status: Public on Mar 05 2021

first: Mary

last: Hummel

address: 303 E. Chicago Ave

institute: Northwestern University

sample_organism:
Homo sapiens
Human betaherpesvirus 5

platform_organism:
Homo sapiens
Human betaherpesvirus 5


title: ChIP-seq analyses for KDM5A, H3K4me3, RNAPII and TAF3 in human multiple myeloma cell line MM.1S

city: Kumamoto

first: Hiroto

last: Ohguchi

state: Kumamoto

address: 2-2-1 Honjo, Chuo-ku

department: IRDA

institute: Kumamoto University

summary: Recent studies have highlighted the oncogenic roles of KDM5A; however, its molecular mechanism has not been fully delineated. Here we identifies KDM5A as a transcriptional activator of MYC target genes in multiple myeloma (MM). Genetic ablation of KDM5A or pharmacologic inhibition of KDM5 by novel inhibitor JQKD82 impairs MM cell growth. Gene expression profile reveals that JQKD82 decreases expression of MYC targets. Expression of MYC targets is similarly downregulated by KDM5A depletion, but little affected by KDM5B knockdown. Mechanistically, KDM5A co-localizes with MYC across the genome, and KDM5A and MYC coordinately promote MYC target gene transcription. The treatment with JQKD82 reduces phosphorylation level of RNA polymerase II (RNAPII), resulting in RNAPII pausing, accompanied by hyper-H3K4me3 status at the MYC loci. Finally, we shows that JQKD82 exerts anti-MM activity and prolongs overall survival in vivo mouse xenograft models. Our results delineate KDM5A function that reinforces MYC transcriptional program, and identify KDM5A as a potential therapeutic target in MM.

overall_design: KDM5A, H3K4me3, RNAPII and TAF3 ChIP samples and their inputs were used for ChIP-seq analyses. MM.1S cells were treated with either JQKD82 or DMSO, or were transduced with either shRNA against KDM5A or luciferase (control) for H3K4me3, RNAPII or TAF3 ChIP-seq.

first: Berkley

last: Gryder


title: RNA-seq analysis after JQKD82 treatment in human multiple myeloma cell line MM.1S

overall_design: MM.1S cells, or MM.1S cells transduced with empty vector or MYC were treated with either JQKD82 or DMSO in duplicate for RNA-seq analysis.


title: Pol2 and H3K27Ac and H3K27me3 occupancy of the viral and host genomes in HCMV infected Kasumi-3 cells at 24 hpi

summary: Purpose: to compare H3K27Ac and H3K27me3 in HCMV infected  and mock infected Kasumi-3 cells at 24 hours post infection and analyze H3K27Ac and H3K27me3 occupancy of the viral genome in GFP+ infected cells

overall_design: Kasumi-3 cells were infected with the TB40/E wtGFP strain of HCMV.  At 4 hr post-infection, virus was inactivated and cells left in culture for additional 20 hours. Infected cells were sortes for GFP expression and live cells. Mock infected cells were sorted for live cells. After fixation, cells were lysed and sonication with Covaris Focused-ultrasonicator at 10% Duty Cycle, 140 peak, 200 cycles per burst, 360 seconds. ChIP was performed by incubating the sonicated sample with Acetyl-Histone H3 (Lys27) (D5E4) XP Rabbit mAb #8173,  Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733 (Cell Signaling Technologies) or Histone H3 antibody #2650 (Cell signaling Technologies) and the  protein A/G agarose beads. Beads were washed and eluted in 200μl of elution buffer (50 mM Tris-HCl at pH 8.0, 10 mM EDTA, 1.0% SDS). The eluates were de-cross-linked and the DNA purified using the Qiagen PCR purification kit. Libraries were prepared with the KAPA HTP Library Preparation Kit multiplexed with NEXTflex DNA Barcodes (Bioo Scientific) and size selected with AMPure XB beads (Beckman Coulter) to have fragments 250-450 bp in length. Library quality control was assessed using Bioanalyzer High Sensitivity DNA Analysis kit (Agilent) and sequencing was performed with Illumina NextSeq technology (50bp single- end reads).

first: Eleonora

last: Forte

first: Matthew

last: Schipma


title: Effects of maternal gestational diet, with or without methionine, on muscle transcriptome of Bos indicus-influenced beef calves following a vaccine-induced immunological challenge

status: Public on Mar 03 2021

city: Madison

first: Francisco

last: Peñagaricano

state: Wisconsin

address: 1675 Observatory Drive

department: Animal and Dairy Sciences

institute: University of Wisconsin-Madison

summary: Maternal nutrition during gestation can cause epigenetic effects that translate to alterations in gene expression in offspring. This 2-year study employed RNA-sequencing technology to evaluate the pre- and post-vaccination muscle transcriptome of early-weaned Bos indicus-influenced beef calves born from dams offered different supplementation strategies from 57 ± 5 d prepartum until 17 ± 5 d postpartum.

overall_design: Seventy-two Brangus heifers (36 heifers/yr) were stratified by body weight and body condition score and assigned to bahiagrass pastures (3 heifers/pasture/yr). Treatments were randomly assigned to pastures and consisted of (i) no pre- or postpartum supplementation (NOSUP), (ii) pre- and postpartum supplementation of protein and energy using 7.2 kg of dry matter/heifer/d of molasses + urea (MOL), and (iii) MOL fortified with 105 g/heifer/wk of methionine hydroxy analog (MOLMET). Calves were weaned on d 147 of the study. On d 154, 24 calves/yr (8 calves/treatment) were randomly selected and individually limit-fed a high-concentrate diet until d 201. Muscle biopsies were collected from the same calves on d 154 and 201 (4 calves/treatment/d/yr) for gene expression analysis using RNA sequencing.

first: Philipe

last: Moriel


title: Expression landscape of human stromal cells in preclinical trials involving chemotherapeutic and histone demethylase KDM4-targeting agents

first: Yu

last: Sun

summary: Aging and age-related pathologies including multiple cancer types can be controlled by specifically targeting senescent cells, or more specifically, their hallmark feature, the senescence-associated secretory phenotype (SASP). Lysine methylation is one of the most common histone posttranslational modifications that regulate chromatin structure in mammalian cells. Changes in histone lysine methylation status have been observed during cancer progression, a consequence of dysregulation of histone lysine methyltransferases and/or their opposing demethylases. KDM4 (or JMJD2) encompasses demethylases that target histone H3 on lysines 9 and 36 sites. Frequently overexpressed in breast, colorectal, lung, prostate, and other tumor types and required for efficient cancer cell proliferation, KDM4 proteins represent novel drug targets. However, emerging studies are beginning to uncover the functional roles of KDM4 in epigenomic regulation during cellular senescence and organismal aging, thus providing a new angle to understand human aging and allowing expansion of the existing list of epigenetic targets, the drugs of which are currently limited to the pharmacological arsenal against DNA methyltransferases and histone deacetylases.

overall_design: Examination of in vivo efficacy of a KDM4 inhibitor, ML324, when combined with conventional chemotherapy in reducing the SASP and promoting tumor regression under prelinical conditions. Experimental mice xenografted with human stromal cells and cancer cells were exposed to mitoxantrone (MIT), ML324 or MIT plus ML324, agents delivered via intraperitoneal injection. Drugs were administered in a metronomic manner, given biweekly from the 3rd week of a 8 week regimen.

first: Boyi


title: Effects of Voluntary Running Wheel Exercise-induced Extracellular Vesicles on Anxiety

status: Public on Mar 12 2021

city: Seoul

first: Kyeong Jin

last: Yoon

address: Gwanak-ro 1

country: South Korea

institute: Seoul National University

summary:
Exercise induces the release of extracellular vesicles (EVs) into the circulation, which transmit signals between organs. EVs contain microRNAs (miRNAs) that inhibit mRNA translation or enhance mRNA degradation. However, the effects of exercise-induced EVs on anxiety remain poorly understood.
We used microarrays to figure out the global alteration of gene expression in the hippocampus underlying stress and sedentary or exercise-induced EVs.

overall_design: We isolated EVs from the sera of mice that were sedentary or that voluntarily exercised. After then, we analyzed changes in mice with 3 weeks of restraint stress-induced anxiety after injection of 20μg amounts of EVs from exercised or sedentary mice into the left cerebral ventricle.

first: Kyeong

first: Hyo

middle: Y

last: Moon


title: Gene expression in chicken skin cells cultured in vitro

city: Vienna

first: Julia

last: Lachner

address: Waehringer Guertel 18-20

country: Austria

institute: Medical University of Vienna

summary: Comparison of gene expression in chicken skin cells cultured in monolayer and in 3D skin equivalents

first: Sophia

last: Derdak

first: Leopold

last: Eckhart


title: The brain transcriptome of the wolf spider, Schizocosa ocreata

city: Tallahassee

first: Michelle

last: Arbeitman

state: FL

address: 1115 W. Call St

department: Biomedical Sciences

institute: Florida State University College of Medicine

summary: The goal of this study was to lay the groundwork for comparative transcriptomics of sex differences in the brain of wolf spiders, a non-model organism of the pyhlum Euarthropoda, by generating transcriptomes and analyzing gene expression. To examine differences in sex-differential gene expression, short read transcript sequencing and de novo transcriptome assembly were performed. Messenger RNA (mRNA) was isolated from dissected brain tissue of male and female subadult and mature wolf spiders (Schizocosa ocreata). The data consist of short read sequences for the two different life stages in each sex. Computational analyses on these data include de novo transcriptome assembly, using Trinity and CAP3 assembly suites, and differential expression analysis using the edgeR package. Sample-specific and combined transcriptomes, gene annotations, and differential expression results are described in this data note and are available from associated database submissions.

overall_design: Sequencing and differential expression analyses were performed using dissected brain tissue of male and female immature (subadult) and mature (adult) wolf spiders (Schizocosa ocreata). The analysis was performed with biological replicates of 3 immature males, 2 immature females, 2 mature males, and 3 mature females.

first: Daniel

last: Stribling

first: Peter

middle: L

last: Chang

first: Justin

last: Dalton

first: Christopher

last: Conow

first: Malcom

last: Rosenthal

first: Eileen

last: Hebets

first: Rita

last: Graze


title: Genome-wide DNA methylation profiles of lymphoblastoid cell lines from sex-matched sibling pairs discordant for autism diagnosis

status: Public on Mar 21 2021

city: Washington

first: Valerie

middle: Wailin

last: Hu

state: DC

department: Biochemistry and Molecular Medicine

institute: The George Washington University School of Medicine and Health Sciences

summary: The goals of this study were to: 1) investigate changes in DNA methylation in lymphoblastoid cell lines (LCL) from individuals with a specific subtype of ASD and their respective non-autistic siblings, 2) relate the differentially methylated genes to ASD-relevant pathways and functions, and 3) explore the potential for class determination (case-control) biomarkers based on methylation signatures.

overall_design: DNA from LCL of 21 sibling pairs (42 samples in all) were analyzed for methylated regions using the MeDIP-chip protocol.  Each sample was represented by MeDIP-enriched DNA and input DNA which were each fragmented, labeled and separately hybridized to Affymetrix Human Promoter 1.0R GeneChips (84 arrays in all)  following the Affymetrix protocols.  Data from the scanned genechips were first analyzed for methylation-enriched regions using Partek's workflow for tiling arrays, which resulted in MAT scores representing methylation-enriched (positive MAT scores) and depleted (negative MAT scores) across the genome as well as determination of average probe intensities across the promoter regions.  The intensities were then imported into Multi-experiment View (MeV) microarray analysis software for further analyses,  These additional data analyses to identify differentially methylated genes for class prediction included Significance Analysis of Microarrays (SAM), Pavlidis Template Matching (PTM), Uncorrelated Shrunken Centroids (USC), and Support Vector Machines (SVM) which were all incorporated into the MeV software package.

middle: W


title: Transcriptional responses of Herbaspirillum seropedicae to environmental phosphate concentration

status: Public on Mar 06 2021

city: San Miguel de Tucumán

first: Mariana

last: Grillo-Puertas

state: Tucumán

address: Chacabuco 461

country: Argentina

institute: INSIBIO-CONICET

summary:
Purpose: The global changes of H. seropedicae SmR1 were evaluated in response to environmental Pi conditions, based on differential intracellular polyP levels
Methods: Three independent samples (biological replicates) of H. seropedicae SmR1 cells grown in NFb5 or NFb50 media after 9 h of growth were used to construct 6 sequencing libraries.The libraries were amplified and enriched using the Ion PI Hi-QTM OT2 200 Kit, and sequenced with the Ion PI Hi-QTM Sequencing 200 kit on an Ion PITM Chip Kit v3. Obtained sequences were trimmed, mapped and analyzed using CLC Genomics Workbench 7.5.1 (Qiagen) against the H. seropedicae SmR1 genome (NC_014323). Differential gene expression was accessed using the DESeq2 package. qRT–PCR validation was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and quantified in triplicate using the Power SYBR-Green PCR MasterMix on a Step-One Plus Real Time-PCR System (Applied Biosystems).
Results: After sequencing, 26 and 35 total million reads were obtained for NFb50 and NFb5 conditions, respectively, and from those, 12 million and 16 million reads were uniquely mapped to the H. seropedicae SmR1 genome. Biological replicates showed a very high level of correlation (r2 > 0.98). Among the 4805 genes of the H. seropedicae SmR1 genome, 3976 genes were expressed considering a read coverage equal or higher than three-fold. 1330 genes show expression levels statistically different (p-value ≤ 0.05) from which 670 had fold changes of 2 or higher and were considered differentially expressed genes (DEG) in NFb50 vs NFb5 transcriptome, being 385 down-regulated and 285 up-regulated. To confirm the differential expression observed by the RNA-seq data, the regulation of some genes was confirmed by RT-qPCR. Molecular and physiological analyses revealed that features related to Pi metabolism, bacterial flagella biosynthesis and chemotaxis, energy production, and polyhydroxybutyrate metabolism were induced in the mentioned condition, while aspects involved in adhesion, and stress response were repressed. Since environmental conditions can influence the effectiveness of the PGPB, enhancement of bacterial robustness to withstand different conditions is an interesting challenge
Conclusions: The present study demonstrated that variations in environmental Pi concentration affect H. seropedicae bacterial traits related to survival and other important physiological characteristics. The obtained data could serve not only to understand the bacterial behavior in respect to changes in rhizospheric Pi gradients, but also as a base to design strategies to improve different bacterial features focusing in biotechnological and/or agricultural purposes.

overall_design: RNA-seq approach comparing of transcriptional profiles of H. seropedicae grown in NFb5 or NFb50 media

first: Emanuel

last: Souza


title: Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes

status: Public on Feb 28 2021

city: Frederick

first: GuoJun

last: Yu

state: MD

address: 560 CHANDLER STREET

department: Cell Biology

institute: National Institutes of Health

summary: Somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt’s lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than the constant (C) region which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout (KO) and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of Pol II and its S2 phosphorylated form at the IgH locus. KO of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of P-TEFb complex) on the V region and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreased SHM and the abundance of Pol II S2P at the IgH locus. With all these factors playing a role in transcription elongation, our studies extend our insights into the chromatin context and dynamics of transcription required for SHM.

overall_design: Examination of Dot1L in chromatin accessibility by ATAC-seq in human B cell line Ramos.

first: Zhi

last: Duan

first: Thomas

last: MacCarthy

first: Matthew

middle: D

last: Scharff


title: Deleterious effects of SARS-CoV-2 infection on human pancreatic cells

city: Los Angeles

first: Dhruv

last: Sareen

state: CA California

address: 8700 Beverly Blvd

department: Board of Governors Regenerative Medicine Institute

institute: Cedars-Sinai Medical Center

summary: In order to study the effects of SARS-CoV-2 on pancreatic cells, iPSCs were differentiated into pancreatic cell and infected with SARS-CoV-2

overall_design: Four conditions were examined, with triplicate samples: Infected cells 1 day post infection, infected cells 3 days post infection, mock cells 1 day after mock infection and mock cells 3 days post mock infection.

first: Hanan

last: Sharuddin

first: Victoria

last: Wang

first: Roberta

middle: S

last: Santos

first: Andrew

middle: R

last: Gross

first: Yizhou

first: Harneet

last: Jawanda

first: Yi

last: Zhang

first: Wohaib

last: Hasan

first: Gustavo

last: Garcia Jr.

first: Vaithilingaraja

last: Arumugaswami


title: Estrogen Receptor-α knockout females, but not Estrogen Receptor-β knockout females, show male-specific liver gene expression

first: Andrea

last: Bilger

state: WI

address: 1111 Highland Avenue

department: McArdle Laboratory for Cancer Research

institute: UW-Madison

summary: Estrogen protects females from hepatocellular carcinoma (HCC). To determine whether this protection is mediated by classic estrogen receptors, we tested HCC susceptibility in estrogen receptor-deficient mice. In contrast to a previous study, we found that diethylnitrosamine induces hepatocarcinogenesis to a much greater extent when females lack Esr1, which encodes Estrogen Receptor-α. Relative to wild-type littermates, Esr1 knockout females developed 9-fold more tumors. Deficiency of Esr2, which encodes Estrogen Receptor-β, did not affect liver carcinogenesis in females. Using microarrays and QPCR to examine estrogen receptor effects on hepatic gene expression patterns, we found that germline Esr1 deficiency resulted in the masculinization of gene expression in the female liver.

overall_design: For microarray analysis, 1.3 µg of total RNA from each sample was used for fluorescent probe preparation, using the Agilent Low Input Linear Amplification and Labeling Kit (Agilent Technologies Inc., Santa Clara, CA). Following linear amplification and dye labeling, the quality of samples was determined by measuring dye incorporation and cRNA concentration using the NanoDrop instrument. In the study examining the effect of deletion of Esr1 specifically in the liver, we examined gene expression in individual animals where probes were labeled with cyanine-3 (Cy-3) and the sex-reference control [pool of ½ B6 female (n=18) and ½ B6 male (n=15)] was labeled with cyanine-5 (Cy-5). In all other gene expression studies, hepatic RNA was prepared from individual mice and RNA pools were made for each group, with approximately the same amount of RNA from each individual, using Cy-5 to label probes and Cy-3 to label the sex-reference control (except for ERalpha WT Male-Rep2 and ERalpha KO Male-Rep2, for which the samples were labeled with Cy3 and the reference was labeled with Cy5). Hybridizations were carried out according to the manufacturer instructions, using the Agilent-014868 Mouse Whole Genome 4 x 44 Oligo Microarray G4122F (Agilent Technologies Inc., Santa Clara, CA), with 1.5 µg of labeled probe and 1.5 µg of labeled sex-reference control. The Agilent Microarray Scanner System G2565BA was used to measure hybridization signals and Agilent Feature Extraction software (version 9.1) was used for analysis.

first: Mara

last: O'Brien

first: Norman

last: Drinkwater


title: RNA sequencing of cancer-associated fibroblasts (CAFs) and normal fibroblasts from a triple-transgenic gastric cancer mouse model

city: Rehovot

first: Ruth

last: Scherz-Shouval

address: 234 Herzl street

department: Biomolecular Science

country: Israel

institute: Weizmann Institute of Science

summary: Gastric cancer is the 3rd most lethal cancer worldwide, and the genomic status of its cancer cells has not translated into effective prognostic or therapeutic strategies. We therefore hypothesize that outcomes may depend on the tumor microenvironment (TME), and in particular cancer-associated fibroblasts (CAFs). However, very little is known about the role of CAFs in gastric cancer. To address this, we map the transcriptional landscape of human gastric cancer stroma by microdissection and RNA sequencing of CAFs from gastric cancer patients. We find a stromal gene signature associated with poor disease outcome. This signature is regulated by the transcription factor Heat Shock Factor 1 (HSF1), that activates CAF-derived extracellular vesicles (EVs) that transfer components including Inhibin Subunit Beta A (INHBA) and Thrombospondin 2 (THBS2), to the TME to promote cancer. Together, our work provides the first transcriptional map of human gastric cancer stroma, and highlights HSF1 and its transcriptional targets as potential diagnostic and therapeutic targets in the genomically stable tumor microenvironment.

overall_design: Gastric cancer was induced in the triple-transgenic iLgr5;GLI2A mice as described in Syu et al, 2016. Mice which developed gastric cancer and control mice were sacrificed, CAFs were isolated from their stomachs and sequenced.

last: Grunberg

first: Gil

last: Friedman

first: Meirav

last: Pevsner-Fischer

first: Yaniv

last: Stein


title: Quantitative in vivo analyses reveal a complex pharmacogenomic landscape in lung adenocarcinoma

city: Stanford

first: Chuan

last: Li

address: 327 Campus Drive

department: Biology

institute: Stanford University

summary: The lack of knowledge about the relationship between tumor genotypes and therapeutic responses remains one of the most important gaps in enabling the effective use of cancer therapies. Here, we couple a multiplexed and quantitative platform with robust statistical methods to enable pharmacogenomic mapping of lung cancer treatment responses in vivo. We uncover a surprisingly complex map of genotype-specific therapeutic responses, with over 20% of possible interactions showing significant resistance or sensitivity. We validate one of these interactions - the resistance of Keap1 mutant tumors to platinum therapy - using a large patient response dataset. Our results highlight the importance of understanding the genetic determinants of treatment responses in the development of precision therapies and define a strategy to identify such determinants.

overall_design: We applied Tuba-seq to assess the genotype-specific therapeutic responses of 11 genotypes of lung tumors to a panel of eight single and combination therapies. KT;Cas9 mice with Lenti-sgTSPool/Cre-initiated lung tumors were treated for three weeks with one of the eight therapies followed by Tuba-seq analysis.

first: Wen-Yang

last: Lin

first: Hira

last: Rizvi

first: Hongchen

last: Cai

first: Christopher

last: McFarland

first: Zoë

middle: N

last: Rogers

first: Maryam

last: Yousefi

first: Ian

middle: P

last: Winters

first: Charles

middle: M

last: Rudin

first: Dmitri

last: Petrov

first: Monte

last: Winslow


title: A Porcine Model of Heart Failure With Preserved Ejection Fraction Induced by Chronic Pressure Overload Characterized by Cardiac Fibrosis and Remodeling

city: Guangzhou

first: Xiang

state: Guangdong

address: 11 fengxin road, guangzhou science city

institute: Guangdong Laboratory Animals Monitoring Institute

summary: In this study, we employed descending aortic constriction (DAC) technique to establish a minipig heart failure model. The cardiac phenotypes, pathological and cellular changes, fibrotic signaling activation, and gene expression profiles have been explored.

overall_design: Total RNAs from the ventricular tissues of Sham (n=3) and DAC (n=3) minipigs were isolated using TRIzol® Reagent. Each group contains three independent samples. Subsequently, a total amount of 3 µg RNA per sample were used for transcriptome sequencing.

first: Weijiang

last: Tan

first: Shuang

last: Zheng

first: Feng

middle: H

last: Yang


title: Dynamic extrinsic pacing of the HOX clock in human axial progenitors control motor neuron subtype specification

status: Public on Mar 02 2021

city: PARIS

first: Stephane

last: NEDELEC

address: 17 rue du fer a moulin

country: France

institute: Institut du Fer à Moulin

summary: Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX genes within axial stem cells that fuel axial embryo elongation. Whether HOX genes sequential activation, the “HOX clock”, is paced by intrinsic chromatin-based timing mechanisms or by temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in human pluripotent stem cells differentiating into spinal cord motor neuron subtypes which are progenies of axial progenitors. We show that the progressive activation of caudal HOX genes is controlled by a dynamic increase in FGF signaling. Blocking FGF pathway stalled induction of HOX genes, while precocious increase in FGF alone, or with GDF11 ligand, accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The HOX clock is thus dynamically paced by exposure parameters to secreted cues. Its manipulation by extrinsic factors alleviates temporal requirements to provide unprecedented synchronized access to human cells of multiple, defined, rostro-caudal identities for basic and translational applications.

overall_design: human ES cells SA001

first: Vincent

last: Mouilleau

first: Margot

last: Jarrige

first: Cécile

last: Martinat

first: Stéphane

last: Nedelec


title: Repetitive in vivo manual loading of the spine elicits cellular responses in porcine  annuli fibrosi

status: Public on Feb 26 2021

city: Neustadt

first: Claudia

last: Klein

address: Höltystr. 10

country: Germany

institute: Friedrich-Loeffler-Institut, Institute of Farm Animal Genetics

summary: Back pain and intervertebral disc degeneration are prevalent, costly, and widely treated by manual therapies, yet the underlying causes of these diseases are indeterminate as are the scientific bases for such treatments.  The present studies characterize the effects of repetitive in vivo manual loads on porcine intervertebral disc using RNA deep sequencing.  A single session of repetitive manual loading applied to the lumbar spine induced both up- and down-regulation of a variety of genes transcribed by cells in the ventral annuli fibrosi.  The effect of manual therapy at the level of loading was greater than at a level distant to the applied load.  Gene ontology and molecular pathway analyses categorized biological, molecular, and cellular functions influenced by repetitive manual loading, with over-representation of membrane, transmembrane, and pericellular activities.  The present studies support previous findings of intervertebral disc cell mechanotransduction, and are the first to report comprehensively on the repertoire of gene targets influenced by mechanical loads associated with manual therapy interventions.  The present study defines the cellular response of repeated, low-amplitude loads on the annuli fibrosi and lays the foundation for future work defining how the intervertebral disc responds to single or low-frequency manual loads typical of those applied clinically.

overall_design:
Examination of a single session of repetitive manual loading applied to the lumbar spine on the ventral annulus fibrosus

Raw data were not provided for GSM5077456, GSM5077457 or GSM5077459.

first: John

last: Matyas

first: Dragana

last: Ponjevic

first: Neil

last: Duncan

first: Gre

last: Kawchuk


title: Transcriptomics of granulosa cells(GCs) from healthy follicles(HFs), early atretic follicles(EFs) and late atretic follicle(LFs) in Chinese buffalo

city: Nanning

first: Yu

last: Pan

state: Guangxi

address: No. 100, Daxuedong Road, Xixiangtang District, Nanning, Guangxi

department: College of Animal Science and Technology

institute: Guangxi University

summary: Compared with granulosa cells from healthy follicles, 2888 (1790 up-regulated, 1098 down-regulated) and 1629 (1230 up-regulated, 399 down-regulated) DEGs (|FC| > 1.75 and P value < 0.05) were identified in granulosa cells of early atretic follicles and late atretic follicle, respectively. Out of these DEGs, 1217 genes were commonly shared among the two comparisons

overall_design: GCs were collected from healthy follicles, early atretic follicles and late atretic follicle  for transcriptome analysis using second-generation high-throughput sequencing

first: Juanru

last: Cheng

first: Yanfei

last: Deng


title: Tissue-specific 5-hydroxymethylcytosine landscape of the human genome

city: Beijing

first: Bo

last: He

state: Beijing

address: Yiheyuan Road 5th.

department: School of Life Science

institute: Peking University

summary: 5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark that regulates gene expression. Charting the landscape of 5hmC in human tissues is fundamental to understanding its regulatory functions. Here, we systematically profiled the whole-genome 5hmC landscape at single-base resolution for 19 types of human tissues. We found that 5hmC preferentially decorates gene bodies and outperforms gene body 5mC in reflecting gene expression. Approximately one-third of 5hmC peaks are tissue-specific differentially-hydroxymethylated regions (tsDhMRs), which are deposited in regions that potentially regulate the expression of nearby tissue-specific functional genes. In addition, tsDhMRs are enriched with tissue-specific transcription factors and may rewire tissue-specific gene expression networks. Moreover, tsDhMRs are associated with single-nucleotide polymorphisms identified by genome-wide association studies and are linked to tissue-specific phenotypes and diseases. Collectively, our results show the tissue-specific 5hmC landscape of the human genome and demonstrate that 5hmC serves as a fundamental regulatory element affecting tissue-specific gene expression programs and functions.

overall_design: We used CATCH-Seq which is a bisulfite-free, base-resolution method for the genome-wide detection of 5hmC, to detect human tissues 5hmC pattern. Our method is based on: (1) selective oxidation of 5hmC to 5fC ; (2) subsequent labeling of the newly generated 5fC; and (3) 5fC labeling adduct caused C-to-T transition during DNA amplification.We then applied CATCH-seq to genomic DNA isolated from human tissues respectively.

first: Chao

last: Zhang

first: Xiaoxue

first: Chengqi

last: Yi


title: MeRIP-sequencing in ESCC cells

first: wei

last: wang

address: 17 Nanli, Panjiayuan, Chaoyang District

department: Thoracic Surgery

institute: National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital

summary: IP with m6A-specific antibodies and Input sequencing for every sample.

overall_design: Examining 2 conditions, with or without METTL3 depletion.


title: RNA sequencing of stroma from good (survival) and bad (lethality) outcome gastric cancer patients is enough to separate patients based on outcome.

first: Ruth

last: Scherz-Shouval

overall_design: Stroma and cancer from gastric cancer patients were captured separately using laser capture microdissection and sequenced.

last: Grunberg

first: Irit

last: Ben-Aharon

first: Tal

last: Goshen-Lago


title: Cholesterol accumulation as a driver of  hepatic inflammation under  translational dietary conditions can be  attenuated by a multicomponent  medicine

status: Public on Mar 01 2021

city: Leiden

first: Martine

middle: C

last: Morrison

address: Sylviusweg 71

department: MHR

institute: TNO

summary: Background: Non-alcoholic fatty liver disease (NAFLD) is a complex multifactorial disorder that is characterised by dysfunctional lipid metabolism and cholesterol homeostasis, and a related chronic inflammatory response. NAFLD has become the most common cause of chronic liver disease in many countries, and its prevalence continues to rise in parallel with increasing rates of obesity. Here, we evaluated the putative NAFLD-attenuating effects of a multicomponent medicine consisting of 24 ingredients: Hepar compositum (HC-24).   Methods: Ldlr-/-.Leiden mice were fed a high-fat diet (HFD) with a macronutrient composition and cholesterol content comparable to human diets for 24 weeks to induce obesity-associated metabolic dysfunction, including hepatic steatosis and inflammation. HC-24 or vehicle control was administered intraperitoneally 3 times/week (1.5 ml/kg) for the last 18 weeks of the study. Histological analyses of liver and adipose tissue were combined with extensive hepatic transcriptomics analysis (next generation sequencing). Transcriptomics results were further substantiated with immunohistochemical and liver lipid analyses.   Results: HFD feeding induced obesity and metabolic dysfunction that was associated with adipose tissue inflammation and increased gut permeability. In the liver, HFD-feeding resulted in a disturbance of cholesterol homeostasis and an associated inflammatory response. HC-24 did not affect body weight, metabolic risk factors, adipose tissue inflammation or gut permeability. While HC-24 did not affect total liver steatosis, there was a pronounced reduction in lobular inflammation in HC-24-treated animals, which was associated with modulation of genes involved in inflammation (e.g. neutrophil chemokine Cxcl1) and cholesterol homeostasis (i.e. predicted effect on 'cholesterol' as an upstream regulator, based on gene expression changes associated with cholesterol handling). These effects were confirmed by immunohistochemical staining of neutrophils and biochemical analysis of hepatic free cholesterol content. Intrahepatic free cholesterol levels were found to correlate significantly with the number of inflammatory aggregates in the liver, thereby providing a potential rationale for the observed anti-inflammatory effects of HC-24.   Conclusions: Free cholesterol accumulates in the liver of Ldlr-/-.Leiden mice under physiologically translational dietary conditions, and this is associated with the development of hepatic inflammation. The multicomponent medicine HC-24 reduces accumulation of free cholesterol and has molecular and cellular anti-inflammatory effects in the liver.

overall_design: Ldlr-/-.Leiden mice (age 13-26wks) were studied in 3 diet/treatment groups. The control group was fed a standard/healthy chow diet for 24 weeks. The two treatment groups were fed a high-fat diet (HFD) with a macronutrient composition and cholesterol content comparable to human diets for 24 weeks to induce obesity-associated metabolic dysfunction, including hepatic steatosis and inflammation. For the two HFD treatment groups, (i) HC-24 or (ii) vehicle control was administered intraperitoneally 3 times/week (1.5 ml/kg) for the last 18 weeks of the study. Histological analyses of liver and adipose tissue were combined with extensive hepatic transcriptomics analysis (t=24wk; next generation sequencing). Transcriptomics results were further substantiated with immunohistochemical and liver lipid analyses.

first: Andrea

last: Mueller

first: Robert

last: Kleemann

first: Eveline

last: Gart

first: Wim

last: van Duyvenvoorde

first: Lars

last: Verschuren

first: Martien

last: Caspers

first: Aswin

last: Menke

first: Natascha

last: Krömmelbein

first: Kanita

last: Salic

first: Yvonne

last: Burmeister

first: Bernd

last: Seilheimer


title: Transcriptomic analysis reveals dynamic progression trajectory and molecular pattern of UV-induced cSCC

city: Shanghai

first: Sibo

last: Zhu

state: China

address: songhulu 2005

institute: Fudan University

summary: In the present study, we aim to identify key genes or pathways in the progression from normal skin to actinic keratoses (AK), then to cutaneous squamous cell carcinoma (cSCC) by transcriptomics analysis. We firstly established a UVR-induced cSCC model using SKH-1 hairless mouse successfully. Then, the transcriptome profiles among normal skin, AK and cSCC were investigated. We also constructed pseudo trajectory and deconvoluted the cell compositions among these three different tissues. A series of bioinformatics and molecular experiments analyses were carried out and we found retinoic acid receptor-related receptor alpha (Rora) was involved in this progression. Our results demonstrated Rora can be a potential therapeutic target for cSCC, which facilitates the treatment of cSCC.

overall_design: Examination of mRNA profiles from normal skin, actinic keratoses and cutaneous squamous cell carcinoma tissues


title: Transcriptomic profile of normal immortalized fallopian tube cells in response to long-term norepinephrine treatment

status: Public on Mar 08 2021

city: Tampa

first: Alvaro

last: Monteiro

address: 12902 Magnolia Dr

institute: H. Lee Moffitt Cancer Center

summary: The goal of this study was to identify transcriptional changes that occur in response to long-term norepinephrine (NE) treatment on normal immortalized fallopian tube cells (iFTSEC283). We treated isogenic cell lines (iFTSEC283 and iFTSEC283p53R175H)  with 10μM NE or vehicle control (water) for 137 days with media change every two days followed by whole-transcriptome RNA seq. We identified 123 differentially expressed genes in 10μM NE vs mock-treated iFTSEC283. In iFTSEC283p53R175H cells, we identified 23 differentially expressed genes in response to chronic NE treatment.

overall_design: There are 12 samples representing 2 cell lines (iFTSEC283 and iFTSEC283 p53R175H), in 2 conditions (10 μM NE and vehicle control) with 3 independent replicates.

first: Sweta

last: Dash

first: Sean

last: Yoder

first: Tania

last: Mesa

first: Andrew

last: Smith

first: Ling

last: Cen

first: Steven

last: Eschrich

first: Guillermo

last: Armaiz-Pena


title: Genomic CpG methylation and TKI-response of the patient with advanced stage lung adenocarcinoma

city: Taichung

first: Wei-Chung

last: Cheng

address: No. 91, Hsueh-Shih Road

department: Research Center for Tumor Medical Science

country: Taiwan

institute: China Medical University

summary: The DNA methylation signature involved 87 differentially methylated regions (DMR) that distinguished patients’ response to EGFR-TKIs. The methylation level of HOXB9 (cg13643585) which was negatively correlated with gene expression and annotated as function of transcription factor displayed 88% and 92% of sensitivity with respect to objective response rate (ORR, OR=9.25, p=0.0002) and disease control rate (DCR, OR=6.64, p=0.0009), respectively. Importantly, HOXB9 gained methylation in patients with progressive disease (PD) relative to partial response (PR) patients specific to those who harbored EGFR-activating mutations (ORR, OR=8.8, p=0.0045; DCR, OR=6.63, p=0.018).

overall_design: The study population consisted of patients with advanced lung adenocarcinoma (stage IIIA to IV) who had received TKI therapy (Table 1) at the Taichung Veterans General Hospital (TCVGH) and National Taiwan University Hospital (NTUH). Fifty patients carried EGFR-activating mutation tumors (28 L858R/L861Q, 20 exon 19 deletion, 2 G719X) and 29 patients were wild type EGFR.


title: Gata2 loss alters epigenetic priming and gene expression profiles in early myeloid and lymphoid progenitors leading to cytopenias [ATAC-seq]

status: Public on Feb 25 2021

city: Boston

first: Leonard

last: Zon

state: MA

address: 1 Blackfan Circle

department: Oncology/Hematology

institute: Boston Children's Hospital

summary: Germline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Due to embryonic lethality of Gata2-/- mice, the role of germline loss of GATA2 has not been studied in adult hematopoiesis. Here, we generated a zebrafish mutant of gata2b, which in adulthood recapitulated the myelomonocytopenia and B cell lymphopenia of GATA2 syndrome. Using single cell epigenetic and transcriptomic analyses, we showed that loss of gata2b alters chromosome accessibility and results in gene expression changes in both early myeloid and lymphoid progenitors. We found differentiation block with skewing away from the monocytic program in myeloid progenitors in gata2bko zebrafish. In lymphoid progenitors, gata2b loss led to increased lymphoid priming but with incomplete B cell lymphopoiesis. These results place GATA2 in critical junctions of lineage specification in adult hematopoiesis.

overall_design: Two approaches of single cell ATAC sequencing were used on unsorted zebrafish kidney marrow cell from wildtype, gata2b heterozygous and gata2b homozygous mutants (exon 2 CRISPR mutants described in this paper). One approach was sci-ATAC sequencing (used for 1 wildtype and 1 gata2b homozygous knockout) and the other was 10x Genomics scATAC sequencing platform (3 wildtype, 2 gata2b heterozygous and 3 gata2b homozygous mutants). The zebrafish underwent cardiac bleeding to reduce amount of peripheral blood in the marrow samples. No other manipulation was performed on the marrow cells. Single nuclei were obtained by the protocol described in the manuscript.

first: Serine

last: Avagyan

first: Sai

last: Ma

first: Song

last: Yang

middle: I


title: Muscle transcriptome profile of Alentejano and Bísaro pig breeds

city: Évora

first: André

middle: Filipe Barreto

last: Albuquerque

address: Rua Ramalho Ortigao, 15

country: Portugal

institute: Universidade de Évora

summary:
Gene expression is one of the main factors to influence meat quality by modulating fatty acid composition and deposition rates in the muscle tissue. This study aimed to explore the transcriptomic universe of the longissimus lumborum muscle in two local pig breeds with distinct genetic background (n=10) using next-generation sequencing technologies and the traditional Real-Time qPCR. RNAseq yielded 49 DEG’s, with 34 overexpressed in the Alentejano (AL) and 15 in the Bísaro (BI) breed. MYH3 was found significantly overexpressed (log2 FC=-1.191, q<0.05) in the leaner BI pigs and is suggested to impact the development of skeletal muscle tissue.
Several candidate genes for lipid metabolism with low counts excluded with RNAseq such as ACLY (p<0.05), ADIPOQ (p<0.01), ELOVL6 (p<0.001), LEP (p<0.05) and ME1 (p<0.05) showed to have an unavoidable relevance through Real-Time quantitation. These results agree with the more adipogenic profile of the AL breed. Adiponectin resistance and higher levels of MAP3K14 may explain an induced insulin resistance, increased inflammatory response and lower loin size.

overall_design: Comparation of the transcriptomic data in muscle tissue (longissimus lumborum) of the two main portuguese pig breeds

first: José

last: Martins

first: Cristina

last: Óvilo


title: Gene expression profile of iPSC-derived dopaminergic neurons from control and Parkinson's disease patients carrying LRRK2 G2019S point mutation

status: Public on Feb 24 2021

city: Hospitalet de Llobregat (Barcelona)

first: Meritxell

last: Pons

address: Avinguda Gran Via, 199-203

institute: IDIBELL

summary:
The goal of this study is to compare the RNA expression profile from human iPSC-derived dopaminergic neurons from control, Parkinson’s disease LRRK2 G2019S and gene-edited isogenic lines (with corrected LRRK2 mutation) to gain insight into the mechanisms underlying neuronal degeneration in PD.
Total RNA was assayed for quantity and quality using Qubit® RNA HS Assay (Life Technologies) and RNA 6000 Nano Assay on a Bioanalyzer 2100.  All the steps were carried out in the ice and using the nCounter nanoString reagent for the Neuropathology panel. The master mix for the hybridization process was prepared by mixing hybridization buffer and reporter CodeSet. A dilution of 10μg/μl for each RNA to be analyzed was prepared and maintained in ice. For each sample, a tube was prepared with 5μl of RNA (10μg/μl) and 8μl of master mix. 2μl of Capture ProbeSet was added in each tube and the hybridization process was started using a PCR machine at 65ºC for at least 16 hours. After this step, the samples were maintained at 4ºC. Once the hybridization process was completed, the hybridized RNA was loaded in the neuropathology cartridge, and the data were available 24 hours later.

overall_design: Parkinson's disease (PD) human iPSC-derived neuronal expression profile was obtained by comparing human iPSC-derived neurons from control, PD LRRK2 G2019S patients and isogenic lines. MRNA profiles were generated using the nCounter nanoString reagent for Neuropathology panel. Control = from one healthy individual;  2 independent differentiations. PD = from 2 PD-LRRK2 G2019S patients; 2/3 independent differentiations; isoPD = from 2 corrected PD-LRRK2 patients; 2/3 independent differentations.


title: PFNF54-Pfs16-GFP-LUC gametocytes treated at early stage or late stage development with compound MMV666810 or MMV674850

status: Public on Feb 19 2021

city: University Park

first: Riette

last: van Biljon

address: W122 Millenium Science Complex

institute: Pennsylvania State University

summary: Plasmodium falciparum gametocytes can often show differential drug responses depending on their stage of development. Here, the transcriptomic response of early stage (Stage II-III) and late stage (Stage IV-V) gametocytes was evaluated following treatment with chemical compounds that show differential killing activity between early and late-stage gametocytes.

overall_design: P. falciparum NF54-Pfs16-GFP-LUC parasites were induced to produce gametocytes by culturing in glucose-deficient media , before removing asexual parasites from the population by daily 5% D-sorbitol treatments from day 1-4 and thereafter culturing the parasites in glucose positive media and grown under stationary conditions for the entire experiment. The compounds were added to the cultures at 10x their previously determined IC50 values on day 5 (early-stage gametocytes) and 10 (late-stage gametocytes) and samples taken at 24 h and 58 h post-treatment. The 12 total samples were hybridized against a Cy3-labeled reference pool of 3D7 mixed stage asexual parasites and NF54-pfs16-GFP-LUC mixed stage gametocytes on a two-color array.  Early gametocyte samples were harvested from culture by 0.01%(w/v) saponin addition  and late-stage samples were harvested via density centrifugation using Nycoprep 1.077 cushions and stored at -80˚C until RNA isolation using TRIZOL reagent along with on-column extraction using the Qiagen RNeasy kit (Qiagen, Germany).

first: Riëtte

middle: A

first: Roelof

last: van Wyk

first: Lindsey

last: Altenhofen

first: Mariëtte

last: van der Watt

first: Janette

last: Reader

first: Jandeli

last: Niemand

first: Manuel

last: Llinás

first: Lyn-Marié

last: Birkholtz


title: Tunicamycin induced ER stress in liver of C57Bl/6N mice

status: Public on Feb 23 2021

first: Peng

address: 1 Xian Nong Tan Street

department: Institute of Materia Medica

institute: Chinese Academy of Medical Sciences and Peking Union Medical College

summary:
ER stress could affect many tissues, especially liver, in which non-alcoholic fatty liver disease, liver steatosis, etc. have been reported relative. But there still lack systematic insight into ER stress in liver, which can be obtained by transcriptomics of the tissue. Here, tunicamycin was utilized to induce ER stress in C57BL/6N mice. And microarray was performed to get the transcriptome alteration.
Microarray of liver from tunicamycin-injected C57Bl/6N mice

overall_design: Liver from tunicamycin- and vehicle- injected C57Bl/6N mice

first: Zijing

last: Wang

first: Yisa

first: Biyu

last: Hou

first: Jialin

last: Sun

first: He

last: Tian

first: Bowen

last: Li

first: Guanghou

last: Shui

first: Xiuying

first: Xinyu

first: Guifen

last: Qiang

first: ChongWee

last: Liew

first: Guanhua

last: Du


title: The DEAD-box RNA helicases RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis

status: Public on Feb 18 2021

city: Geneva

first: Martina

last: Valentini

address: 1, rue Michel Servet

department: Department of Microbiology and Molecular Medicine

country: Switzerland

institute: University of Geneva

summary: Purpose: We wanted to determine the transcriptome profile of PAO1 wild type, PAO1ΔrhlE1, PAO1ΔrhlE2 and PAO1ΔrhlE1ΔrhlE2 mutants on swarming conditions. Method: Cells were harvested using the RNA bacteria protect solution (QIAGEN). Total RNAs was extracted and purified using Monarch RNA isolation kit (NEB), treated with DNase I (Promega) three times to remove contaminating genomic DNA and re-purified again using phenol-chloroform. The removal of ribosomal RNA was performed using the Ribo-Zero Bacteria Kit (Illumina) and cDNA libraries were generated with the ScriptSeq v2 Kit (Illumina) . The samples were sequenced in single end mode on an Illumina HiSeq 4000 device and mRNA reads were trimmed and mapped to the NC_002516.2(PAO1) reference genome from NCBI using Stampy pipeline with defaut settings.

overall_design: PAO1 wild type, PAO1ΔrhlE1, PAO1ΔrhlE2 and PAO1ΔrhlE1ΔrhlE2 mutants were grown in twelve 12-cm square swarming plates at 37°C. Samples from 6 plates were pooled and two replicates of each strain were therefore obtained. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Kit (Illumina). Then, libraries were prepared using the Illumina TruSeq stranded mRNA kit and validated on the Bioanalyzer 2100 (Agilent). Samples were sequenced using the Illumina HiSeq 2000, 100 bp single end read at the iGE3 genomics platform of the University of Geneva.

first: Stéphane

last: Hausmann

first: Diego

last: Gonzalez

first: Johan

last: Geiser


title: Gata2 loss alters epigenetic priming and gene expression profiles in early myeloid and lymphoid progenitors leading to cytopenias [RNA-seq]

first: Leonard

last: Zon

overall_design: Single cell RNA sequencing were performed on sorted zebrafish kidney marrow cell from wildtype, gata2b heterozygous and gata2b homozygous mutants (exon 2 CRISPR mutants described in this paper), n=3 each genotype. The zebrafish underwent cardiac bleeding to reduce amount of peripheral blood in the marrow samples. Cells were sorted on BDAria2 sorter into 1x PBS with 0.04% BSA. The goal of sorting was to enrich for no mature myeloid and erythroid cell types, comprising <50% of the adult marrow cells.

first: Serine

last: Avagyan

first: Song

last: Yang


title: Transcriptome analysis of barley root meristem under Al and low pH stress

city: Bialystok

first: Karolina

last: Chwialkowska

address: Kilinskiego 1

country: Poland

institute: Medical University of Bialystok

summary: Aluminum (Al) is the most common metal in the Earth’s crust and Al toxicity is considered to be the most harmful abiotic stress in acidic soils that today comprise more than 50% of the world’s arable lands. The first symptom of Al toxicity is the reduction of root growth, resulting in decreased water and nutrients uptake, plant growth retardation, and finally, yield reduction. Barley (Hordeum vulgare L.), which is the fourth cereal crop in regards to cultivation area and production tonnage, belongs to crops most sensitive to toxic aluminum ions in low pH soils. We present the RNA-seq transcriptome analysis of root meristems of barley seedlings grown in hydroponics at optimal pH (6.0), low pH (4.0), and low pH with Al (10 µM of bioavailable Al3+ ions). Two independent experiments were conducted: with short-term (24 h) and long-term (7 days) Al treatment. Interestingly, in the short-term experiment, more genes were differentially expressed between root meristems grown at pH=6.0 and pH=4.0, than between those grown at pH=4.0 with and without Al treatment. The upregulated genes that were overrepresented at conditions of low pH, compared to optimal pH, were associated with response to oxidative stress, cell wall organization, and iron ion binding. Among genes downregulated by low pH were mainly those related to chromatin organization. These results show that low pH itself is a severe stress for barley plants. Among genes upregulated by short Al treatment, overrepresented were those related to response to stress condition and calcium ion binding. After 7 days of hydroponics, the number of DEGs between hydroponics at pH=4.0 and 6.0 were still high but lower than in the short-term experiment, which suggests that plants partially adapted to the low pH.  Interestingly, 7 day Al treatment caused massive changes in the transcriptome profile compared to the condition of low pH alone. Over 4 000 genes were upregulated and almost 2 000 genes were downregulated by long-term Al stress. These DEGs were related to e.g. stress response, cell wall development and metal ion transport. Based on our results we can assume that both, Al3+ ions and low pH are harmful to barley plants.Additionally, we phenotyped in detail the root system of barley seedlings grown in the same hydroponic conditions for 7 days at pH=6.0, pH=4.0, and pH=4.0 with Al. The results correspond to transcriptomic data and show that low pH itself is a stress facor that causes a significant reduction of root growth and the addition of aluminum further increases this reduction. It should be underlined that in the acidic arable lands, plants are exposed simultaneously to both of these stresses (low pH and Al), as Al becomes soluble at pH below 5.5. The presented transcriptome analysis may help to find potential targets for breeding barley plants more tolerant to such conditions.

overall_design: We examined the influence of low pH and aluminum on transcriptome of barley root meristems in two independent hydroponic experiments (short- and long-term). Root meristems from at least 8 plants from one hydroponic container were considered as one repetition (with an average of 5 root meristems per plant). Each experimental combination (optimal pH=6, low pH=4 and Al treated)  was analyzed in three replicates in both experiments.

last: Chwiałkowska

first: Miriam

last: Szurman


title: Differentially expressed lncRNA in biological premature ovarian insufficient(bPOI)

status: Public on Feb 20 2021

city: jinan

first: Duan

address: Wenhua Xi Road

institute: Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University

summary: We have completed the lncRNA expression profiling of human granulosa cells to reveals the differentially expressed lncRNAs involved in POI.

overall_design: In this study, we applied lncRNA sequencing in ovarian granulosa cells from patients with bPOI and normal people.

first: Yingying

last: Qin

first: Shidou

last: Zhao

first: Xiaoyan

last: WANG


title: Functionally Distinct Pomc-Expressing Neuron Subpopulations in Hypothalamus Revealed by Intersectional Targeting

city: Cologne

first: Nasim

last: Biglari

address: Gleueler Str. 50

institute: Max Planck Institute for Metabolism Research

summary: We report differences in translational profiles between POMCLepr+ and POMCGlp1r+ neurons.

overall_design: 6 hypothalami from male and female (3m, 3f) POMCDre LeprCre ROSA26lSlrSrEGFPL10a+/- and 12 hypothalami from male and female (6m, 6f) POMCDre Glp1rCre ROSA26lSlrSrEGFPL10a+/- mice were pooled for RNA extraction. Mice were rapidly decapitated. The arcuate nucleus (ARC) was dissected manually, snap frozen in liquid Nitrogen and stored at -80°C. RNA was extracted from the samples using the RNA extraction protocol (see details below). The pooled hypothalami, served as the input and was subjected to immunoprecipitation of the EGFP-tagged ribosomes using an anti-EGFP antibody. RNA concentrations were measured by a Qbit machine, finally, mRNA sequencing was performed on input and pulldown RNA samples.

first: Paul

last: Klemm

first: Jens

last: Brüning


title: G2/M DNA synthesis at transcription start sites because of RNA transcription persistence during DNA replication [G2M async Brdu Seq]

status: Public on Feb 17 2021

city: Birmingham

first: Marco

last: Saponaro

address: Vincent Drive

department: Institute of Cancer and Genomic Sciences

country: United Kingdom

institute: University of Birmingham

summary: RNA transcription and DNA replication both use DNA as a substrate, that however can be engaged only by one of them at any given time. Consequently, conflicts between transcription and replication significantly affect genome stability and human health. To understand the reciprocal interplay between transcription and replication in human cells, we performed a genome-wide analysis of the two processes throughout S-phase. We found that transcription persists during replication, with direct consequences for replication progression through genes and sites of DNA damage. Importantly however, we also found that transcription start sites will not be replicated together with the surrounding regions, and remain under-replicated throughout S-phase. DNA synthesis across transcription start sites occurs specifically when cells enter mitosis, because the RNA Polymerase II is removed at that point from the majority of the genes. Intriguingly, this G2/M DNA synthesis is not associated with increased DNA damage and occurs at very high frequency.

overall_design: Genome-wide DNA synthesis profile of BJ-hTERT and U2OS cells in G2/M phase generated by BrdU-seq

first: Jianming

last: Wang

first: Patricia

last: Rojas


title: Expression data from normal human cerebellum

city: Toronto

first: Michael

middle: David

last: Taylor

state: Ontario

address: 555 University Avenue

department: Division of Neurosurgery

institute: SickKids

summary: Affimetrix Human Gene 1.1 ST Array profiling of 13 normal human cerebellum samples. Total RNA from 8 fetal brains and 5 adult brains was obtained from the Biochain company.

overall_design: Total RNA from normal human cerebellum has been obtained from the Biochain company for hybridization on Affimetrix microarrays.


title: G2/M DNA synthesis at transcription start sites because of RNA transcription persistence during DNA replication [Chip-Seq H2AX]

overall_design: Genome-wide profiles of H2AX for the normalisation of γ-H2AX levels in asynchronous BJ-hTERT cells  treated with the indicated siRNA.


title: G2/M DNA synthesis at transcription start sites because of RNA transcription persistence during DNA replication [REPLI-Seq async]

overall_design: Genome-wide DNA replication of BJ-hTERT by BrdU-seq, with cells sorted in 5 fractions based on their DNA content


title: White matter aging drives microglial diversity

city: Munich

first: Ozgun

last: Gokce

address: Feodor-Lynen-Strasse 17

institute: Institute for Stroke and Dementia Research

summary: Aging results in both grey and white matter degeneration, but the specific microglial responses are unknown. Using single-cell RNA sequencing from white and grey matter separately, we identified white matter associated microglia (WAM), which share parts of the disease-associated microglia (DAM) gene signature and are characterized by the activation of genes implicated in phagocytic activity and lipid metabolism. WAM depend on triggering receptor expressed on myeloid cells 2 (TREM2) signaling and are aging dependent. In the aged brain, WAM form independently of apolipoprotein E (APOE), which is in contrast to mouse models of Alzheimer’s disease, in which microglia with WAM gene signature are generated prematurely and in an APOE-dependent pathway similar to DAM. Within the white matter, microglia frequently cluster in nodules, where they are engaged in clearing degenerated myelin. Thus, WAM may represent a potentially protective response required to clear degenerated myelin accumulating during white matter aging and disease.

overall_design: Study of microglia in aging and in a tissue specific manner (white and grey matter) using Smart-seq2 and 10x scRNA-seq technologies. We also analysed, 5xFAD mice (AD model) with Smart-seq2, and Apoe-KO with 10x transcriptomics. Additionally we studied the impact of Actinomycin-D during tissue dissociation on gene expression in microglia using Smart-seq2.

first: Simon

last: Besson-Girard

first: Tugberk

last: Kaya

first: Lu

last: Liu

first: Hao

last: Ji

first: Fumere

last: Usifo

first: Mikael

last: Simons


title: Gata2 loss alters epigenetic priming and gene expression profiles in early myeloid and lymphoid progenitors leading to cytopenias

first: Leonard

last: Zon

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

overall_design: Refer to individual Series


title: The histone reader PHF7 cooperates with the SWI/SNF complex at cardiac super-enhancers to promote direct cardiac reprogramming

city: Dallas

first: kenian

last: chen

state: TX

address: 5323 Harry Hines Boulevard

department: Population and Data Science

institute: UTsouthwestern

summary: Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through the overexpression of the transcription factors Gata4, Mef2c, and Tbx5 (GMT), and later, Hand2 (GHMT) and Akt1 (AGHMT) were found to further enhance this process. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogram adult fibroblasts. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor. Mechanistically, PHF7 localizes to cardiac super-enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, increases chromatin accessibility and transcription factor binding at these sites. Importantly, PHF7 is the first epigenetic factor found to achieve efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic modifiers, such as PHF7, in harnessing chromatin remodeling complexes to overcome critical barriers to direct cardiac reprogramming.

overall_design: To understand the mechanism of PHF7 in reprogramming, we profiled the binding sites of PHF7 through PHF7 ChIP-Seq in day 2 iCLM MEFs both on its own (PHF7) and in the context of reprogramming (5F+PHF7). We further profiled chromatin accessibility through ATAC-Seq in day 2 iCLM MEFs in AGHMT (5F) and AGHMT+PHF7 (5F+PHF7) samples. Global changes in transcription were profiled by RNA-Seq in day 7 TTF iCLMs.

first: Glynnis

last: Garry

first: Kenian

last: Chen

first: Eric

last: Olson


title: G2/M DNA synthesis at transcription start sites because of RNA transcription persistence during DNA replication [Chr-RNA-seq]

overall_design: Genome-wide chromatin-bound RNA sequencing profile of BJ-hTERT cells throughout S phase generated by NEXT-seq in two biological repeats


title: G2/M DNA synthesis at transcription start sites because of RNA transcription persistence during DNA replication [ChIP-seq]

overall_design: Genome-wide profiles of γ-H2AX of asynchronous BJ-hTERT cells treated with indicated siRNA for 72 h and generated by NEXT-seq in one or two biological repeats


title: The transcriptional profiles of chronic HBV patients and asymptomatic carriers

status: Public on Feb 16 2021

city: Hangzhou

first: Jian

last: Yao

state: Zhejiang

address: 79th Qingchun Rd

institute: Zhejiang University

summary: The study aims at investigating the specifical transcriptional signatures of chronic HBV patients

overall_design: We applied microarray analysis of the whole genome transcriptome of peripheral blood mononuclear cells (PBMCs) taken from chronic HBV patients and asymptomatic carriers as control.

first: Hongyan

last: Diao