title: Transcriptomics reveal strain-specific metabolic strategies for acid resistance and gamma-aminobutyric acid (GABA) production in Levilactobacillus brevis.

status: Public on Aug 07 2021

city: Madison

first: Bryan

last: Zabel

state: wi

address: 3329 Agriculture Dr

department: GEM

institute: International Flavors & Fragrances Inc

summary:
Background: Of the many neurotransmitters in humans, gamma-aminobutyric acid (GABA) shows potential for improving several mental health indications such as stress and anxiety. The microbiota-gut-brain axis is an important pathway for GABAergic effects, as microbially-secreted GABA within the gut can affect host mental functionhealth outcomes. Understanding the molecular characteristics of GABA production by microbes within the gut can offer insight to novel therapies for mental health.
Results: Three strains of Levilactobacillus brevis with syntenous glutamate decarboxylase (GAD) operons were evaluated for overall growth, glutamate utilization, and GABA production in typical synthetic growth media supplemented with monosodium glutamate (MSG). Levilactobacillus brevis Lbr-6108 (Lbr-6108) and Levilactobacillus brevis Lbr-35 (Lbr-35)  had similar growth profiles but differed significantly in GABA secretion and acid resistance. Lbr-6108 produced GABA early, within the growth phase, and produced significantly more GABA than Lbr-35 and the type strain Levilactobacillus brevis ATCC 14689 after the stationary phase. The global gene expression during GABA production was determined by RNA sequencing at several timepoints. The GAD operon, responsible for GABA production and secretion, activated in Lbr-6108 after only six hours of fermentation and continued throughout the stationary phase. Furthermore, Lbr-6108 activated many different acid resistance mechanisms concurrently, which contribute to acid tolerance and energy production. In contrast, Lbr-35, which has a genetically similar GAD operon, including two copies of the GAD gene, showed no upregulation of the GAD operon, even when cultured with MSG.
Conclusions: This study is the first to evaluate whole transcriptome changes in L. brevis during GABA production over multiple timepoints. The concurrent expression of multiple acid-resistance mechanisms reveals niche-specific metabolic functionality between common human commensals and highlights the complex regulation of GABA metabolism in this important microbial species. Furthermore, the increased and rapid GABA production of Lbr-6108 highlights the strain’s potential as a therapeutic and the overall value of screening microbes for effector molecule output.

overall_design: Gene expression of Lbr-35 and Lbr-6108 timeseries grown in media with and without additional monosodium glutamate.

first: Wesley

last: Morovic

first: Svetlana

last: Gerdes

first: Derek

last: Nedveck

first: Matthew

last: Poore


title: Pro-BMP9 signalling in human pulmonary arterial endothelial cells after 5 hours treatment

status: Public on Aug 08 2021

city: Cambridge

first: Wei

last: Li

address: Hills Road

department: Medicine

country: United Kingdom

institute: University of Cambridge

summary: This study was designed to investigate the global gene expression of circulating, prodomain bound form of BMP9 (pro-BMP9) in human pulmonary arterial endothelial cells (PAECs) because previous microarray experients have used the growth factor domain alone. We are interested in its signalling capacity in pulmonary vascular endothelial cells using the physiological circulating form and at physiologically circulating concentrations.  Also, this study reports data 5 hours after the treatment to look at the secondary genes regulated by BMP9 signalling.

overall_design: Four independent PAECs (i.e. isolated from four different individuals) were purchased from Lonza. Each line was grown up and seeded separately for the treatment by pro-BMP9, containing 0.4 ng/ml of final growth factor domain. Treatment was carried out for 5 hours at 37 C to allow the detection of the genes indirectly regulated by pro-BMP9.

first: Richard

middle: M

last: Salmon

first: Nicholas

middle: W

last: Morrell


title: Transcriptome Analysis in a Mouse Model of Premature Aging of Dentate Gyrus: Rescue of Alpha-Synuclein Deficit by Virus-Driven Expression or by Running Restores the Defective Neurogenesis

status: Public on Aug 06 2021

city: Udine

first: Simone

last: Scalabrin

state: UD

address: via Jacopo Linussio 51

country: Italy

institute: IGA Technology Services

type: Expression profiling by high throughput sequencing

summary: To identify genes responsible for stem and progenitor cells maintenance, we sought here to find genes underlying premature neural aging, and whose deregulated expression could be rescued by running. Through RNA sequencing we analyzed the transcriptomic profiles of the dentate gyrus isolated from Btg1 wild-type or Btg1 knockout adult (two-month-old) mice submitted to physical exercise or sedentary. In Btg1 knockout mice, 545 genes were deregulated, relative to wild-type, while 2081 genes were deregulated by running. We identified 42 genes whose expression was not only down-regulated in the dentate gyrus of Btg1 knockout, but was also counter-regulated to control levels by running in Btg1 knockout mice, vs. sedentary.

first: Laura

last: Micheli

first: Teresa Maria

last: Creanza

first: Manuela

last: Ceccarelli

first: Giorgio

last: D'Andrea

first: Giacomo

last: Giacovazzo

first: Nicola

last: Ancona

first: Roberto

last: Coccurello

first: Raffaella

last: Scardigli

first: Felice

last: Tirone


title: Expression profiling of E. coli in three media treated with antibiotics

status: Public on Aug 05 2021

city: San Diego

first: Anand

last: Sastry

state: CA

address: 9500 Gilman Dr

department: Bioengineering

country: USA

institute: University of California, San Diego

summary: RNA sequencing was performed on E. coli K12 MG1655 on three media (M9, CA-MHB, R10LB) treated with four antibiotics (Ciprofloxacin, Trimethoprim-sulfamethoxazole, Ceftriaxone, Meropenem) at their media-specific MIC90s

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: E. coli was grown to mid-log phase and treated with antibiotics for 30 min with shaking. Total RNA was sampled from independent duplicate cultures

middle: V

last: Dillon

last: Szubin

first: Ying

last: Hefner

first: Sibei

last: Xu

first: Bernhard

middle: O

last: Palsson


title: LMO2 is essential to maintain the ability of progenitors to differentiate into T-cell lineage in mice

city: Isehara

first: Hiroyuki

last: Hosokawa

state: Kanagawa

address: 143 Shimokasuya

department: Department of Immunology

country: Japan

institute: Tokai University School of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Notch signaling primarily determines T-cell fate. However, the molecular mechanisms underlying the maintenance of T-lineage potential in pre-thymic progenitors remain unclear. Here, we established two Ebf1-deficient pro-B cell lines, with and without T-lineage potential. The latter expressed lower levels of Lmo2; their potential was restored via ectopic expression of Lmo2. Conversely, the CRISPR/Cas9-mediated deletion of Lmo2 resulted in the loss of the T-lineage potential. Introduction of Bcl2 rescued massive cell death of Notch-stimulated pro-B cells without efficient LMO2-driven Bcl11a expression but was not sufficient to retain their T-lineage potential. Pro-B cells without T-lineage potential failed to activate Tcf7 due to DNA methylation; Tcf7 transduction restored this capacity. Moreover, direct binding of LMO2 to the Bcl11a and Tcf7 loci was observed. Altogether, our results highlight LMO2 as a crucial player in the survival and maintenance of T-lineage potential in T-cell progenitors via the regulation of the expression of Bcl11a and Tcf7.

overall_design: ChIP-seq; Ten million of ProB(+) cells were fixed with 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min at RT followed by additional 10 min with addition of formaldehyde up to 1%.  The reaction was quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed with HBSS (Gibco). Pelleted nuclei were dissolved in lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8) and PIC) and sonicated on SFX150 (Branson) for 6 cycles of 20sec sonication followed by 1min rest, with 30% amplitude.  Six ug of anti-Lmo2 Abs (a mixture of 2 ug of NB110-78626 (Novus), 2 ug of ab91652 (Abcam) and 2 ug of AF2726 (R&D systems)) were pre-bound to Dynabeads Protein A/G (Invitrogen) and then added to the diluted chromatin complexes in parallel aliquots.  The samples were incubated overnight at 4 °C, then washed and eluted for 6 h at 65 °C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 ug/ml proteinase K).  Precipitated chromatin fragments were cleaned up using PCR purification Kit (Qiagen).  ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (E6240, NEB) and sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt.

first: Ken-Ichi

last: Hirano

first: Maria

last: Koizumi

first: Yusuke

last: Endo

first: Katsuto

last: Hozumi


title: Gene expression profile of human mesenchymal stromal cells (MSCs) exposed to hypoxic and pseudohypoxic preconditioning

status: Public on Aug 04 2021

city: Warsaw

first: Katarzyna

last: Zielniok

address: Nowogrodzka 59

department: Department of Clinical Immunology

country: Poland

institute: Medical University of Warsaw

summary: We performed gene expression profiling by RNA sequencing (RNA-Seq) to fully characterize the gene regulation effect induced by exposure of MSCs to two hypoxic preconditioning methods. We determined the gene expression profile of 6 populations of human MSCs isolated from bone marrow and preconditioned for 6h in 2% O2 (hypoxia) or with 40μM Vadadustat (pseudohypoxia), compared to control cells cultured in 21% O2. There were 5 populations isolated from males and 1 population isolated from a female. The mean age of the BM-MSCs donors was 50.5 years and the median was 44 years. RNA-Sequencing was performed using the Illumina platform. For each library an average of 16 - 20 million read pairs were generated. Quality control checks of the sequencing raw data was conducted with FastQC, while adapter-trimming was performed with BBDUK2. The relative expression of transcripts were quantified for each donor using the Salmon program v0.8.1. Fastq files were mapped to the Homo_sapiens.GRCh37 reference genome using HISAT2 software version 2.1.0. The tximport pipeline was used to import transcript abundance datasets for the differential gene expression analysis for three groups: Control vs. Vadadustat, Control vs. Hypoxia and Vadadustat vs. Hypoxia by DESeq2 software version 1.14.1. Our results provide insight into the transcriptomic effects of these two methods of hypoxic preconditioning of MSCs.

overall_design:
mRNA profiles of human MSCs preconditioned in hypoxia and with Vadadustat
*** Raw data not submitted due to patient privacy concerns (currently under IRB review) ***

first: Anna

last: Burdzinska

first: Victor

last: Murcia Pienkowski

first: Agnieszka

last: Koppolu

first: Malgorzata

last: Rydzanicz

first: Radoslaw

last: Zagozdzon

first: Leszek

last: Paczek


title: Genes differentially expressed between NEUROG3-Venus positive and negative hiPSC line differentiated to pancreatic endocrine progenitors [RNA-Seq]

city: Illkirch

first: Valerie

last: SCHREIBER

address: 1 rue Laurent Fries

country: France

institute: IGBMC

summary: Despite this critical role in islet cell development, the precise function and downstream genetic programs regulated directedly by NEUROG3 remain elusive. We therefore mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (iPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. To this aim, we generated a novel hiPSC line (NEUROG3-HA-P2A-Venus), where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique, an alternative method to CHIP-seq allowing transcription factor profiling from a low cell number, to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) differentiated from this hiPSC line. To optimize the stringency and relevance of NEUROG3 binding sites, we focused our analysis on regions identified both with HA and NEUROG3 antibodies and integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs and their NEUROG3-dependence. Mapping of NEUROG3 genome occupancy in PEPs uncovers an unexpectedly broad, direct control of the endocrine gene regulatory network (GRN) and raises novel hypotheses on how this master regulator controls islet and beta cell differentiation.

overall_design: The hiPS SB AD3.1 cell line (Van de Bunt et al, Islets, 8(3), 83-95 (2016) has been edited using the CRISPR/Cas9 technology to C-terminally tag NGN3 with a 3HA epitope and P2A-3NLS-Venus. The generated NEUROG3-HA-P2A-Venus#31 clone was differentiated to pancreatic endocrine progenitors (PEP, day 13, end of stage 5) according to the protocol described by Petersen et al, Stem cell reports:1-37 (2017). Venus-positive and negative cells were purified at PEP stage (day 13) by FACS for RNA-seq analyses.

last: Schreiber

first: Gérard

last: Gradwohl

first: Sara

last: Jiménez

first: Aline

last: Meunier


title: Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network

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

overall_design: Refer to individual Series


title: Single-cell analysis of cell fate bifurcation in the chordate Ciona

city: Kansas City

first: Konner

last: Winkley

state: MO

address: 2401 Gillham Road

department: Genomic Medicine Center

institute: Children's Mercy Research Institute

summary: Inductive signaling interactions between different cell types are a major mechanism for the further diversification of embryonic cell fates. Most blastomeres in the model chordate Ciona robusta become restricted to a single predominant fate between the 64-cell and mid-gastrula stages. The deeply stereotyped and well-characterized Ciona embryonic cell lineages allow the transcriptomic analysis of newly-established cell types very early in their divergence from sibling cell states without the pseudotime inference needed in the analysis of less synchronized cell populations. This is the first ascidian study to use droplet scRNAseq with large numbers of analyzed cells as early as the 64-cell stage when major lineages such as primary notochord first become fate restricted. We identify 59 distinct cell states, including new subregions of the b-line neural lineage and the early induction of the tail tip epidermis. We find that 34 of these cell states are directly or indirectly dependent on MAPK-mediated signaling critical to early Ciona patterning. Most of the MAPK-dependent bifurcations are canalized with the signal-induced cell fate lost upon MAPK inhibition, but the posterior endoderm is unique in being transformed into a novel state expressing some but not all markers of both endoderm and muscle. Divergent gene expression between newly bifurcated sibling cell types is dominated by upregulation in the induced cell type. The Ets family transcription factor Elk1/3/4 is uniquely upregulated in nearly all the putatively direct inductions. Elk1/3/4 upregulation together with Ets transcription factor binding site enrichment analysis enables inferences about which bifurcations are directly versus indirectly controlled by MAPK signaling. We examine notochord induction in detail and find that the transition between a Zic/Ets-mediated regulatory state and a Brachyury/FoxA-mediated regulatory state is unexpectedly late. This supports a ‘broad-hourglass’ model of cell fate specification in which many early tissue-specific genes are induced in parallel to key tissue-specific transcriptional regulators via the same set of transcriptional inputs.

overall_design: Whole-embryo single-cell RNA sequencing of Ciona embryos

first: Wendy

last: Reeves

first: Michael

last: Veeman


title: SARS-CoV-2 disease severity and transmission efficiency is increased for airborne compared to fomite exposure in Syrian hamsters.

city: Hamilton

first: Craig

last: Martens

state: MT

address: 903 S. 4th St

department: DIR

institute: NIAID

summary: Transmission of SARS-CoV-2 is driven by contact, fomite, and airborne transmission. The relative contribution of different transmission routes remains subject to debate. Here, we show Syrian hamsters are susceptible to SARS-CoV-2 infection through intranasal, aerosol and fomite exposure. Different routes of exposure present with distinct disease manifestations. Intranasal and aerosol inoculation causes severe respiratory pathology, higher virus loads and increased weight loss. In contrast, fomite exposure leads to milder disease manifestation characterized by an anti-inflammatory immune state and delayed shedding pattern. Whereas the overall magnitude of respiratory virus shedding is not linked to disease severity, the onset of shedding is. Early shedding is linked to an increase in disease severity. Airborne transmission is more efficient than fomite transmission and dependent on the direction of the airflow. Carefully characterized SARS-CoV-2 transmission models will be crucial to assess potential changes in transmission and pathogenic potential in the light of the ongoing SARS-CoV-2 evolution

overall_design: Hamster lung samples infected with SARS-CoV-2 1 day or 4 days after infection comparing routes of infection; arerosol, formite, intranasal, plus control.  29 samples total, 3 to 4 replicates per condition.

first: Julia

middle: R

last: Port

first: Claude

middle: K

last: Yinda

first: Irene

last: Owusu

first: Myndi

last: Holbrook

first: Robert

last: Fischer

first: Trenton

last: Bushmaker

first: Victoria

middle: A

last: Avanzato

first: Jonathan

middle: E

last: Schulz

first: Neeltje

last: van Doremalen

first: Chad

middle: S

last: Clancy

first: Vincent

middle: J

last: Munster


title: Genome wide profiling of NEUROG3 occupancy in human pancreatic endocrine progenitors [CUT&RUN]

first: Valerie

last: SCHREIBER

overall_design: The hiPS SB AD3.1 cell line (Van de Bunt et al, Islets, 8(3), 83-95 (2016) has been edited using the CRISPR/Cas9 technology to C-terminally tag NGN3 with a 3HA epitope and P2A-3NLS-Venus. The generated NEUROG3-HA-P2A-Venus#31 clone was differentiated to pancreatic endocrine progenitors (PEP, day 13, end of stage 5) according to the protocol described by Petersen et al, Stem cell reports:1-37 (2017). Venus-positive cells were purified at PEP stage (day 13) by FACS and NEUROG3-bound genes isolated by CUT&RUN (Hainer and Fazzio, Current Protocols 126 (1):e85 (2019), using antibodies recognizing either NEUROG3 or the 3HA tag. CUT&RUN with anti-H3K27me3 and H3K4me3 antibodies was done to follow chromatin state. Donkey anti sheep and rabbit-anti-mouse IgG were used as negative controls.

last: Schreiber

first: Tao

last: Ye

first: Gérard

last: Gradwohl

first: Sara

last: Jiménez

first: Reuben

last: Mercier

first: Bernard

last: Jost


title: Anti-CD4 treatment affects TCR repertoire of CD8+ T cells

status: Public on Aug 03 2021

city: Goyang-si

first: Chungyong

last: Han

state: Gyeonggi-do

address: 323 Ilsan-ro, Ilsandong-gu

country: South Korea

institute: National Cancer Center

summary: Anti-CD4 monoclonal antibody, a prominent immunomodulatory agent, elicits robust anti-tumor immunity in various cancers by increasing tumor-infiltrating lymphocytes and promoting CD8+ T cell reactivity against tumor cell-derived antigens. We conducted TCR repertoire analysis of anti-CD4-exposed endogenous CD8+ T cells to investigate the expansion pattern of the cell population.

overall_design: The model of mouse melanoma (B16F10) was used to study anti-CD4-induced change of lymphoid cells. We designed a regimen that utilizes cyclophosphamide treatment, adoptive transfer of tumor-specific T cells, and anti-CD4 treatment, a combination that synergistically increases the efficacy of adoptive T cell therapy. Three days after the B16F10 challenge, C57BL/6 mice sequentially received cyclophosphamide, ex vivo-primed Pmel-1 cells (melanoma-specific TCR-transgenic CD8+ T cells), and IL-2. A transient treatment (on day 10, 17, and 24) of anti-CD4 started seven days after cyclophosphamide treatment. Twenty-five days after the tumor challenge, cells from lymphoid tissues of the mice were subjected to TCR repertoire analysis. Two groups (3 and 5 mice, respectively) received cyclophosphamide/Pmel-1 or cyclophosphamide/Pmel-1/anti-CD4.  For each mice, endogenous CD8+ T cells were isolated and used in the analysis.

first: Seon-Hee

last: Kim


title: A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5) [WGS-Seq]

city: Shanghai

first: Shijun

last: Shen

state: Shanghai

address: 1239 Siping Road, Shanghai, P.R. China

department: Bioninformatics

country: China

institute: Tongji University

type: Genome variation profiling by high throughput sequencing

summary: Gametocytogenesis, the process by which malaria parasites produce sexual forms that can infect mosquitoes, is essential for the transmission of malaria. A transcriptional switch of the pfap2-g gene triggers sexual commitment, but how the complex multi-step process is precisely programed remains largely unknown. Here, by systematic functional screening of a panel of ApiAP2 transcription factors, we identify six new apiAP2 members associated with gametocytogenesis in Plasmodium falciparum. Among these, PfAP2-G5 (PF3D7_1139300) was found to be indispensable for gametocytogenesis. This factor suppresses the transcriptional activity of the pfap2-g gene via binding to both the upstream region and exonic gene body, the latter is linked to the maintenance of local heterochromatin structure, thereby preventing initiation of sexual commitment. Removal of this repressive effect through pfap2-g5 knockout disrupts the asexual replication cycle and promotes sexual commitment accompanied by upregulation of pfap2-g expression. However, the gametocytes produced fail to mature fully. Further analyses show that PfAP2-G5 is essential for gametocyte maturation, and causes the down-regulation of pfap2-g and a set of early gametocyte genes activated by PfAP2-G prior to gametocyte development. Collectively, our findings reveal a regulation cascade of gametocyte production in malaria parasites, and provide a new target for transmission blocking interventions.

overall_design: Parasites were harvested to perform next-generation whole genome sequencing assay using  genomic DNA.

first: Xiaomin

last: Shang

first: Cizhong

last: Jiang

first: Qingfeng

last: Zhang


title: Melatonin reverses mitochondria dysfunction and oxidative stress-induced apoptosis of Sudan I-exposed mouse oocytes Exposure to Sudan I compromises oocyte quality via the induction of mitochondrial dysfunction and oxidative stress in mice

city: 南京

first: 春华

last: 邢

state: 江苏

address: 江苏省南京市玄武区孝陵卫街道

institute: 南京农业大学

summary: Sudan I is one of the industry dyes and widely used in cosmetics, wax agent, solvent and textile. Sudan I has multiple toxicity such as carcinogenicity, mutagenicity, genotoxicity and oxidative damage. However, Sudan I has been illegally used as colorant in food products, triggering worldwide attention about food safety. Nevertheless, the toxicity of Sudan I on reproduction, particularly on oocyte maturation is still unclear. In the present study, using mouse in vivo models, we report the toxicity effects of Sudan I on mouse oocyte. The results reflect that Sudan I exposure disrupts spindle organization and chromosomes alignment as well as cortical actin distribution, thus leading to the failure of polar body extrusion. Based on the transcriptome results, it is found that the exposure of Sudan I leads to the change in expression of 764 genes. Moreover, it’s further reflected that the damaging effects of Sudan I are mediated by the destruction of mitochondrial functions, which induces the accumulated ROS to stimulate oxidative stress-induced apoptosis, while in vivo supplementation of melatonin efficaciously suppresses mitochondrial dysfunction and the accompanying apoptosis, thus reverses oocyte meiotic deteriorations. Collectively, our results prove the reproduction toxicity of Sudan I for the exposure of Sudan I reduces the oocyte quality, and demonstrate a feasible solution to mitigating Sudan I-induced meiotic deteriorations.

overall_design: Transcriptomic analysis of oocytes control, Sudan I-exposed, and melatonin rescue groups. was carried out using a protocol for RNA-Seq. The constructed library was sequenced with Geekgene and Gene Denovo platform.

first: Shao-Chen

last: Sun

first: Yu


title: ATAC-seq, H3K9ac ChIP-seq and 4sU-seq in WT, SAGA and ATAC mutant mouse embryonic stem cells

city: Illkirch CEDEX

first: Veronique

last: Fischer

address: 1 Rue Laurent Fries

summary: This SuperSeries is composed of the ATAC-seq, H3K9ac ChIP-seq and 4sU-seq SubSeries.


title: Newly synthesized RNA analysis in WT, SAGA and ATAC mutant mouse embryonic stem cells

summary: To analyse the genome-wide impact of inactivation of the ATAC or SAGA coactivator complexes on RNA polymerase II (Pol II) transcription, we purified newly synthesized RNA from mutant mouse embryonic stem (ES) cell lines in which subunits of ATAC (Yeats2, Zzz3) or SAGA (Supt7l) are inactivated or depleted. We also performed this analysis in mutant cell lines in which a subunit (Tada3) of the shared histone acetyltransferase activity of these two complexes is depleted. Newly synthesized RNA was purified following the 4sU labelling method (more details in extract protocol). For two wildtype samples, the total RNA input was also analysed.

overall_design: Total and newly synthesized RNA from wildtype (WT) and mutant mouse ES E14 cell lines. Samples were sequenced in three independent sequencing projects over the course of three years (project 1: VQFR1-12, project 2: VQFR13-18, project 3: VQFR25-28) RNA from two independent clones per cell line were sequenced.

first: Matthieu

last: Jung

first: Damien

last: Plassard

first: Bernardo

last: Reina San Martin

first: Laszlo

last: Tora

first: Didier

last: Devys


title: A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5) [ChIP-Seq HM]

overall_design: The WT and  pfap2-g5_trun::WT lines at trophozoite (30-40hpi) stage were harvested to perform ChIP assay using  antibody against H3K9ac or H3K9me3.  The input material served as the control.


title: H3K9ac ChIP-seq in WT, SAGA and ATAC mutant mouse embryonic stem cells

summary: Assess H3K9ac levels in WT and mutant ES E14 strains using ChIP-seq. Mutant strains are depleted for subunits of the coactivator complexes SAGA (Supt7l KO) or ATAC (AID-Yeats2) or of HAT subunits (AID-Tada3, Tada2a+Tada2b KO) using constitutive knock-out (KO) or the auxin-inducible degron (AID) system.

overall_design: ChIP-seq was performed with 49 ug of mouse chromatin and 1 ug of Drosophila S2 cell spike-in chromatin with 2 ug of H3K9ac antibody (Abcam, Cat#ab4441) in one replicate.

first: Tao

last: Ye

last: Stierle


title: ATAC-seq in WT, SAGA and ATAC mutant mouse embryonic stem cells

summary: Assess chromatin accessibility in WT and mutant ES E14 strains using ATAC-seq. Mutant strains are depleted for subunits of the coactivator complexes SAGA (Supt7l KO) or ATAC (Yeats2, Zzz3) using constitutive knock-out (KO) or the auxin-inducible degron (AID) system.

overall_design: ATAC-seq was performed on 100.000 cells in two replicates (on two different days) with two independent clones. Samples 1-6 = day 1, Samples 7-12 = day 2.


title: CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression [UMI-4C]

status: Public on Aug 02 2021

city: Seville

first: José

middle: M.

last: Santos-Pereira

address: Avda. Manuel Siurot s/n

country: Spain

institute: IBiS, Universidad de Sevilla-CSIC

summary: CTCF is an 11-zinc-finger DNA-binding protein that acts as a transcriptional repressor and insulator, as well as an architectural protein required for 3D genome folding. CTCF mediates long-range chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures composed of genomic regions with high contact frequency. Although CTCF is essential for cycling cells and developing embryos, its in vitro removal has only modest effects over gene expression, challenging the generally accepted idea that TADs facilitate enhancer-promoter interactions within gene regulatory landscapes. However, the effects of an altered CTCF-mediated chromatin structure on gene regulation in vivo are poorly understood. Here, we have generated a ctcf knockout mutant in zebrafish that allows us to monitor the effect of CTCF loss of function during embryo patterning and organogenesis. CTCF absence leads to a loss of chromatin structure in zebrafish embryos and affects the expression of thousands of genes, among them many developmental genes. In addition, we show that chromatin accessibility, both at CTCF sites and at developmental cis-regulatory elements (CREs), is severely compromised in ctcf mutants. Probing chromatin interactions from developmental genes at high resolution, we further demonstrate that promoters fail to fully establish long-range contacts with their associated regulatory landscapes, leading to altered gene expression patterns during development. Therefore, our results demonstrate that CTCF and TADs are essential to finetune gene expression during embryonic development, providing the structural basis for the establishment of developmental gene regulatory landscapes.

overall_design: UMI-4C assays in wild-type and ctcf zebrafish mutants, at developmental stage 48 hpf

first: Martin

last: Franke

first: Elisa

last: De la Calle-Mustienes

first: Ana

last: Neto

first: María

last: Almuedo-Castillo

first: Ibai

last: Irastorza-Azcárate

first: Rafael

middle: D

last: Acemel

first: Juan

last: Tena

middle: L

last: Gómez-Skarmeta


title: CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression [ATAC-seq]

overall_design: ATAC-seq assays in wild-type and ctcf zebrafish mutants, at developmental stages 24 hpf and 48 hpf, with and without injection of tp53 morpholino


title: CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression [Hi-ChIP]

overall_design: Hi-C assays in wild-type and ctcf zebrafish mutants, at developmental stage 48 hpf


title: CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression [RNA-seq]

overall_design: RNA-seq assays in wild-type and ctcf zebrafish mutants, at developmental stages 24 hpf and 48 hpf, with and without injection of tp53 morpholino


title: A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5) [ChIP-Seq TF]

first: Shijun

last: Shen

overall_design: The PfAP2-G5-GFP::WT line at trophozoite (30-40hpi) and G0-G1 gametocyte (D5) stages, the PfAP2-G-GFP::WT and  PfAP2-G-GFP::pfap2-g5_trun lines at trophozoite (30-40hpi) stage were harvested to perform ChIP assay using  antibody against GFP or IgG. There are two biological replicates for each of the parasite lines. The input and IgG material served as the control.

first: Xiaomin

last: Shang

first: Cizhong

last: Jiang

first: Qingfeng

last: Zhang


title: A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5)


title: A transcriptional repressor programs gametocytogenesis in human malaria parasites (PfAP2-G5) [RNA-Seq]

overall_design: We carried out strand-specific RNA-seq libraries followed by deep sequencing (RNA-seq) to explore the transcriptional profile of the pfap2-g5_trun, pfap2-g5_dDBD, pfap2-g5_dDBD::pfap2-g_ko  and WT-3D7 lines (control). We carried out strand-specific RNA-seq libraries followed by deep sequencing (RNA-seq) to explore the transcriptional profile of the pfap2-g5_trun,  pfap2-g4_trun and WT-NF54 (control)  lines.


title: Transcriptomic analysis of ChAdOx/MVA P1A vaccine-induced P1A-35-43-specific CD8+ T-cells by single-cell RNA-sequencing

status: Public on Aug 01 2021

city: Oxford

first: James

last: McAuliffe

state: Oxfordshire

address: Old Road Campus Research Building, Roosevelt Drive

department: Ludwig Institute for Cancer Research,

institute: University of Oxford

summary: This study aimed to analyze the transcriptional profiles of ChAdOx/MVA vaccine induced P1A-35-43-specific CD8+ T-cells and how this is affected by both tissue location and anti-PD-1 treatment. 15V4T3 tumour-bearing DBA/2 mice were vaccinated with ChAdOx/MVA P1A with or without anti-PD-1 treatment. Following treatment, spleens and tumours were collected, processed to single-cell suspension and P1A-35-43-specific CD8+ T-cells were then isolated by MHC-class I multimer staining and FACS. Single-cell RNA-seq experiments were performed using the 10X Genomics platform to analyze P1A-specific CD8+ T-cell transcriptional profiles. Results of this transcriptomic analysis showed that cells segregated into distinct phenotypic subtypes reflecting either effector or stem-like states, the distribution of which was dependent on tissue of origin and treatment status. These results provide an in depth phenotypic analysis of MAGE-type-antigen-specific CD8+ T-cells induced by ChAdOx/MVA vaccination and how phenotype is influenced by physiological location and immune checkpoint blockade treatment.

overall_design: 15V4T3-tumour-bearing mice were treated with either ChAdOx/MVA P1A vaccination, or with ChAdOx/MVA P1A + anti-PD-1 combination. Following treatment, P1A-35-43-specific CD8+ T-cells were isolated from spleens and tumours by MHC-class I multimer staining and FACS sorting. Single-cell RNA-seq experiments were performed using the 10X Genomics platform to analyze the transcriptional profile of P1A-specific CD8+ T-cells.

first: Hok

middle: F

last: Chan

first: Andrei

last: Ramirez-Valdez

first: Carol

last: Leung


title: The single-cell RNA sequencing for α-gustducin-positive taste cells

city: Fukuoka-city

first: Shingo

last: Takai

state: Fukuoka

address: 3-1-1, Maidashi, Higashi-ku,

department: Graduate School of Dental Science

institute: Kyushu University

summary: Taste buds are complex sensory organs that are embedded in the epithelium of fungiform papillae (FP) and circumvallate papillae (CV). The sweet, bitter, and umami taste are sensed by type II taste cells that expressed taste receptors (Tas1rs and Tas2rs) which coupled with taste G-protein α-gustducin. Recent studies revealed that the taste response profiles of α-gustducin-expressing cells are different between FP and CV. We applied the high-throughput single-cell RNA-sequencing combined with fluorescence-activated cell sorting (FACS) to profile the transcriptome of the α-gustducin-expressing taste cells in both fungiform and circumvallatae papillae with transgenic mice expressing green fluorescent protein (GFP).

overall_design:
Transcriptome profile of 90 α-gustducin-positive taste cells of α-gustducin-GFP mice that were collected by fluorescence-activated cell sorting. Single-cell capture and cDNA synthesis were performed by the BD Rhapsody Single-Cell Analysis System (BD). Total cDNA was amplified by the TAS-Seq method (Immunogenetics). Sequencing was performed using Novaseq6000 (Illumina). Raw reads were aligned to the mouse reference sequencing RNA using the Bowtie2 program. Quality control was performed using the DropletUtils package. The expression data matrix was adjusted using distribution-based error correction, which is manufacturer-developed algorithms correcting for sequencing errors.
Downstream analyses were performed using the Seurat v2.3.4 R package. The data matrix contained cells derived from FP or CV were integrated. Cells with 500 < of genes, > 0.6% of mitochondrial genes, > 0.1% of Ribosome RNA, and 0.9 < of log3 value of Gnat3 ,that encodes α-gustducin, expression level were further processed.

first: Yu

last: Yamada

first: Noriatsu

last: Shigemura


title: Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs.

city: Bronx

first: Thomas

last: Meier

state: New York

address: 1300 Morris Park Avenue

department: Department of Anatomy & Structural Biology

institute: Albert Einstein College of Medicine, INC

summary: Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (~0.1% of total arginine), Rme2a was highly abundant (~1.1%), and Rme2s was intermediate (~0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

overall_design: Examination of 1 week PRMT inhibition in A549

first: Maxim

middle: I

last: Maron

first: David

last: Shechter


title: Analysis of locus-specific long-range chromatin interactions in BCL11A WT and KO HUDEP-2 cells by CAPTURE

status: Public on Jul 30 2021

city: Memphis

first: Jian

last: Xu

state: Tennessee

address: 262 Danny Thomas Place, MS 345

department: Pathology

institute: St. Jude Children's Research Hospital

summary: We determined the locus-specific long-range chromatin interactions associated with the human beta-globin gene cluster in BCL11A wild-type and knockout HUDEP-2 cells

overall_design: CAPTURE-3C-seq experiments were performed using 3 to 5 million wild-type (WT) or BCL11A knockout HUDEP-2 cells. Three biological replicates were merged for WT or BCL11A knockout HUDEP-2 cells, respectively. Only significant long-range DNA interactions from the following captured bait regions with BF score ≥ 20 were used for quantitative analyses: HBB, HBG1/2, HBD-3.5kb (containing sgRNAs targeting HBD-1kb, HBD-1.5kb and HBD-3.5kb regions), and upstream enhancer locus control (HS1-5) regions.

first: Yong

first: Yuannyu

last: Liu

first: Stuart

middle: H

last: Orkin

first: Vijay

middle: G

last: Sankaran


title: Transcriptome analysis of Epicardial Adipose Tissue from obese, diabetic and lean individulas

city: London

first: Paula

last: Longhi

address: William Harvey Research Institute

institute: Queen Mary, University of London

summary: The overall goal of this studyis to compare the transcription differences of epicardial adipose tissue between between lean and obese and Type 2 diabetic individuals

overall_design: Epicardial adipose tissue was obtained during open heart surgery. Transcriptome of epicardial adipose tisue was generated by deep sequencing, using Illumina PE150 (30M reads)

first: Vishal

last: Vyas


title: Transcriptomic analysis of 15V4T3 tumours from ChAd/MVA vaccine + anti-PD-1 treated mice by RNA-sequencing

status: Public on Jul 31 2021

first: James

summary: This study aimed to analyse the effect of immunotherapeutic treatment with ChAdOx/MVA MAGE vaccine and anti-PD-1 on the transcriptional mRNA profiles of murine 15V4T3 tumours. 15V4T3 tumour-bearing mice were treated with either a PBS control, anti-PD-1 inhibitor (3-doses of 100 µg 3-days apart), ChAdOx/MVA P1A vaccine (10^7 IU / 10^6 PFU doses 1-week apart), or with a combination of vaccine + anti-PD-1. Following treatment, RNA was isolated from tumour tissue samples and then tumour transcriptomic gene expression profiles were analysed by RNA-sequencing. Sequenced reads were mapped to the mouse genome GRCm38.  A counts matrix reporting the numbers of reads mapping to each gene was then generated, and abundance measurements performed using TMM-normalized (EdgeR) counts data to quantify gene expression levels. 14937 expressed transcripts were identified as expressed above a minimum threshold. Results from the transcriptomic analysis indicated that ChAdOx/MVA P1A vaccination had a profound effect on the gene expression profiles of 15V4T3 tumours. Approximately 1250 differentially expressed genes were identified in tumours from vaccinated compared to unvaccinated mice using a threshold for differential expression of fold change > 2 and Padj < 0.05. Furthermore, gene expression signatures corresponding to a CD8+ T-cell driven inflammatory response and an IFNg response were found to be highly enriched in tumours from vaccinated mice. Such gene expression signatures have been found in other studies to be predictive of response to immunotherapy and anti-PD-1 immune checkpoint blockade. GSVA further confirmed the enrichment of gene sets corresponding to immune activation, a pro-inflammatory response and T-cell activity in the mRNA expression data from tumours of vaccinated mice compared to unvaccinated. Taken together these findings show that a ChAdOx/MVA vaccine targeting a MAGE type antigen can generate and drive tumour gene expression patterns associated with a T-cell inflammatory response and positive response to cancer immunotherapy.

overall_design: 15V4T3-tumour-bearing mice were treated with a PBS control, anti-PD-1 inhibitor, ChAdOx/MVA P1A vaccine, or with vaccine + anti-PD-1 combination. Following treatment tumours were excised and tumour-RNA isolated. Tumour mRNA expression profiles were then analyzed by RNA-sequencing. n=12 samples total, 3 from each experimental treatment group.

first: Hok-Fung


title: Transcriptome sequencing (RNA-Seq) of PAT cells in CAD and control patients for exploring the association between CAD and PAT

first: Kailei

last: Shi

address: Yanan west road NO.168

institute: Huadong Hospital Affiliated to Fudan University

summary: Coronary artery disease (CAD) poses a worldwide health threat. Compelling evidence shows that pericardial adipose tissue (PAT), a brown-like adipose adjacent to the external surface of the pericardium, is associated with CAD. However, the specific molecular mechanisms of PAT in CAD are elusive.  For characterizing human PAT and explore its association with CAD, the transcriptome characteristics were assessed in 5 CAD patients and 4 controls via RNA-sequencing.

overall_design: mRNA profiles of PAT cells in CAD and control patients were generated by deep sequencing, using Illumina NovaSeq 6000.

first: Mingxuan

last: Li


title: 5CAPture-seq: a method for enriching full-length cDNA and identifying 5′ capped nucleotides

city: Khlong Nung

first: Pavita

last: Kaewprommal

state: Pathum Thani

department: NBT

country: Thailand

institute: NSTDA

summary: Current transcriptomic methods for mapping 5′-ends of eukaryotic RNA employ only 5′-end enriched samples. The absence of a matched unenriched control limits the ability to distinguish genuine 5′ ends from incomplete cDNAs. We developed a transcriptomic method based on sequencing of cDNA enriched for full-length fragments and an unenriched control. From the mapped sequence reads, enrichment scores are calculated for all transcribed nucleotides and a statistical model of the enrichment constructed. We tested the method in the human malaria parasite Plasmodium falciparum. Data were obtained from ring, trophozoite and schizont stages of the parasite intra-erythrocytic development cycle. Two groups of 5′ capped nucleotides were assigned by unsupervised clustering. The first group contains sites located mostly outside of annotated protein-coding exons in regions of high local nucleosomal occupancy, but low occupancies of ribosome and elongating RNA polymerase II. The majority of sites in the second group are intra-exonic and show different patterns, most notably a peak of ribosome occupancy centered on the 5′ end position and a biased representation among codons possibly prone to ribosome stalling. Our method can be used to annotate 5′ capped nucleotides, including intra-exonic nucleotides that can be distinguished from incomplete cDNA artifacts.

overall_design: Total RNA was extracted from synchrononous cultures of Plasmodium falciparum. Parasites were harvested at ring, trophozoite and schizont stages. Two independent cultures were performed. cDNA from each RNA sample was synthesized and split into two pools. One pool was enriched for full-length cDNA, whereas another was processed as an unenriched control sample. Enriched and unenriched cDNAs were converted to Illumina sequencing libraries for transcriptomic sequencing.

first: Philip

last: Shaw

first: Jittima

last: Piriyapongsa

first: Chayaphat

last: Wongsombat

first: Chadapohn

last: Chaosrikul

first: Krirkwit

last: Teeravajanadet

first: Manon

last: Boonbangyang

first: Chairat

last: Uthaipibull

first: Sumalee

last: Kamchonwongpaisan

first: Sissades

last: Tongsima


title: Anti-CD4 treatment increases tumor-suppressive IL18Rαhi CD8+ T cells

first: Chungyong

last: Han

summary: Anti-CD4 monoclonal antibody, a prominent immunomodulatory agent, elicits robust anti-tumor immunity in various cancers by increasing tumor-infiltrating lymphocytes and promoting CD8+ T cell reactivity against tumor cell-derived antigens. We conducted single-cell transcriptome analysis of anti-CD4-exposed lymphoid cells to investigate the detailed mechanism.

overall_design: The model of mouse melanoma (B16F10) was used to study anti-CD4-induced change of lymphoid cells. We designed a regimen that utilizes cyclophosphamide treatment, adoptive transfer of tumor-specific T cells, and anti-CD4 treatment, a combination that synergistically increases the efficacy of adoptive T cell therapy. Three days after the B16F10 challenge, C57BL/6 mice sequentially received cyclophosphamide, ex vivo-primed Pmel-1 cells (melanoma-specific TCR-transgenic CD8+ T cells), and IL-2. A transient treatment (on day 10, 17, and 24) of anti-CD4 started seven days after cyclophosphamide treatment. Twenty-five days after the tumor challenge, cells from lymphoid tissues of the mice were subjected to single-cell transcriptome analysis. Two groups (3 mice each) received cyclophosphamide/Pmel-1 or cyclophosphamide/Pmel-1/anti-CD4.  For each group, endogenous CD8+ T cells from three mice were pooled and used in the analysis.

first: Seon-Hee

last: Kim


title: The induction of preterm labor in Rhesus macaques is determined by the strength of immune response to intrauterine infection

city: Los Angeles

first: Suhas

last: Kallapur

address: 10833 Le Conte Avenue

institute: UCLA

summary: We demonstrated that the intensity of the immune response to LPS and E. coli regulates the induction of preterm labor in Rhesus macaques

overall_design: RNAseq of chorioamnion-decidua in a Rhesus model of intrauterine infection/inflammation (3 ctrl, 3 LPS 48h; 3 E coli, 2 E coli +Abx)

first: Monica

last: Cappelletti

first: Pietro

last: Presicce

first: Faiyang

last: Ma

first: Paranthaman

last: Senthamaraikannan

first: Lisa

last: Miller

first: Matteo

last: Pellegrini

first: Myung

last: Sim

first: Alan

last: Jobe

first: Senad

last: Divanovic

first: Sing

last: Way

first: Claire

last: Chougnet


title: Characterization of growth and development of sorghum genotypes with differential susceptibility to Striga hermonthica

city: Davis

first: Dorota

last: Kawa

address: 451 Health Sciences Dr.

department: Plant Biology and Genome Center

institute: University of California

summary: Variation in strigolactone composition in sorghum root exudates underlies its resistance to parasitic weed, Striga hermonthica. Root exudates of the Striga susceptible variety Shanqui Red (SQR) contain primarily 5-deoxystrigol, which has a high efficiency of inducing Striga germination. SRN39 roots primarily exude orobanchol, leading to reduced Striga germination and making this variety resistant to Striga. This structural diversity in exuded strigolactones is determined by the polymorphism in the LGS1 (LOW GERMINATION STIMULANT 1) locus. Yet, the effects of the lgs1 mutation as well as the consequences of the vast genetic diversity between SQR and SRN39 have not been addressed in terms of growth and development. In response to this knowledge gap, we demonstrate additional consequences of LGS1 loss-of-function by phenotypic and molecular characterization. A suite of genes related to metabolism was differentially expressed between SQR and SRN39. Increased levels of gibberellin precursors in SRN39 were accompanied with its slower growth rate and developmental delay and we observed an overall increased SRN39 biomass. The slow-down in growth and differences in transcriptome profiles of SRN39 were strongly associated with plant age. Additionally, analyses of multiple LGS1 loss-of-function genotypes indicated that strigolactone stereochemistry influences root system architecture. To summarize, we demonstrated that the consequences of lgs1 mutation reach further than the changes in strigolactone profiles in root exudates and translates to alteration in growth and development.

overall_design: Root transcriptome profiling of two sorghum varieties Shanqui Red and SRN39 at 28 and 35 day old stage. Three to four biological replicates and three technical replicates for each RNA sample.

first: Siobhan

middle: M

last: Brady


title: Genome-wide enhancer analysis reveals the role of AP-1 transcription factor in head and neck squamous cell carcinoma [RNA-Seq]

city: Wuhan

first: Chen-Yu

last: Wang

state: Hubei

address: 299 Bayi Road, Wuchang District

institute: Wuhan University

summary: Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer. It causes significant morbidity and mortality, with a low 5-year survival rate. we used RNA-Seq to analyze the tumor and normal tissues of the head of the Tgfbr1/Pten 2cKO mouse model. RNA-Seq analysis found enhanced keratinization, cell proliferation, angiogenesis, inflammation and immune response, TNF signaling pathway, NF-kappa B signaling pathway, and p53 signaling pathway in tumor tissues，which ultimately contribute to HNSCC progression.

overall_design: mRNA profiles of the tumor and normal tissues of the head of the Tgfbr1/Pten 2cKO mouse model

first: Chuan

last: Gao

first: Ji

last: Chen

first: Qing-Lan

last: Li

first: Lu

last: Zhang

first: Min

last: Wu

first: Zhi-Jun

last: Sun

first: Lian-Yun


title: SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation

city: Cambridge

first: Brian

middle: Yee Hong

last: Lam

address: Box 289 Addenbookes Hospital, Hills Road

department: Metabolic Research Laboratories

institute: University of Cambridge

summary: Macrophages exhibit a spectrum of activation states ranging from classical to alternative activation1. Alternatively activated macrophages are involved in diverse pathophysiological processes such as confining tissue parasites2, improving insulin sensitivity3 or promoting an immune tolerant microenvironment that facilitates tumour growth and metastasis4. Recently, the role of metabolism in the regulation of macrophage function has come into focus as both the classical and alternative activation programmes require specific regulated metabolic reprogramming5. While most of the studies regarding immunometabolism have focussed on the catabolic pathways activated to provide energy, little is known about the anabolic pathways mediating macrophage alternative activation. In this study, we show that the anabolic transcription factor sterol regulatory element binding protein 1 (SREBP1) is activated in response to the canonical Th2 cytokine interleukin 4 (IL-4) to trigger the de novo lipogenesis (DNL) programme, as a necessary step for macrophage alternative activation. Mechanistically, DNL consumes NADPH, partitioning it away from cellular antioxidant defences and raising ROS levels. ROS serves as a second messenger, signalling sufficient DNL, and promoting macrophage alternative activation. The pathophysiological relevance of this mechanism is validated by showing that SREBP1/DNL is essential for macrophage alternative activation in vivo in a helminth infection model.

overall_design: Bone-marrow derived macrophages feom 6 different animals per genotype - WT or Macrophage-SCAP-KO - were treated or not with interleukin 4 (IL4) for 24h

first: Guillaume

last: Bidault

first: Samuel

last: Virtue

first: Kasparas

last: Petkevicius

first: Helen

last: Jolin

first: Aurélien

last: Dugourd

first: Anne-Claire

last: Guénantin

first: Jennifer

last: Leggat

first: Betania

last: Mahler-Araujo

middle: Y

first: Marcella

last: Ma

first: Martin

last: Dale

first: Stefania

last: Carobbio

first: Arthur

last: Kaser

first: Padraic

last: Fallon

first: Julio

last: Saez-Rodriguez

first: Andrew

middle: N

last: McKenzie

first: Antonio

last: Vidal-Puig


title: BLM helicase negatively regulates a DNA damage dependent CDX2 inducible microRNA signature which targets BRCA1

city: New Delhi

first: Sagar

last: Sengupta

address: Aruna Asaf Ali Marg

department: Signal Transduction Laboratory-2

country: India

institute: National Institute of Immunology

type: Non-coding RNA profiling by high throughput sequencing

summary: In trying to understand how DNA damage modulates miR expression by high level of endogenous DNA damage, we carried out small RNA sequencing with two pairs of isogenic cell lines generated from two BS patients. For this, miRNA from immortalized cells from GM03509 complemented with either GFP-BLM (Clone 4.3.4) or GFP (Clone 100) or immortalized cells from another BS patient GM08505 complemented with either GFP-BLM or GFP were profiled by Next Generation Sequencing.

overall_design: We performed Next Generation sequencing to compare miRNA profiles of GM03509 GFP-BLM Clone 4.3.4, GM03509 GFP Clone 100 , GM08505 GFP-BLM and GM08505 GFP cell lines.

first: Arnab

last: Mukhopadhyay

first: Awadhesh

last: Pandit


title: Genome-wide enhancer analysis reveals the role of AP-1 transcription factor in head and neck squamous cell carcinoma [ChIP-Seq]

first: Chen-Yu

last: Wang

summary: Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer. It causes significant morbidity and mortality, with a low 5-year survival rate. we used ChIP-Seq to analyze the tumor and normal tissues of the head of the Tgfbr1/Pten 2cKO mouse model.  we use H3K4me1 ChIP-Seq and H3K27ac ChIP-Seq, combined with transcriptome analysis, to find different active enhancers in tumor and normal tissues.

overall_design: Examination of 2 different histone modifications in tumor and normal tissues of the head of the Tgfbr1/Pten 2cKO mouse model

first: Chuan

last: Gao

first: Ji

last: Chen

first: Qing-Lan

last: Li

first: Lu

last: Zhang

first: Min

last: Wu

first: Zhi-Jun

last: Sun

first: Lian-Yun


title: Bcl6 controls meningeal Th17-B cell interaction in murine neuroinflammation

city: Muenster

first: I-Na

last: Lu

state: NRW

address: Domagkstraße 20

department: Department of Pediatric Hematology and Oncology

country: Germany

institute: University Children's Hospital Münster

summary: Ectopic lymphoid tissue containing B cells forms in the meninges at late stages of human multiple sclerosis (MS) and when neuroinflammation is induced by interleukin (IL)-17 producing T helper (Th17) cells in rodents. B cell differentiation and the subsequent release of class-switched immunoglobulins have been speculated to occur in the meninges, but the exact cellular composition and underlying mechanisms of meningeal-dominated inflammation remain unknown. Here, we performed in depth characterization of meningeal vs. parenchymal Th17-induced rodent neuroinflammation. The most pronounced cellular and transcriptional differences between these compartments was the localization of B cells exhibiting a follicular phenotype exclusively to the meninges. Correspondingly, meningeal but not parenchymal Th17 cells, acquired a B cell-supporting phenotype and resided in close contact with B cells. This preferential B cell tropism for the meninges and the formation of meningeal ectopic lymphoid tissue was partially dependent on the expression of the transcription factor Bcl6 in Th17 cells, that is required in other T cell lineages to induce isotype class-switching in B cells. A function of Bcl6 in Th17 cells was only detected in vivo and was reflected by the induction of B cell-supporting cytokines, the appearance of follicular B cells in the meninges, and of immunoglobulin class-switching in the CSF. We, thus, identify the induction of a B cell-supporting meningeal microenvironment by Bcl6 in Th17 cells as a novel mechanism controlling compartment-specificity in neuroinflammation.

overall_design: Single-cell RNA sequencing and TCR-sequencing of spinal cord (SC) meninges and parenchyma in AT-EAE recipient mice (Th17-adoptive transfer EAE; recipients of wt-Th17 vs. Bcl6KO-Th17 cells); bulk-RNA seq of Vβ11+ wt-Th17 vs. Vβ11+ Bcl6KO-Th17 cells

first: Maike

last: Hartlehnert

first: Xiaolin

first: Anna-Lena

last: Börsch

first: Michael

last: Heming

first: Gerd

last: Meyer zu Hörste


title: Qualitative changes in activated T cells induced by checkpoint blockade in vivo

city: Birmingham

first: David

last: Bending

address: IBR

department: Institute of Immunology and Immunotherapy

institute: University of Birmingham

summary: Here we perform QuantSeq 3' mRNA sequencing of RNA extracted from flow sorted splenic T cell from Tg4 Nr4a3-Tocky Tiger mice that had been immunised with 80ug of 4Y MBP peptide for 24 hours. After 24 hours mice were re-stimulated with 8ug 4Y MBP in the presence of isotype, anti-PD1 or anti-Lag3 antibodies for 4 hours. The results show that anti-PD1 but not anti-Lag3 imparts a unique transcriptional signature on responding T cells, indicative of stronger TCR signalling.

overall_design: 3 experimental groups in biological duplicates. Tg4 Nr4a3-Tocky Tiger mice were immunised with 80ug of 4Y MBP peptide s.c. for 24 hours. After 24 hours, mice were injected i.p. with 800ug of either 1) Isotype, 2) anti-PD1(clone 29F.1A12) or 3) anti-Lag3 (clone CB97W) antibodies. 30 minutes later 8ug of 4Y MBP peptide was administered s.c. and 4hrs later spleens removed. CD4+ Nr4a3-TimerBlue+TimerRed+ T cells were flow sorted from spleens for analysis of RNA


title: Comparison of genes differentially expressed between Medicago R108  and abi3 mutants (NF3185  and NF6003) during different stages of seed development (16, 24, 32 and 40 DAP)

status: Public on Jul 29 2021

city: Beaucouze

first: Julia

last: Buitink

address: 42 rue Georges Morel

department: BAP

institute: INRAE

summary: ABI3 is a B3-domain transcription factor that acts as a master regulator of seed maturation. To identify genes that are regulated specifically by ABI3 in the model legume Medicago R108 during seed development, using the Medicago NimbleGen chip, a transcriptomic analysis was performed between abi3 mutants (NF3185 and NF6003) and R108 wild-type at 4 stages of seed development.

overall_design: Two-condition experiment, mutant versus Wt. Biological replicates: 3 control replicates (Wt), 3 treatment replicates (Homozygous mutants) and 4 stages of seed development for NF6003 mutant. 2 control replicates (Wt), 2 treatment replicates (Homozygous mutants) and 3 stages of seed development for NF3185 mutant. Independently grown and harvested. One replicate per array. Dye swap method.

last: Lalanne

first: Jérôme

last: Verdier


title: Periplaneta americana gut transcriptomics under germ-free, gnotobiotic and coventionalized (wild-type) reared conditions

city: Columbus

first: Aturo

last: Vera Ponce de Leon

state: Ohio

department: EEOB

institute: The Ohio State University

summary:
Background: Germ-free or axenic organisms are valuable tools for studying immunity, digestion, and development in different hosts. Although most of these studies have been conducted on mice, recently, germ-free invertebrate models (e.g. Drosophila and Apis) are used due to their easy husbandry, low cost for production, maintenance and the high number of individuals per generation they produce. However, a limitation of using these insects is the simple bacterial community present in their guts. The gut of the American cockroach Periplaneta americana displays a complex gut bacterial community composed of hundreds of species. Using P. americana, we developed a germ-free omnivorous invertebrate model to investigate how gut bacteria stimulate and shape normal gut development and metabolism. To determine if the insect host is directly affected by the presence of specific members of their bacterial community, gnotobiotic cockroaches were generated by inoculating a set of various P. americana gut-endemic Gram-negative (Bacteroidetes; n=11) and Gram-positive (Firmicutes; n=2) bacterial strains into germ-free insects. Additionally, we were able to recover the ‘normal’ bacterial-induced gut phenotype by co-housing germ-free cockroaches with wildtype P. americana to produce gut-bacteria conventionalized insects.  Changes in gene expression profiles from two distinct regions (midgut and hindgut) of P. americana guts were quantified by RNA-Seq analysis of the germfree, gnotobiotic and conventionalized insects.
Basic transcriptomics description: High-resolution transcriptome profiling of germ-free, gnotobiotic, and conventionalized treated P. americana midgut and hindguts.  Ca. 43 million reads were obtained for each treatment. A de-novo assembly of all sequence reads was performed by Trinity assembler. Transcriptome assembly yielded 369,082 gene models and 554,155 isoforms. After running Trinotate pipeline, 65,047 (12 %) these transcripts matched an annotated product in at least one of the reference databases used (Uniprot, pfam, KEGG, COG). Additionally, 1,008 putative bacterial genes were annotated in the P. americana genome and ultimately excluded from these analyses. After bacteria decontamination, 553,147 assembled isoforms were used for transcript quantification and differential expression analysis using the DESeq2 pipeline. DESeq2 analysis detected 6,730 and 3,958 differentially expressed transcripts among the germ-free, gnotobiotic and conventionalized treatments in P. americana hindgut and midgut, respectively.

overall_design: Differential expression analysis was performed with DESeq2 pipeline, briefly, treatments were classified depending tissue (midgut M or hindgut H) and germ-free, gnotobiotic or conventionalized conditions.

first: Arturo

first: Sabree

last: Zakee


title: Antigen calibration of T Cell Receptor signalling strength and T cell differentiation in vivo

summary: Here we perform QuantSeq 3' mRNA sequencing of RNA extracted from flow sorted splenic T cell from Tg4 Nr4a3-Tocky Tiger mice immunised with a high or low dose of MBP 4Y peptide, enabling identification of time and dose dependent transcriptional signatures

overall_design: There are 7 experimental groups in biological triplicate from Tg4 Nr4a3-Tocky Tiger mice: 1) Control group, unimmuinsed splenic T cells. 2) 0.8ug 4Y MBP peptide immunised T cells isolated at 4hrs. 3) 0.8ug 4Y MBP peptide immunised T cells isolated at 12hrs. 4) 0.8ug 4Y MBP peptide immunised T cells isolated at 24hrs. 5) 80ug 4Y MBP peptide immunised T cells isolated at 4hrs. 6) 80ug 4Y MBP peptide immunised T cells isolated at 12hrs. 7) 80ug 4Y MBP peptide immunised T cells isolated at 24hrs


title: Modeling Hepatoblastoma

city: Cincinnati

first: Bruce

last: Aronow

state: OH

address: 240 Albert Sabin Way

department: Biomedical Informatics, Developmental Biology

institute: Cincinnati Children's Hospital Medical Center

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

overall_design: Refer to individual Series


title: Gene expression data of four cancer cell lines (A375, A549, MCF7, and PC3) treated with KR-37524

city: Daejeon

first: Gyutae

last: Lim

state: Not US or Canada

address: 141 Gajeong-ro, Yuseong-gu

institute: KRICT

summary: KR-37524, known as a Urotensin-II receptor antagonist, has low usability due to its low affinity. For this reason, gene expression information was obtained by treating KR-37524 in four cell cancer cell lines to find a new target. The change in gene expression profile was measured and compared with the LINCS L1000 database and used to find a new target.

overall_design: Six sheets of each cell line were seeded in a 60mm dish and incubated for 24 hours. After 24 hours, DMSO and KR-37524 were diluted 1000-fold in medium and the cells were treated. After 6 hours, the medium was removed and cells were obtained by treatment with 1ml of TRIzol. Cells were disrupted by pipetting and stored in a deep freezer.

first: ChaejJo

first: JeongjHyun

last: Lee

first: ByungjHo

first: JaejYong

last: Ryu

first: Kwang-Seok

last: Oh


title: Genome-wide enhancer analysis reveals the role of AP-1 transcription factor in head and neck squamous cell carcinoma

first: Chen-Yu

last: Wang


title: Modeling Hepatoblastoma: Identification of Distinct Tumor Cell Populations and Key Genetic Mechanisms through Single Cell Sequencing (bulk)

summary: Hepatoblastoma (HB) is the most common primary liver malignancy of childhood. However, molecular investigations of the disease are limited and effective treatment options are lacking. The use of patient derived xenografts (PDX) to study biology and treatment strategies of HB has proven to be a useful tool.   There is currently a knowledge gap in the investigation of key driver cells of HB in PDX models. Key driving pathways of HB tumor including WNT, AP-1, Hedgehog, Notch and MAPK pathways and genes such as GPC3, DLK1 and HMGA2 have been identified in primary HB tumor and PDX as integral players in HB tumor growth. Cell clusters have been defined with distinct roles in tumor development. Cell populations with initiating, angiogenic (endothelial), maintenance, and progression signatures have been identified in one HB patient tumor and corresponding PDX tumor. Critical pathways combined with identification of distinct cell populations within HB tumor will allow for investigation of novel treatment strategies in vitro and in vivo.

overall_design: Hepatoblastoma tumor was implanted into NSG mice and surgically separated into new mice to generate sequential passages of tumor (F). PDX tumor was compared to parent tumor as well as backgorund liver to characterize maintenance of tumor phenotype over time. Two background liver, two patient HB tumor samples and six PDX passages per patient tumor (12 total). Six additional PDX samples were examined to see response to chemo treatment: three control and three treated with irinotecan.

first: Alexander

last: Bondoc

first: Kathryn

last: Glaser

first: Kang

last: Jin

first: Charissa

last: Lake

first: Stefano

last: Cairo

first: James

last: Geller

first: Gregory

last: Tiao


title: Differences in Biological Basis and Its Effects on Vascular Endothelium in Dyslipidemic Mice with the Phlegm-Dampness Retention Syndrome and the Spleen and Kidney Yang Deficiency Syndrome Based on RNA-seq of Vascular Endothelial Macrophages

status: Public on Jul 28 2021

city: Bejing

first: Chao

last: Ye

address: No. 5, Haiyangcang, Dongcheng District, Beijing

department: Dongzhimen Hospital

institute: Beijing University of Chinese Medicine

summary:
Objective: This study aimed to identify the differentially expressed genes and related biological processes in dyslipidemia with the Phlegm-Dampness Retention (PDR) syndrome and the Spleen and Kidney Yang Deficiency (SKYD) syndrome using transcriptomic analysis.

Methods: Ten ApoE knockout (ApoE-/-) mice were used for the establishment of dyslipidemic disease-syndrome models via multifactor-hybrid modeling, with 5 in the the PDR group and 5 in the SKYD group. Five C57BL/6J mice were employed as normal controls (NC) group, which is not included in the current records.. Test model quality. Aortic endothelial macrophages in mice were screened using flow cytometry. Transcriptomic analysis was performed for macrophages using RNA-Seq.

Results: The quality assessment of the disease-syndrome model showed that TG, TC, and LDL-C levels significantly increased in the PDR and SKYD groups versus the NC group (P < 0.05). Combined with HE staining of aorta, the disease model was successfully established. The quality assessment of the syndrome models showed that mice in the PDR group presented with typical manifestations of the PDR syndrome, and mice in the SKYD group had the related manifestations of the SKYD syndrome, indicating that the syndrome models were successfully constructed.  After comparing the differentially expressed gene (DEG) expressions in macrophages in dyslipidemia mice with different syndromes, 4142 genes were identified with statistical significance (P < 0.05). The Gene Ontology (GO) analysis for the DEGs showed that biological process of difference between PDR group and SKYD group include both adverse and protective processes were included.

Conclusion: The DEGs between the PDR syndrome and the SKYD syndrome indicate different biological mechanisms between the onset of the two syndromes. They have distinctive biological processes, including adverse and protective processes, corresponding to the invasion of pathogenic factors into the body and the fight of healthy qi against pathogenic factors, respectively, in the TCM theory.  Our results have demonstrated the biological evidence behind ‘treating the same disease with different treatments’ in TCM.

overall_design: Cross-sectional study；ApoE knockout (ApoE-/-) mice；

first: Jing

last: Chen

first: Zheng

last: Yang

first: Tieshan

first: Bing

last: Xu

first: Pengyang

last: Li

first: Shan

last: Zhang

first: Xiaolin

last: Xue


title: Modeling Hepatoblastoma: Identification of Distinct Tumor Cell Populations and Key Genetic Mechanisms through Single Cell Sequencing (scRNA-seq)

first: Bruce

last: Aronow

overall_design: To examine HB pathways and define cell populations in a heterotopic PDX model, we implant patient source tumor into the intrascapular fat pad of female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. PDX tumor was compared to background liver and primary HB tumor and evaluated using single cell RNA sequencing (scRNAseq). 26,886 cells across 3 samples were collected after preprocessing and quality control.


title: Olfactory bulb gene expression profiling upon loss of Sox9 in adult astrocytes

city: Houston

first: Brittney

last: Lozzi

state: TX

address: 1 Baylor Plaza

department: Cell & Gene Therapy

institute: Baylor College of Medicine

summary: The goal of this study is to determine how the loss of the transcription factor Sox9 affects the molecular profiles of adult astrocytes from the olfactory. We performed RNA-sequencing on wildtype and Sox9 knockout (KO) astrocytes from the olfactory bulb and analyzed the molecular signatures of Sox9 KO astrocytes compared to the wildtype control.

overall_design: AAV-GFAP-iCre-P2a-TurboRFP was injected into the olfactory bulbs of Sox9fl/fl mice or C57BL/6J mice to generate astrocyte-specific Sox9 knockout and control mice. FAC sorted astrocytes from the olfactory bulbs were collected in triplicate from our viral Sox9 KO mouse line and control mice 4 weeks after the viral injection. Sox9 KO and control astrocytes were molecularly profiled using RNA-sequencing.

first: Kevin

last: Ung

first: Teng-Wei

last: Huang

first: Benjamin

last: Deneen

last: Arenkiel


title: Bulk RNA sequencing of acetylcholine-synthesizing macrophages from subcutaneous adipose tissue

city: Ann Arbor

first: Shanshan

last: Liu

state: Michigan

address: 210 Washtenaw Avenue

institute: University of Michigan

summary: We aimed to study the transcriptomic profile of acetylcholine-synthesizing adipose macrophages. Using a ChAT-eGFP reporter mouse (ChAT, or choline acetyltransferase, is the main enzyme for acetylcholine biosynthesis) acutely exposed to cold temperature, we used FACS to isolate ChAT-eGFP+ and ChAT-eGFP- macrophages from inguinal subcutaneous white adipose tissue (IWAT). We subjected these cells to bulk RNA-seq to better understand the transcriptomic landscape of cholinergic macrophages under cold stress and their role in beige fat thermogenesis.

overall_design: Comparison of ChAT-eGFP+ and ChAT-eGFP- macrophages FACS-isolated from IWAT of cold-exposed ChAT-eGFP reporter mice

first: Jun

last: Wu

first: Alexander

last: Knights

first: Heejin

last: Jun


title: Microarray analysis of long noncoding RNA expression profile in HeLa cells infected Chlamydia trachomatis

city: Hengyang

first: Yating

last: Wen

state: Hunan

address: No. 28, Changsheng West Road

institute: University of South China

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: Chlamydia trachomatis (C. trachomatis) is an intracellular bacterium, and is one of the main pathogens that cause sexually transmitted infections worldwide. Long non-coding RNAs (lncRNAs) have become vital regulators in many biological processes. However, few studies have shown that lncRNAs take part in the pathogenesis of C. trachomatis. Here, we used microarrays to study the expression profiles of lncRNAs and mRNAs in HeLa cells at 12, 24, and 40 hours pot-infection (hpi). Our study provides evidence that lncRNAs are involved in the interaction between C. trachomatis and hosts.

overall_design: Microarray expression profiling of lncRNAsand mRNAs were conducted using RNA extracted from C. trachomatis-infected HeLa cells at 12, 24, and 40 hpi and non-infected HeLa cells.

first: Zhongyu

first: Fangzhen

last: Luo

first: Lanhua

last: Zhao

first: Shengmei

last: Su

first: Wenbo

last: Lei

first: Yi

first: Keliang

last: Shi


title: Down-regulation of Polo-like kinase 4 (PLK4) induces G1 arrest via activation of the p38/p53/p21 signalling pathway in bladder

city: Shen Zhen

first: Haiyan

last: Sun

state: Guang Dong

address: 47 Youyi Road, Luohu District

institute: The Third Affiliated Hospital of Shenzhen University (LuoHu Hospital Group)

summary: We demonstrate that Polo-like kinase 4(PLK4) is overexpressed in human bladder cancer (BC) cell lines and tissues, and its overexpression correlates with poor prognosis. PLK4 inhibition suppresses BC cell growth and induces G1 phase arrest via activating p38/p53/p21 pathway in vitro and in vivo. The data suggest that PLK4 might serve as a novel molecular target for BC treatment.

overall_design: Examination of mRNA profiles of shNC and shPLK4 in bladder cancer cells (5637 and MGHU3)

first: Ziyi

last: Yang


title: Yeast three hybrid experiment

city: SEATTLE

first: Wei

last: Zhou

state: WA

address: William H. Foege Hall, 3720 15th Ave NE

department: genome sciences

institute: University of Washington

summary: Expanding the binding specificity for RNA recognition by a PUF domain

overall_design: We used next generation sequencing to score the binding activity of each variant for its ability to bind to an RNA sequence containing each possible RNA base at the cognate position, which identified many variants with highly repeat-specific interactions.

first: Daniel

last: Melamed

first: Marvin

last: Wickens

first: Stanley

last: Fields


title: Hofbauer cells spread Listeria monocytogenes among placental cells and undergo pro-inflammatory reprogramming while retaining production of tolerogenic factors

status: Public on Jul 27 2021

first: Amy

last: Hite

state: OH - Ohio

address: 1800 Cannon Drive 250 Lincoln Tower

department: Biomedical Informatics

summary: Pregnant women are highly susceptible to infection by the bacterial pathogen Listeria monocytogenes leading to miscarriage, premature birth, and neonatal infection. It is thought that L. monocytogenes breaches the placental barrier by infecting trophoblasts at the maternal interface. However, the fate of L. monocytogenes within the chorionic villi and how infection reaches the fetus are unsettled. Hofbauer cells (HBCs) are fetal placental macrophages and the only leukocytes residing in healthy chorionic villi, forming a last immune barrier protecting fetal blood from infection. Little is known about HBCs’ antimicrobial responses to pathogens. Here, we present the first study of L. monocytogenes interactions with human HBCs isolated from healthy term placentas. Control untreated (5 h and 24 h) HBCs, 24 h LPS/IFNgamma-stimulated, and 5 h and 24 h L. monocytogenes-infected HBCs, were lysed with TRIzol. RNA was isolated followed by on-column DNase treatment and ribo-depletion. RNA-seq libraries were made, pooled, and sequenced on an Illumina NovaSeq SP flow cell in paired-end 150 bp format to a read yield between 70 – 80 million reads (equivalent to 35 – 40 million clusters). The RNA-seq data analysis showed that HBCs undergo pro-inflammatory reprogramming upon L. monocytogenes infection and following stimulation by the potent M1-polarizing agents LPS/IFNgamma. Of particular interest, infected HBCs also upregulated genes encoding numerous pro-inflammatory chemokines known to promote placental infiltration by maternal leukocytes. However, HBCs maintained expression of a collection of tolerogenic genes accompanied by secretion of tolerogenic cytokines, consistent with their tissue homeostatic role in prevention of fetal rejection.

overall_design: 12 samples from 4 different conditions with 3 replicates per sample. Each sample divided across 2 sequencing lanes. Conditions were compared pairwise

last: Webb

first: Siavash

last: Azari

first: Lauren

last: Johnson

first: Stephanie

last: Seveau


title: A 4-gene signature associated with recurrence risk in patients with low and intermediate-risk endometrial cancer

city: Barretos

first: Rui

last: Reis

state: SP

address: Antenor Duarte Viléla Street, 1331

department: Molecular Oncology Research Center

country: Brazil

institute: Barretos Cancer Hospital

summary:
The molecular profile of endometrial cancer has become an important tool in determining patient prognosis and their optimal adjuvant treatment. In addition to The Cancer Genome Atlas (TCGA), simpler tools have been developed, such as the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE). We attempted to determine a genetic signature to build a recurrence risk score in patients diagnosed with low- and intermediate-risk endometrial cancer.
A case-control study was conducted. The eligible patients were women diagnosed with recurrence low- and intermediate-risk endometrial cancer between January 2009 and December 2014 at a single institution; the recurrence patients were matched to two nonrecurrence patients with the same diagnosis by age and surgical staging. Following RNA isolation of 51 cases, 17 recurrence and 34 nonrecurrence patients, the expression profile was determined using the nCounter® PanCancer Pathways Panel, which contains 770 genes.
The expression profile was successfully characterized in 49/51 (96.1%) cases. We identified 12 genes differentially expressed between the recurrence and nonrecurrence groups. The ROC curve for each gene was generated, and all had AUCs higher than 0.7. After backward stepwise logistic regression, four genes were highlighted: FN1, DUSP4, LEF1, and SMAD9. The recurrence risk score was calculated, leading to a ROC curve of the 4-gene model with an AUC of 0.93, sensitivity of 100%, and specificity of 72.7%.
We identified a four-gene signature that is associated the risk of recurrence in patients with low- and intermediate-risk endometrial cancer. This finding suggests a new prognostic factor in this poorly explored group of patients with endometrial cancer.

overall_design: Overall, 49/51 patients (17 recurrence and 34 non-recurrence) were analyzed. Nonrecurrence patients with the same histopathological diagnosis were matched to recurrence cases in a 2:1 ratio by age and FIGO (International Federation of Gyneocologic and Obstetrics) staging (IA and IB)

first: Diocesio

last: de Andrade

first: Luciane

last: da Silva

first: Ana

middle: C

last: Laus

first: Marcos

last: de Lima

first: Gustavo

last: Berardinelli

first: Vinicius

first: Graziela

last: de Macedo Matsushita

first: Murilo

last: Bonateli

first: Aline

middle: V

first: Adriane

last: Evangelista

first: Jesus

last: P Carvalho

first: Ricardo

last: dos Reis


title: Rewiring of human neurodevelopmental gene regulatory programs by Human Accelerated Regions (HARs)

status: Public on Jul 24 2021

city: Boston

first: Janet

last: Song

state: Massachusetts

address: 3 Blackfan St

institute: Boston Children's Hospital

summary: Human Accelerated Regions (HARs) are the fastest-evolving regions of the human genome and many are hypothesized to function as regulatory elements that drive human-specific gene regulatory programs. We interrogate the in vitro enhancer activity of >3,100 HARs in massively parallel reporter assays (MPRAs), demonstrating that many HARs appear to act as neural enhancers and that sequence divergence at HARs has largely augmented their neuronal enhancer activity.

sample_organism:
Homo sapiens
Mus musculus
unidentified plasmid

overall_design: Massively parallel reporter assays (MPRAs) of HARs in human and mouse neural cell lines

first: Christopher

middle: A

last: Walsh

first: Kelly

last: Girskis


title: Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

city: Zürich

first: Puneet

last: Sharma

state: Zürich

address: Otto-Stern-Weg 3

department: Institute of Biochemistry

country: Switzerland

institute: ETH

summary: Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

sample_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae
Candida albicans
Homo sapiens

overall_design: Comprehensive analysis of the effect of CHX on ribosome profiling experiments in different yeasts and mammalian cells. Libraries were generated with a highly standardized and optimized protocol to maximize comparability and to deduce CHX-specific biases.

first: Benedikt

last: Nilges

first: Jie

last: Wu

first: Sebastian

last: Leidel


title: Next generation sequencing  of TFE3-translocation renal cell carcinoma and adjacent normal tissue transcriptomes

city: Shanghai

first: Ning

last: He

address: No. 11, Lane 100, Banxia Road, Pudong New Area, Shanghai, China

institute: GloriousMed Technology Co., Ltd.

summary:
Purpose: The goals of this study are using whole-exome and RNA sequencing technologies on 63 cases with TFE3-tRCC to explore the molecular characteristics of TFE3-tRCC and provide potential effective therapeutic strategies
Methods: Total RNA was isolated from each sample (63 tumor samples and 14 paired adjacent normal samples) using Qiagen RNeasy formalin-fixed paraffin-embedded (FFPE) Kit (Qiagen, Hilden, Germany). Strand-specific RNA sequencing libraries were generated using the Whole RNA-seq Lib Prep kit for Illumina (ABclonal, China). Final libraries were sequenced at the Novogene Bioinformatics Institute (Beijing, China) on an Illumina Hiseq X10 platform by a 150bp paired-end reads. The raw RNA-sequencing reads were filtered by FastQC, Reads were aligned using STAR v2.7.0f with default parameters to the Ensembl human genome assembly GRCh37.
Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling.
Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

overall_design: Renal carcinoma mRNA profiles of 63 tumor tissue with TFE3 fusion and 14 adjacent normal tissue

first: Guangxi

last: Sun


title: RNF5 Regulation of RBBP4 Defines Acute Myeloid Leukemia Growth and Susceptibility to Histone Deacetylase Inhibitors

status: Public on Jul 25 2021

city: Rockville

first: Tongwu

last: Zhang

state: MD

address: 9609 Medical Center Drive

department: Division of Cancer Epidemiology & Genetics

institute: National Cancer Institute

summary:
To investigate a potential role for RNF5 in AML, we monitored changes in gene expression profiles in multiple AML cell lines after RNF5 knock-down.
PolyA RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module, and bar-coded libraries were constructed using the NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA). Libraries were pooled and single end-sequenced (1×75) on the Illumina NextSeq 500 using the High output V2 kit (Illumina, San Diego, CA). Quality control was performed using Fastqc (v0.11.5, Andrews S. 2010)), reads were trimmed for adapters, low quality 5′ bases, and minimum length of 20 using CUTADAPT (v1.1). The number of reads per sample and the number of aligned reads suggested that read quality and number were good and that the data were valid for analysis. High-quality data were then mapped to human reference genome (hg19) using STAR mapping algorithm (version 2.5.2a). FeatureCounts implemented in Subread (v1.50) was used to count the sequencing reads from mapped BAM files. Analyses of differentially expressed genes (DEGs) were subsequently performed using a negative binomial test method (edgeR) implemented using SARTools R Package.
A list of the DEGs was observed and pathway analysis was performed using IPA (http://www.ingenuity.com) based on the following criteria:  |log2(fold change)| >0.4 and p value < 0.05.

overall_design: RNA was prepared 5 days following RNF5 knockdown, and triplicates were used for RNAseq analysis.

first: Ali

last: Khateb

first: Anagha

last: Deshpande

first: Ze’ev

last: Ronai


title: A223 bintrafusp alfa non-responders and responders

status: Public on Jul 23 2021

city: Auroa

first: Xiao Jing

last: Wang

state: CO

department: Pathology

institute: University of Colorado AMC

summary: Mice bearing murine A223 SCC tumors were treated with bintrafusp alfa, targeting both TGF-beta and PD-L1, then processed for single-cell sequencing to identify cell population signaling changes that characterize responders to therapy.

overall_design: Murine A223 tumors from two non-responders and two responders to bintrafusp alfa were analyzed.

first: Xiao-Jing

first: Alexander

last: Strait


title: Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis

status: Public on Jul 26 2021

first: Hui

last: Pan

address: 1 Joslin Place

department: Bioinformatics and Biostatistics Core

institute: Joslin Diabetes Center

summary: Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.

overall_design: All experimental procedures were approved by the Washington University and Joslin Diabetes Center Animal Studies Committees and conducted in accordance with the Public Health Service Policy for the Humane Care and Use of Laboratory Animals. Male C57BL/6J mice were obtained from the Jackson Laboratory (JAX 000664). For in vivo knockdown experiments, locked nucleic acids (LNA, Qiagen) targeting SNORA73 or GFP (control) were injected intraperitoneally into 8-week-old mice at a dose of 3 mg/kg every other day for 3 injections. Animals were euthanized 2 days after the final injection for analyses. Input RNA for transcriptome studies was recovered from liver tissue lysed in polysome buffer using Trizol LS and RNeasy mini kit (Qiagen) including on column DNase digestion. For translatome studies, RNA was recovered from corresponding polysome fractions by Trizol-LS extraction, followed by chloroform-isopropanol-ethanol precipitation, heparinase treatment (New England Biolabs, 15 minutes, 37°C), and RNA cleanup step using RNA Clean & Concentrator (Zymo). Quality of RNA samples was assessed by Agilent Bioanalyzer. Sequencing libraries were prepared using Kapa mRNA Hyper Prep (Roche) according to the manufacturer’s protocol. Briefly, total RNA samples (100-1000 ng) were polyA-selected and reverse transcribed into double-stranded cDNA. High-throughput sequencing was performed using a NovaSeq 6000 Instrument (Illumina) at The Molecular Biology Core Facilities (MBCF) at Dana-Farber Cancer institute (DFCI). RNA-seq raw reads were 100bp reversely stranded paired-end reads.

first: Arthur

last: Sletten

first: Jean

middle: E

last: Schaffer


title: Overexpression of MtABI3 splicing forms by ectopically expressing  cDNA sequences in transgenic Medicago truncatula hairy roots generated from an abi3 mutant background (mutant NF6003)

first: Julia

last: Buitink

institute: INRAE

summary: ABI3 is a B3-domain transcription factor that acts as a master regulator of seed maturation. To identify genes that are regulated specifically by different splicing forms in the model legume Medicago truncatula, Medicago hairy roots obtained from an abi3 mutant background (NF6003) were generated using Agrobacterium rhizogenes transformed with the three cDNA sequence of the ABI3 gene splicing forms of Medicago. Using the Medicago NimbleGen chip, a transcriptomic analysis was performed to identify differentially expressed genes compared to the GUS expressed control.

overall_design: Two-condition experiment, GUS versus the three ABI3 splicing form sequences. Biological replicates: 3 control replicates (35S::GUS), 3 treatment replicates for each splicing form (35S::SF1 and 35S::SF3),and only two replicates for SF2 (35S::SF2),  independently grown and harvested. One replicate per array. Dye swap method.

first: David

last: Lalanne


title: Evaluation of Integrin aE-positive and negative cells

city: South San Francisco

first: Jason

last: Hackney

state: CA

address: 1 DNA Way

department: Department of Bioinformatics and Computational Biology

institute: Genentech, Inc.

summary: Etrolizumab, a humanized monoclonal antibody that selectively binds the b7 subunit of the heterodimeric integrins a4b7 and aEb7, is currently in development for patients with moderate-to-severely active UC. Integrin aE forms a heterodimer with b7; and aEb7 interacts with E-cadherin to mediate retention of aEb7+ cells in the intestinal epithelium. Baseline integrin aE levels have been identified as a potential predictive biomarker for etrolizumab response. Additional markers, including granzyme A, have also been identified and are being developed in partnership with Roche Molecular as potential predictive biomarkers for etrolizumab along with integrin aE. Extensive work has been done to characterize gut aE+ T cells by immunohistochemistry, flow cytometry and qPCR. We hope to use this project to validate differences between aE+ and aE- cell populations using CD4, CD8 and granzyme A expression as controls. The results from this project will be used to pilot single cell sequencing of CD3+aE+ cells isolated from the gut mucosa to identify cellular heterogeneity within aE+ cells under normal and inflammatory conditions. Materials and methods: Mucosal lymphocytes were isolated using a collagenase digestion from surgical specimens taken during routine gastrointestinal resections of diverticulitis and ulcerative colitis patients. CD45+TCRab+TCRgd- cells were sorted into aE+ and aE- groups.

overall_design:
RNA was isolated from CD45+TCRab+TCRgd- T cells sorted into aE+ and aE- subsets. **Raw data not provided for patient privacy reasons**
Raw data not provided due to patient privacy concerns

first: Ryan

last: Ichikawa


title: Etrolizumab treatment in Crohn's disease (Bergamot) cohort 1 RNA-seq

summary: Etrolizumab is an investigational monoclonal antibody that binds the β7 subunit of α4β7 and αEβ7 integrins. The relative contribution of α4β7 and αEβ7 to gut lymphocyte trafficking remains to be characterized. Here we show that αEβ7 is highly expressed on human intestinal CD8+ cytotoxic intraepithelial lymphocytes (IELs) and a small subset of proinflammatory CD4+ T cells. Dual blockade of α4β7 and αEβ7 reduced CD8+ T cell accumulation in the gut to a greater extent than single blockade of either pathway using a photo-convertible mouse model. αEβ7 blockade decreased T cell-epithelial interactions, increased the migratory speed and promoted egress of activated T cells. In Crohn’s disease patients treated with etrolizumab, a reduction of inflammatory genes and cytotoxic IEL gene signatures was observed. Concurrent blockade of α4β7 and αEβ7 increases reduction of cytotoxic IELs and inflammatory T cells in the gut mucosa through a stepwise inhibition of cell migration and tissue retention. The "SAMPLE_ID" sample characteristic is a sample identifier internal to Genentech.

overall_design:
RNA isolated from intestinal biopsies from a phase 3 randomized double-blind placebo-controlled 14-week trial of etrolizumab in Crohn's disease
Raw data not provided due to patient privacy concerns

first: Mary

last: Keir