title: Behavioral and transcriptional response to selection for olfactory behavior in Drosophila

first: Stephanie

last: Rollmann

state: OH

department: Biological Sciences

institute: University of Cincinnati

summary: The detection, discrimination, and behavioral responses to chemical cues in the environment can have marked effects on organismal survival and reproduction, eliciting attractive or aversive behavior. To gain insight into mechanisms mediating this hedonic valence, we applied thirty generations of divergent artificial selection for Drosophila melanogaster olfactory behavior. We independently selected for positive and negative behavioral responses to two ecologically relevant chemical compounds: 2,3-butanedione and cyclohexanone. We also tested the correlated responses to selection by testing behavioral responses to other odorants and life history traits. Measurements of behavioral responses of the selected lines and unselected controls to additional odorants showed that the mechanisms underlying responses to these odorants are, in some cases, differentially affected by selection regime and generalization of the response to other odorants was only detected in the 2,3-butanedione selection lines. Food consumption and lifespan varied with selection regime and, at times, sex. An analysis of gene expression of both selection regimes identified multiple differentially expressed genes. New genes and genes previously identified in mediating olfactory behavior were identified. In particular, we found functional enrichment of several gene ontology terms, including cell-cell adhesion and sulfur compound metabolic process, the latter including genes belonging to the glutathione S-transferase family. These findings highlight a potential role for glutathione S-transferases in the evolution of hedonic valence to ecologically relevant volatile compounds and set the stage for a detailed investigation into mechanisms by which these genes mediate attraction and aversion.

overall_design:
Whole-head mRNA profiles of Drosophila melanogaster artificially selected lines were generated by RNA sequencing, using Illumina HiSeq 4000. Two biological replicates for each of three replicate lines were generatated for high and low behavioral responses to 2,3-butanedione, cyclohexanone, and an unselected control, totalling 30 samples.

The sample 'characteristics: selection treatment' = PI+ indicates selection for attraction to the odorant, while PI- indicates selction for repulsion to the odorant.

first: Elizabeth

last: Brown

first: Alexandra

middle: R

last: Elchert

last: Layne


title: RNA Expression Profile of HBV-infected Primary Human Hepatocytes treatment by Interferon subtypes.

status: Public on Apr 20 2020

city: Shanghai

first: Yang

last: Wang

address: Dongan road 131

country: China

institute: Fudan University

summary: Microarray-dependent transcriptional analysis of interferon stimulated genes treated with different kind of interferon subtypes in HBV-infected PHH.

overall_design: HBV infected PHH treated with IFN-α2 or IFN-α14 at 2 ng/ml or not treated, time point 6 h.

first: Yaming

last: Li

first: Jieliang

last: Chen

first: Zhenghong

last: Yuan


title: Transcriptional profiling of Granny Smith apple peel (Malus x domestica Borkh) at harvest between contrasted years for scald incidence

city: Beaucouzé

first: Mathilde

last: ORSEL

address: 42, rue Georges Morel

country: France

institute: IRHS

summary: Granny Smith apples (Malus x domestica Borkh) were harvested at early physiological mature stage in 2014, 2015 and 2017 and phenotyped for scald incidence after long time cold storage. Differential analyses were set-up for peel samples collected at harvest between fruit batches with low or high scald incidence.

overall_design: The experimental design includs 6 samples collected over 3 years on 2 different plots, and set up in 3 comparisons between low and high scald samples. Each comparison included a technical repetition with dye swap. Each sample included apple peel from 20 fruits.

first: Mathieu

last: Marc

first: Maryline

last: Cournol

first: Sandrine

last: Balzergue

first: Muriel

last: Bahut

first: Sandra

last: Pelletier

last: Orsel


title: Expression data of J3T-1, J3T-2, J3T-1shA, J3T-2A glioma

city: Okayama

first: Yuji

last: Matsumoto

address: 2-5-1 Shikata, kita-ku

department: Neurological Surgery

country: Japan

institute: Okayama University

summary:
We have previously established two sibling glioma subclones, J3T-1 and J3T-2, showing distinct invasive and angiogenic phenotypes. J3T-1, expressing high annexin A2, demonstrates robust angiogenesis and tumor invasion around neovasculature. J3T-2, expressing low annexin A2, demonstrates diffuse invasion along white matter tracts. Knockdown of annexin A2 in J3T-1 (J3T-1shA) resulted in diffuse invasion pattern, and overexpression of annexin A2 in J3T-2 (J3T-2A) showed prominent angiogenesis.
We used microarrays to identify genes which promote the phenotypic transition regulated by annexin A2.

overall_design: Total RNA of J3T-1, J3T-2, J3T-1shA, and J3T2A were isolated, and each gene expression profile was investigated using Affymetrix Canine Genome 2.0 Array.

first: Tomotsugu

last: Ichikawa

first: Satoshi

last: Inoue

first: Manabu

last: Onishi

first: Yoshihiro

last: Otani


title: Transcriptome analysis of the perivascular adipose tissue in peripheral artery disease patients

city: Milano

first: Gualtiero

middle: Ivanoe

last: Colombo

address: Via Carlo Parea, 4

country: Italy

institute: Centro Cardiologico Monzino IRCCS

summary: Perivascular adipose tissue (PVAT) is thought to play a role in vascular homeostasis and in the pathogenesis of diseases of large vessels. We tested the hypothesis that locally restricted transcriptional profiles characterize the PVAT localized at the site of the obstruction of the abdominal aorta in peripheral artery disease (PAD) patients. By a genome-wide approach and a paired-samples design, we investigated the PVAT transcriptome of 11 PAD patients with occlusive and with stenotic abdominal aortic lesions. We performed a data adjustment step using the DaMiRseq R/Bioconductor package, to remove the effect of confounders as produced by high-throughput gene expression techniques. We compared PVAT of the distal versus the proximal aorta of each patient to limit the effect of inter-individual variability, using the limma R/Bioconductor package. We did not find consistent differences in PVAT gene expression clearly distinguishing the two PVAT of the same patient. However, we found significant differences by comparing patients with total occlusive versus those with stenotic abdominal aortic lesions. We dissected putative mechanisms associated with PVAT involvement in PAD patients with total occlusive and with stenotic abdominal aorta lesions through a functional enrichment network analysis: cholesterol, sterol and alcohol biosynthetic process were enriched in patients with total occlusive lesions, whereas pathways recalling the structure maintenance and remodeling of the vessels were associated with patients with stenotic lesions. Our results would suggest that the PVAT transcriptome at the distal aorta site is associated with the degree of the atherosclerotic burden in PAD patients and that this effect can probably account for a diffuse (systemic) process affecting homogenously the PVATs spanning along the distal aorta rather than a singular specific trait.

overall_design: A paired-sample study design was used to reduce the problem of inter-individual variations. Eleven patients with PAD were consecutively enrolled. Diverse adipose tissue (AT) depots were safely obtained sequentially, in the following order: subcutaneous abdominal fat, omental-visceral fat, periaortic fat obtained from the aortic neck proximal to the lesion, and periaortic fat surrounding the lesion. RNA was extracted from 50-100 mg of frozen AT samples. RNA samples of poor quality and yield were discarded.

first: Luca

last: Piacentini

first: Josè

middle: P

last: Werba

first: Elisa

last: Bono

first: Mattia

last: Chiesa

first: Claudio

last: Saccu

first: Elena

last: Tremoli

first: Rita

last: Spirito

middle: I


title: Tissue specific light response during Arabidopsis de-etiolation

city: Rishon LeZion,

first: Yogev

last: Burko

address: 68 HaMaccabim Rd.,

department: Plant Biology

country: Israel

institute: Volcani Institute

type: Expression profiling by high throughput sequencing

summary: The time between seed germination and the development of the first true leaves is crucial for plants success.  Depending on the environmental light conditions a plant must decide which developmental program to use. If light is limited, the seedling will use etiolated growth characterized by an elongated hypocotyl and tightly-closed, underdeveloped cotyledons, thus allowing it to reach a light source as quickly as possible. In contrast, seedlings that are grown in unlimited light will use de-etiolation growth, that is characterized by inhibited hypocotyl growth and unhooked, unfolded and fully expanded cotyledons. These seedlings become photosynthetic and increase their chances for reproduction. Interestingly, the outcome of the light signal varies between the seedling’s different organs, e.g. hypocotyl growth is inhibited by light whereas cotyledon expansion is induced. Separating the cotyledons and hypocotyls of de-etiolated seedlings during their first 12 hours of white light exposure, enabled us to perform individual transcriptome analyses of each organ and to examine what causes this variation in light responsiveness.

overall_design: mRNA from cotyledons and hypocotyls of three-days-old old dark growing seedlings (dark) which moved to simulated white light (Blue, Red and low Far-red Led light – 100 µmol m−2 s−1) for 15 minutes, 30 minutes, one hour, three hours, six hours or 12 hours.

first: Joanne

last: Chory

first: Ullas

last: Pedmale


title: Chimeric activators and repressors define HY5 activity and a feedback control mechanism in Arabidopsis (HY5_ChIPseq)

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Transcription regulation requires many protein interactions on chromatin, and only a subset of transcription factors have well-defined activation or repression domains. The Arabidopsis transcription factor HY5 controls critical growth-related gene expression programs during plant development, but it’s primary activity in regulating transcription remains unclear. To address this question, we generated constitutive repressor and  activator HY5 fusion proteins to direct the expression of HY5 target genes. We used RNA-seq, ChIP-seq, and multiple phenotypes to demonstrate that HY5 depends on accessory factors to promote transcription and identify high confidence direct targets of HY5. We suggest that this strategy can be used broadly to define the transcription regulation activity and direct targets of transcription factors. Interestingly, this approach also revealed a mechanism by which HY5 promotes the accumulation of its own negative regulators. We show that HY5 directly regulates components of the COP1 E3-ubiquitin ligase complex, and  by uncoupling this feedback loop we can induce partial de-etiolation in the dark. This provides a system by which plants can quickly repress growth upon light exposure. Lastly, we show that modulating this system can generate significant phenotypic diversity and provide proof of concept that these fusion proteins can modulate growth in tomato,  opening a novel path toward selecting desirable traits in crop species.

overall_design: We performed whole-genome chromatin immunoprecipitation with sequencing (ChIP-Seq) analysis on 35S:FLASH-HY5,  35S:FLASH-HY5-VP16 and 35S:FLASH-HY5-SRDX during de-etiolation (dark+2.5h in simulated white light (blue, red and far-red LED lights- 100 µmol m−2 s−1 )

first: Joseph

last: Ecker

first: Mark

last: Zander


title: Chimeric activators and repressors define HY5 activity and a feedback control mechanism in Arabidopsis (HY5_RNAseq)

overall_design: mRNA from 3days old dark growing seedlings and dark + 1.5h , 4h simulated white light (blue, red and far-red LED lights) of WT, hy5, 35S:FLASH-HY5, 35S:FLASH-HY5-VP16, 35S:FLASH-HY5-SRDX  was sequenced to analyze the genes regulated by HY5.


title: Kinome expression profiling of muscle invasive bladder cancer (MIBC) patients

city: Birmingham

first: Sooryanarayana

last: Varambally

state: Alabama

address: 1824 6th Avenue S

department: Pathology

country: USA

institute: University of Alabama

summary: Muscle invasive bladder cancer (MIBC) is one of the aggressive cancer with limited treatment options. Targeted gene expression profiling in normal and tumor samples from MIBC patients could aid in identification of potential therapeutic targets. Thus we used kinase specific panel to study their expression pattern and to identify targetable genes.

overall_design: Tumor and adjacent normal macrodissected tissues were obtained from 24 MIBC patients. RNA isolated from tissues were profiled on the nCounter Human Kinase Panel.

first: G

last: Sonpavde

first: S


title: RNA sequencing of CD4+ and CD8+ subsets of human engineered regulatory T cell lines expressing FOXP3, FOXP+Helios-FL, FOXP3+Helios-Δ3b

city: Kansas City

first: Mary

middle: A

last: Markiewicz

state: KS

address: 3901 Rainbow Blvd. MS 3029

department: Microbiology, Molecular Genetics & Immunology

institute: University of Kansas Medical Center

summary: We report RNA sequencing data for three human engineered regulatory T cell lines expressing FOXP3, FOXP+Helios-FL, FOXP3+Helios-Δ3b.  From total RNA from 3 biological replicates from each cell line, we generated RNA Sequencing data that compared gene expression between CD4+ and CD8+ cells for each cell line. RNA Sequencing demonstrated that the addition of full-length Helios changed gene expression in cellular pathways and the Treg signature compared to FOXP3 alone or the other major Helios isoform.

overall_design: Examination of CD4+ and CD8+ cells from three human engineered regulatory T cells cell lines.


title: Genome-wide transcriptome analysis of PAK4 inhibition/knockdown in human bladder cancer cell line VMCUB1

summary: Using multi-omic analyses, we identified PAK4 (P21 (RAC1) Activated Kinase 4) amplification and overexpression of Kinase PAK4 in a subset of bladder cancers. We confirmed the role of PAK4 in bladder cancer cell proliferation and invasion by in vitro experiments. Furthermore, our studies showed that PAK4 inhibitor is effective in curtailing bladder cancer cell growth.  In order to understand the effect of PAK4 inhibition and knockdown on bladder cancer transcriptome, we performed RNA-sequencing of PAK4 siRNA transfected and PAK4 inhibitor (PF-3758309) treated bladder cancer cells (VM-CUB1). Analyses led to identification of direct targets of PAK4 in bladder cancer and associated pathways.

overall_design: Total RNA were separately isolated from a) VM-CUB1 cells treated with 5 nM PAK4 inhibitor (PF-3758309, Selleck Chemicals) and incubated for 4 days, b) VM-CUB1 cells treated with DMSO for 4 days, c) VM-CUB1 cells transfected with PAK4 siRNA using Lipofectamine RNAiMAX Reagent (Life Technologies) and b) VM-CUB1 cells transfected with non-targeting siRNA.

first: Darshan

last: Chandrashekar


title: Transcriptome analysis of ITGA3+ and ITGA3- from expanded CB cells

city: Montreal

first: Guy

last: Sauvageau

state: QC

address: 2950, Chemin de Polytechnique

country: Canada

institute: Institute for Research in Immunology and Cancer

summary: Cell purification technology combined with whole transcriptome sequencing and small molecule agonist of hematopoietic stem cell self-renewal has allowed us to identify ITGA3 as a surface maker that defines a rare subpopulation of human cells which is highly enriched for stem cell activity in vivo. ITGA3-positive cells (within the EPCR+CD90+CD133+CD34+CD45RA- fraction) exhibit a robust multi-lineage differentiation potential and serial reconstitution in immunocompromised mice. In culture, most if not all of the HSC activity is detected in the ITGA3+ subset, arguing for the stability of this marker on the surface of cultured cells, a feature not found with more recently described markers such as CD49f. Functionally ITGA3 is essential for human HSC activity in vivo. Its exploitation should open new possibilities in our effort to understand the molecular bases behind HSC self-renewal.

overall_design: Examining 2 cellular subsets: ITGA3+ and ITGA3- derived form EPCR+CD90+CD133+CD34+CD45RA- cord blood cells after 7 day expansion in UM171. A 3rd cell fraction (EPCR+CD34+) was sorted as a control

last: Tomellini

first: Bernhard

last: Lehnertz


title: Targeting the scaffolding role of LSD1(KDM1A) poises acute myeloid leukemia cells for Retinoic Acid induced differentiation [RNA-seq]

city: milano

first: Saverio

last: Minucci

address: via Adamello 16

institute: European Institute of Oncology

summary: The histone demethylase LSD1 is deregulated in several tumors, including leukemias, providing the rationale for the clinical use of LSD1 inhibitors . In acute promyelocytic leukemia (APL), pharmacological doses of retinoic acid (RA) induce differentiation of APL cells through degradation of the PML-RAR oncogene. APL cells are resistant to LSD1 inhibition or knock-out, but LSD1 inhibition sensitizes them to physiological doses of RA without altering the stability of PML-RAR, and extends survival of leukemic mice upon RA treatment. Non-enzymatic activities of LSD1 are essential to block differentiation of leukemic cells, while the combination of LSD1 inhibitors (or LSD1 knock-out) with low doses of RA releases a differentiation-associated gene expression program, not strictly dependent on changes in histone H3K4 methylation (known substrate of LSD1). An integrated proteomic/epigenomic/mutational analysis showed that LSD1 inhibitors alter the recruitment of LSD1-containing complexes to chromatin through inhibition of the interaction between LSD1 and GFI1, a relevant transcription factor in hematopoiesis.

overall_design: Examination of NB4 cells treated as indicated in each sample versus cells trated with DMSO (vehicle).  MC_2580 = LSD1 inhibitor

first: Roberto

last: Ravasio

last: Ceccacci

first: Pierluigi

last: Rossi


title: Targeting the scaffolding role of LSD1(KDM1A) poises acute myeloid leukemia cells for Retinoic Acid induced differentiation [ChIP-seq]

overall_design: H3K4me1, H3K4me2, H3K4me3 and H3K27ac ChIP-seq on NB4 cells treated with DMSO, MC_2580 (LSD1 inhibitor), RA 0.01 µM, RA 1µM and MC_2580 + RA 0.01µM.  Series includes anti-LSD1 chIP on DMSO and MC_2580-treated NB4 cells and GFI1 ChIP-seq on untreated cells.

first: Elena

first: Amir

last: Hosseini


title: Histone crotonylation and its readers control virulence-related biology of a major human commensal and pathogen.

city: Clayton

first: Traude

last: Beilharz

state: VIC

address: Wellington Rd

country: Australia

institute: Monash University

summary: PAT-seq approach was utilised to determine the gene expression  of C. albicans. Biological duplicates of wild type and taf14∆∆ mutant samples were grown to log phase, with or without calcofluor white treatment (20 µg/ml, 2 h).

overall_design: Analysis of RNA-seq in C. albicans

first: Ana

last: Traven

first: Qi

last: Wang

first: Jiyoti

last: Verma

first: Michael

last: See

middle: H


title: A novel positive feed-back loop mediated by the membrane-associated transcription factor NTL3  is important for heat stress tolerance in rice

first: Xue

middle: Huan

last: Liu

state: Shanghai

address: NO.2005 Songhu Road

department: School of Life Sciences

summary: Global warming has great impacts on plant growth and development. Heat shock transcription factors are the master regulators of heat stress response to alleviate protein misfolding in the cytosol in plants. However, how plants deal with accumulation of misfolded proteins in the endoplasmic reticulum (ER) under heat stress conditions is less understood, especially in crops such as in rice. Here we report a positive feed-back loop mediated by the membrane-associated transcription factors NTL3 and bZIP74 in heat stress response in rice. In response to heat stress, the ER-membrane-associated bZIP74 is activated and up-regulate the expression of NTL3 in rice; NTL3 encodes an ER-membrane-associated transcription factor that is activated and regulate downstream genes including bZIP74 involved in protein folding and reactive oxygen species scavenging. Loss-of-function mutations of NTL3 are sensitive to heat stress while inducible expression of the processed form of NTL3 increases heat stress tolerance in rice seedlings. Our work reveals the important role of NTL3 in ER stress response for heat stress tolerance in rice.

overall_design: Rice seedling (8 days after germination) mRNA profiles of wild type (WT) and NTL3-edited plants were generated with or without heat treatment by deep sequencing, in triplicate, using Illumina HiSeq 2000.

first: Xue-huan

first: Jian-Xiang


title: Targeting the scaffolding role of LSD1(KDM1A) poises acute myeloid leukemia cells for Retinoic Acid induced differentiation

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

overall_design: Refer to individual Series


title: Epigenome analysis of pediatric bithalamic diffuse gliomas

status: Public on Apr 19 2020

city: Stanford

first: David

middle: A.

last: Solomon

state: CA

address: 300 Pasteur Dr, Lane L216

institute: Stanford University School of Medicine

type: Methylation profiling by genome tiling array

summary: Genome-wide DNA methylation profiling of 10 pediatric bithalamic diffuse gliomas. The Illumina Infiunium 450k Human DNA Methylation Beadchip (n=4) or the Infinium EPIC 850k Human DNA Methylation Beadchip (n=6) was used to obtain DNA methylation profiles across approximately 450,000 or 850,000 CpG sites of genomic DNA extracted from formalin-fixed, paraffin-embedded tumor tissue of 10 pediatric bithalamic diffuse gliomas.

overall_design: Bisulfite converted genomic DNA extracted from the 10 tumor samples was hybridized to either the Illumina Infinium 450k Human DNA Methylation BeadChip or the Infinium EPIC 850k Human DNA Methylation Beadchip


title: Deep proteomic analysis of murine erythroid differentiation

city: New Haven

first: Vince

last: Schulz

state: CT

address: 333 Cedar St. LMP 4085

department: Department of Pediatrics

institute: Yale University

summary: Deciphering cellular proteomes is critical for our understanding of erythropoiesis. To better comprehend commonly used models of erythropoiesis, we obtained absolute quantification of proteins expressed in cultured primary murine bone-marrow derived erythroblasts throughout erythroid differentiation. These data were compared to proteomes from Friend murine erythroleukemia (MEL), G1ER, and MEDEP cells. Overall, more than 7200 proteins were quantified in at least one of these models of murine erythropoiesis. Protein expression during differentiation of MEDEP cells was most similar to that of differentiating primary murine erythroid cells, while proteomes of Friend MEL and G1ER cells were more distantly related. Comparison of the proteomes of murine and human erythroblasts revealed species-linked variability, but these differences were not as dramatic as those observed comparing human and murine transcriptomes during erythroid differentiation. To functionally validate the differences between transcriptional and translational control, heme synthesis was inhibited in MEDEP cells and the effects on the transcriptome and the proteome examined. While heme deficiency had minimal global effect on the cellular proteome, globin proteins were significantly down regulated, supporting observations heme regulation of globin synthesis is tightly regulated at the protein level to avoid deleterious over production of globin chains. As predicted, heme deficiency led to up regulation of -aminolevulinic acid synthase 2 (Alas2) protein. Interestingly, Alas1 was upregulated at both the transcriptional and protein levels. Characterization of cellular proteomes during erythropoiesis has potential to yield insight into mechanistic principles of human disease, as well as reveal novel therapeutic targets.

overall_design: MEDEP cells were subject to differentiation with EPO, and samples were obtained at 0, 12, 24, 36, 48 and 72  hours. MEDEP cells were also treated for 12hrs with Epo in the presence of succinyl acetone (SA) to block heme synthesis. Three biological replicates were obtained for each sample type.

first: Emilie-Fleur

last: Gautier

first: Marjorie

last: Leduc

first: Meriem

last: Ladli

first: Vincent

first: Carine

last: Lefevre

first: Michaela

last: Fontenay

first: Catherine

last: Lacombe

first: Frédérique

last: Verdier

first: François

last: Guillonneau

first: Narla

last: Mohandas

first: Patrick

middle: G

last: Gallagher

last: Mayeux


title: Microarray analysis of gene expression in rice lacking cytosolic glutamine synthetase 1;1 and 1;2 grown under the ammonium sufficient condition

city: Yokohama

first: Miyako

last: Kusano

state: Kanagawa

address: 1-7-22, Suehiro, Tsurumi

institute: RIKEN CSRS

summary:
Nitrogen (N) is a critical limiting element in the provision of important components, e.g., chlorophylls, to plants. Organic N synthesized by glutamine synthetase (GS) may help regulate metabolic networks for plant growth and development. However, how multiple isoforms of cytosolic GSs act for metabolic regulation remain unclear. Using the knockout mutants, Osgs1;1 and Osgs1;2, we compared the effects of two cytosolic GSs on metabolism and cell differentiation in rice roots using reverse genetic-, metabolite- and transcript profiling- and microscopic analyzes together with integrated metabolite-transcript network analysis.
We used microarrays to detail the global programme of gene expression in rice Osgs1;1 and Osgs1;2 roots.

overall_design: Seventeen root samples were analyzed; three biological replicates were used for each tissue except for NC2373 homozygote (n = 2).

first: Atushi

last: Fukushima

first: Tomoko

last: Nishizawa


title: Systemic inflammation drives rapid conversion of conventional CD4+ T cells to Treg in vivo

city: San Diego

first: Joseph

middle: Samuel

last: Dolina

state: California

address: 9420 Athena circle

institute: La Jolla Institute for Allergy and Immunology

summary: The immune system faces a task that approximates cognition in its complexity as it processes a multitude of intrinsic and extrinsic signals and integrates these into responses of the appropriate class, specificity, magnitude, and duration for a given threat, with the host’s life often depending on the outcome. CD4+ T lymphocytes occupy a unique role in the generation and regulation of immunity within this context and influence multiple innate and adaptive cell types. With respect to CD8+ cytotoxic T lymphocytes (CTL), CD4+ T cells function early in the response as ‘helpers’ (TH) to increase its magnitude and functionality, and later as regulatory cells (Treg) to restore homeostasis and avoid immune pathology. Using a Listeria monocytogenes (Lm) infection model, we probed whether the conditions of initial pathogen encounter could influence the generation of TH versus Treg. At low dose infection, CD4+ T cells ‘help’ CTLs via CD40-CD40L signaling, while high dose infection induces rapid polyclonal conversion to functional FoxP3+CD25+ Treg within 24 hours. These findings resolve long-standing questions regarding the requirement for TH and reveal a remarkable degree of plasticity in the function of CD4+ T cells, which can be quickly converted to Treg in vivo in response to acute inflammation.

overall_design: Differential gene expression analysis of total RNA isolated from splenic CD4+eGFP− (Tconv) and CD4+eGFP+ (Treg) from Foxp3eGFP mice following low dose and high dose Lm ΔactA-Ova infections.

middle: S

first: Joey

last: Lee

first: Lara

last: Labarta-Bajo

first: Sumetha

last: Kannan

first: Jason

last: Greenbaum

first: Zbigniew

last: Mikulski

first: Nawal

last: Bahia El Idrissi

first: Margot

middle: J

last: Pont

first: Sandy

middle: L

last: Rosales

first: Gregory

last: Seumois

first: Pandurangan

last: Vijayanand

first: Michael

last: Croft

first: Stephen

last: Schoenberger


title: Differential CpG methylation in peripheral naive and memory CD4+T-cells, and monocytes in early rheumatoid arthritis patients

city: Leeds

first: Frederique

last: Ponchel

state: England

address: St. James’s University Hospital

department: Leeds Institute of Rheumatic and Musculoskeletal Medicine

country: United Kingdom

institute: University of Leeds

summary:
The genetic risk associated with rheumatoid arthritis (RA) includes genes regulating DNA methylation and many T-cells genes with a strong MHC-association, pointing to immuno-genetic mechanisms as disease triggers. We aim to identify methylation change in naive and memory CD4+T-cells and monocytes in the early stage of disease, to gain insight to disease pathology.
DNA-methylation was explored using a genome wide array (DNA methylation Illumina 450K bead chip array).  Differential methylation in promoters of many genes was associated with several disease relevant pathways notably in naive CD4+T-cells. This pointed to the IL6/JAK1/STAT3 signalling cascade linking into inflammatory mechanisms and TNF signalling as well as the engagement of naive CD4 T-cells intoTh17 differentiation and the implication of several Interferon response genes.

overall_design: 10 early RA patients and 6 Healthy controls were selected. PBMC were sorted for 3 cell subsets: naive CD4+T-cells, memory CD4+T-cells, and monocytes. DNA was extracted from each sample, then bisulfite converted and used on an Illumina Infinium 450k Human Methylation array: 46 methylation profiles were retained after quality controls assessments.

first: Rujiraporn

last: Pitaksalee

first: Agata

middle: N

last: Burska

first: Philip

last: Chambers

first: Shoaib

last: Ajaib

first: Rekha

last: Parmar

first: Xuanxiao

last: Xie

first: Katarina

last: Mydlova

first: Joshua

last: Rogers

first: Jagtar

last: Nijjar

first: Alastair

last: Droop

first: Iain

middle: B

last: McInne

first: Paul

last: Emery

first: Richard

last: Hodgett


title: Dysfunction of hair follicle mesenchymal progenitors contributes to age related hair loss

city: Calgary

first: Jeff

last: Biernaskie

state: Alberta

address: 3330 Hospital Drive NW

department: Comparative Biology and Experimental Medicine

institute: University of Calgary

summary: Advanced age drives the progressive degeneration of mammalian hair follicles (HF) and termination of its regeneration cycle. The role of hair follicle dermal stem cell (hfDSC) in this age-associated HF dysfunction remains largely unexplored. Here, we first characterize a novel function of hfDSCs in regenerating the full dermal papilla (DP) after laser ablation. Next, we performed long-term fate mapping, age-dependant in vivo clonal analysis and in vitro proliferation assay on hfDSCs to describe the age-related defects in mesenchymal stem cell self-renewal and differentiation capabilities. Moreover, single-cell RNA-sequencing of the HF mesenchymal population mapped the intrinsic transcriptional changes that may drive their age-related dysfunction. We highlight the requirement of hfDSCs for normal HF regeneration. Our findings characterize a novel function of mesenchymal stem cells, and how this crucial cell becomes dysfunctional with age to drive age-related hair loss.

overall_design: We enriched for mesenchymal cells within the mammalian hair follicle for analysis. We first generated α-SMACreERT2:ROSAYFP transgenic mice, and treated the mice with tamoxifen at 3 and 4 days of age to permanently label the hair follicle dermal stem cells and their progeny. The mice were aged to either young (2mo and 12mo) aged (18mo) cohorts before isolation. To isolate cells of the hair follicle mesenchyme, we utilized Fluorescence activated cell sorting (FACS). We collected two populations of cells for all samples, to a total of 10,000 cells per age cohort (N=2 per cohort). First, we collected 5000 cells that were YFP+ and (Itga9+ or Cd200+), then we collected 5000 cells that were Cd26+ and (Itga9+ or Cd200+). The two populations were mixed to generate two replicates of young (2mo), one replicate of 12mo and one replicate for 18mo. Samples were processed according to 10X Genomics ChromiumTM Single Cell 3’ Reagent Guidelines v2 or v3 Chemistry as per the manufacturer’s protocol. Single cells were sorted into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed by Genome Quebec using Illumina HiSeq4000. We recovered 2707cells for sample 1, 1906 for sample 2, 6998 for sample 2 and 5415 for sample 3. Our estimated doublet rate was approximately ≈3% due to loading of 10,000 cells and we achieved ~50,0000-100,000 reads/cell.

first: Wisoo

last: Shin

first: Nicole

last: Rosin

first: Holly

last: Sparks

first: Sarthak

last: Sinha

first: Waleed

last: Rahmani

first: Nilesh

last: Sharma

first: Matt

last: Workentine

first: Sepideh

last: Abbasi

first: Elodie

last: Labit

first: Jo

last: Stratton


title: Early commitment genes drive fast, irreversible commitment to human embryonic stem cell differentiation [GATA3_ChIP-seq]

city: London

first: Silvia

middle: DM

last: Santos

address: 1 Midland Road

institute: The Francis Crick Institute

summary: The goal of this study was to characterise the binding sites of GATA3 in human embryonic stem cells (hESCs) after exposure to the pro-differentiation growth factor BMP4.

overall_design: ChIP-Seq of GATA3 was performed on cultured hESCs grown in mTeSR1 with or without treatment with BMP4 for 6 hours.

first: Santos

last: Silvia


title: Comparative transcriptome analysis between two tomato materials under cold stress

city: Harbin

first: Jian

last: Wang

address: 600 Changjiang Road

institute: Northeast Agricultural University

summary: RNA sequencing was performed to investigate the the response mechanism of tomato response to cold stress.“Micro-TOM-EX” is the 'Micro-TOM' plants overexpressing a GATA transcription factor gene.

overall_design: mRNA profiles of different tomato leaf samples were generated by deep sequencing. The sequence reads that passed quality filters were analyzed at the transcript .

first: Tingting

last: Zhao

first: Xiangyang

last: Xu


title: Early commitment genes drive fast, irreversible commitment to human embryonic stem cell differentiation [SMAD_ChIP-seq]

summary: The goal of this study was to characterise the binding sites of SMAD1 in cultured human embryonic stem cells (hESCs) immediately after exposure to the pro-differentiation growth factor BMP4.

overall_design: ChIP-Seq of SMAD1 was performed on the cultured hESC line H1 grown in mTeSR1 with or without treatment with BMP4 for 60 minutes.


title: Early commitment genes drive fast, irreversible commitment to human embryonic stem cell differentiation (RNA-seq data set 2)

summary: The goal of this study was to characterise the earliest transcriptional changes that occur in human embryonic stem cells (hESCs) immediately after exposure to various pro-differentiation stimuli.

overall_design: RNA-Seq of mRNA was performed on cultured hESCs treated with either BMP4 or growth factor cocktails designed to direct differentiation to a particular germ lineage (endoderm, ectoderm, trophectoderm) for 0, 20, 40, or 60 minutes. Experiments were performed in triplicate.


title: HNF1A-AS1, as competing endogenous RNA (ceRNA), is involved in VEGF-C-mediated PI3K signaling pathway in gastric cancer

city: Jinan

first: Peng

last: Gao

address: Wen Hua Xi Road 44

institute: Shandong University

summary: To investigate the effect of lncRNA on theVEGF-C-mediated cancer development , genome-wide lncRNA  were analyzed by Arraystar Human LncRNA  microarray V3.0 upon knockdown VEGF-C in MKN-45gastric cancer cells.

overall_design: The MKN-45 gastric cancer cells were transfected with si-VEGF-C or si-NC. Total RNA was extracted 48h after the transfection and the differentially expressed genes were analyzed byArraystar Human LncRNA  microarray V3.0.

first: Gao

last: Peng

first: Hai-ting

last: Liu


title: RNA sequencing of miRNA in plasma from myocardial ischemia and reperfusion mice and sham mice

status: Public on Apr 18 2020

city: Baltimore

first: Sheng

state: MD

address: 660 W Redwood Street, Howard Hall 564

department: Anesthesiology

institute: University of Maryland Baltimore

type: Non-coding RNA profiling by high throughput sequencing

summary:
Purpose: The aim of this study is to compare the plasma miRNA profile between mice subject to myocardial ischemia and reperfusion and mice subject to sham operation.
Methods: 8 to 10-week old C57BL/6 mice underwent myocardial ischemia and reperfusion (MIR) or Sham operation. Plasma RNA was isolated using Trizol LS reagent 4h post-surgery. NGS cDNA libraries were prepared using Norgen Biotek Small RNA Library Prep Kit. Library quality was validated prior to sequencing on an Illumina NextSeq 500 platform.

overall_design: 8 to 10 weeks old male C57BL/6 mice were subject to myocardial ischemia and reperfusion (8 mice) or sham surgery (6 mice). Plasma was collect for RNA isolation 4 hour post-surgery. cDNA librarues were sequenced using Illumina NextSeq 500 platform.

first: Yang

last: Yang

first: Wei

last: Chao


title: Early commitment genes drive fast, irreversible commitment to human embryonic stem cell differentiation (RNA-seq data set 1)

overall_design: RNA-Seq of mRNA was performed on cultured hESCs treated with either BMP4 or growth factor cocktails designed to direct differentiation to a particular germ lineage (endoderm, ectoderm, trophectoderm) for 20, 40, or 60 minutes, and compared to untreated (pluripotent) hESCs. Experiments were performed in triplicate.


title: Elongation factor ELOF1 drives transcription-coupled repairand prevents genome instability [DRB-TTchem-seq]

first: Richard

middle: James

last: Mitter

state: LONDON

department: Bioinformatics & Biostatistics

summary: Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA Polymerase II (Pol II), causing transcription inhibition and transcription-replication conflicts. Cells have evolved intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions, however the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR/cas9 screen, we identified elongation factor ELOF1 as an important new factor in the damage-induced transcription stress response. ELOF1 has an evolutionary-conserved role in TC-NER where it promotes the recruitment of the TC-NER factors UVSSA and TFIIH and facilitates Pol II ubiquitylation to efficiently repair TBLs. Importantly, ELOF1 has an additional role in protecting cells from transcription-mediated replication hindrance, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage by two distinct mechanisms.

overall_design:
Determining the elongation rates in HCT116 wt, ELOF1 KO, CSB KO or ELOF1 KO+ ELOF1-GFP cells using DRB-TTchem-seq.
transient transcriptome sequencing (TT-seq); 3.5 hrs pre-treatment with 100uM DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole), followed by release from DRB inhibition for 10, 20, 30 or 40 min (including 10 min 1mM 4-thiouridine pulse)

first: Marit

middle: E

last: Geijer

first: Simona

last: Cugusi

first: Jesper

middle: Q

last: Svejstrup

first: Jurgen

last: Marteijn


title: β-catenin is a node at orchestrating distinct aspects of epithelial-mesenchymal transition (EMT) and malignant mammary tumor progression

city: Basel

first: Ravi Kiran Reddy

last: KALATHUR

state: Basel

address: Mattenstrasse 28

department: Departement Biomedizin

country: Switzerland

institute: University of Basel

summary: Canonical Wnt signaling plays crucial roles in the progression of many cancer types. In canonical Wnt signaling, β-catenin acts as a controller determining the transcriptional output: via its N-terminus it recruits the signaling coactivators Bcl9 and Bcl9/9l and via the C-terminus it interacts with the general transcriptional machinery. In the intestine, the C terminal output is essential, while the N-terminus controls only a subset of target genes. In breast cancer, the relative contribution of b-catenin’s different output is not known. Also not known is the functional contribution of β-catenin’s a role in cadherin-mediated cell adhesion, a function which confounds the analysis of conventional loss of function models. To address these unknowns, we combined the MMTV-PyMT mouse model of metastatic breast cancer with mouse lines carrying mutations in β-catenin that abolish its function completely or specifically target the N or C-terminal transcriptional outputs. Notably, the complete lack of β-catenin resulted in apoptosis of mammary tumor cells in vivo and in vitro. In contrast, the loss of β-catenin’s transcriptional functions did not provoke apoptosis but did diminish cell proliferation, epithelial-mesenchymal transition (EMT) and cell migration in vitro. This was also reflected in a reduction in primary tumor growth, tumor invasion, and metastasis formation in vivo. Whole transcriptome analysis identified subsets of genes, which were specifically regulated either by β-catenin’s N or C-terminal activities. Intriguingly, the N-terminal output was critical for TGFb-induced EMT and metastasis formation. The observations from the mouse models correlated with expression studies in human breast cancers.

overall_design: RNA-Seq of β-cateninfl/fl  ( WT) and β-cateninD164A/-,β-cateninΔC/- and β-catenindm/- mutant cell lines on treatment with TGFb or Wnt3a was performed in biological duplicates

first: David

last: Büchel

first: Ravi

last: Kiran Reddy Kalathur

first: Gerhard

last: Christofori


title: H2A.Z is dispensable for both basal and activated transcription in post-mitotic mouse muscles [ATAC-Seq - muscle]

city: Lyon

first: Thomas

last: Simonet

address: Faculté de Médecine Lyon Est, 8 Avenue Rockefeller

institute: Université de Lyon

summary: While the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription.

overall_design: ATAC-seq on WT and H2A.Z double KO mouse muscle nuclei, in triplicate

first: Belotti

last: Edwige

first: Lacoste

last: Nicolas

first: Simonet

last: Thomas


title: Identification of genetic biomarker signatures associated with cannabis pharmacokinetics and tolerability

city: Toronto

first: George

middle: S.

last: Charames

state: ON

address: 600 University Ave, Ste 6-500

department: Pathology and Lab Medicine

institute: Mount Sinai Hospital

summary: Medical cannabis has been increasingly prescribed for a range of conditions including epilepsy, chronic neuropathic pain, and chemotherapy-induced nausea and vomiting. The benefits and possible adverse events of medical-grade cannabis products vary between patients, suggesting that genetics may play a role in the pharmacokinetics of the cannabinoids, yet regulatory restrictions have led to limited clinical studies. This study is aimed at identifying a genetic signature that is predictive of the pharmacokinetics of tetrahydrocannabinol (THC), the principal intoxicating chemical compound derived from cannabis.  We have identified 55 variants among 38 genes that were overrepresented in either the Low-THC or High-THC groups.

overall_design: Saliva samples were obtained from 28 healthy subjects who consented to the genetics sub-study after participating in a controlled pharmacokinetic study on a THC extract capsule. Subjects were grouped as ‘Low-THC’ or ‘High-THC’ based on the profile of their plasma THC concentration over various timepoints following intake of 5 mg or 10 mg THC under fed and fasting conditions. DNA, extracted from their saliva, was then analyzed using Illumina's Infinium DrugDev microarray.

first: Andrew

last: Wong

first: Shannon

last: O'Hearn

first: Angelo

last: Fefekos


title: Expression data from adult wild-type and transgenic zebrafish revealed Oligo-Fucoidan Prevents Radiation induced fibrosis and secondary tumors in zebrafish

city: Zhunan Town

first: Chiou-Hwa

last: Yuh

state: Miaoli County

address: #35 Keyan Road

country: Taiwan

institute: National Health Research Institutes

summary:
Radiotherapy (RT) often causes unwanted side effects such as radiation-induced fibrosis (RIF) and second malignancies (SM). Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has many biological effects including anti-inflammation and anti-tumor. In the present study, we investigated the radioprotective effect of Oligo-Fucoidan (OF) using zebrafish animal model.
Using an oligo-fucoidan oral gavage in wild-type and adult transgenic zebrafish, we then used microarrays to detail the global programme of gene expression after fucoidan treatment and identified distinct classes of up- and down-regulated genes during this process.

overall_design: Forty wild-type (WT) 5-month-old adult fish were divided into two group treated with γ-irradiation (R) and one group were fed with oligo-fucoidan for one month followed by 40 Gy γ-irradiation exposure (OF+R), two batches of WT fish were performed, the first is treated with 40 Gy and the second is treated with 10Gy. Three-month-old female HBx,Src and HBx,Src,p53–/+ transgenic zebrafish were diet-induced obesity (DIO) for two months by approximately 6.9 mg and 83 mg cysts/fish/day respectively, and then 5-month-old adult fish were divided into two group treated with γ-irradiation (R), and one group of fish oral feding with oligo-fucoidan for one month followed by γ-irradiation exposure (OF+R). The γ-irradiation exposure for HBx,Src and HBx,Src,p53–/+ were 40Gy and 10Gy respectively. OF (300 mg/kg) was administrated by oral gavage for one month starting at 4 months of age for one month. The fish were sacrificed and the hepatic tissue was immediately frozen in liquid nitrogen and stored at −80 °C for later RNA extraction and hybridization on Affymetrix microarrays.

first: Szu-Yuan

last: Wu

first: Wan-Yu

last: Yang

first: Chun-Chia

last: Cheng

first: Ming-Chen

last: Hsiao

first: Kuan-Hao

last: Lin


title: Identification and elimination of the platelet-derived transcripts contamination in circulating tumor cell single-cell RNA-seq study

city: Shanghai

first: Sibo

last: Zhu

state: China

address: songhulu 2005

institute: Fudan University

summary: Single-cell RNA sequencing (scRNA-seq) holds great potential to revolutionize our understanding of cancer biology by discovering biomarkers and biological functions from circulating tumor cells (CTCs). However, the contamination from blood cells is one of the main challenges in CTC studies. Through investigation of recently published studies, we found that several previously reported CTC “biomarkers” were apparently contaminated by blood-derived transcripts such as HBB, HBA1/2, ITGA2B, and LCP1. Through re-analysis of several published CTC and cell line datasets, we revealed that CTC “biomarkers” were not only highly expressed in blood, but showed a high correlation between CTC and blood. We sequenced transcriptome of seven blood components from a healthy donor, finding that the platelet exhibited the highest probability to confound CTC data in terms of expression profiling and correlation analysis. Using CTC spike-in and recovery assay and we showed that the platelet induced artifacts could be reproduced in vitro. Surprisingly, the reproduced artifacts were also highly expressed in the platelet data but not in the other six blood components. We performed SNP calling on recovered CTC simulators, finding cross-contamination of exogenous transcripts. Furthermore, we applied a multiple linear regression model to estimate the platelet confounding index, showing that previously published CTC datasets were confounded with 13%~26% of platelet fractions and 7.9%~17% platelet cloaking judged by fluorescence flow cytometry. We devised a red blood cell lysis based protocol that better removed platelet compared to several platelet inhibitory reagents and kept transcriptomic profile undisturbed in CTC scRNA-seq. This work not only demonstrated the potential blood cell confounding factors in CTC studies, validated them in the past published datasets, and reproduced the artifacts in CTC simulation assay, but also devised an easily applicable method to remove the platelet cloaking, guaranteeing the reliability of CTC scRNA-seq research.

overall_design: CTC mimics were spiked into blood, treated by platelet removal reagents and recovered for scRNA-seq

first: Leming

last: Shi


title: Pharmacologic IRE1/XBP1s Activation Confers Targeted ER Proteostasis Reprogramming

status: Public on Apr 17 2020

city: La Jolla

first: R. Luke

last: Wiseman

address: 10550 North Torrey Pines Road

department: Molecular Medicine

institute: The Scripps Research Institute

summary: Activation of the IRE1/XBP1s signaling arm of the unfolded protein response (UPR) is a promising strategy to correct defects in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. However, no pharmacologic activators of this pathway identified to date are suitable for ER proteostasis remodeling through selective activation of IRE1/XBP1s signaling. Here, we use high-throughput screening to identify non-toxic compounds that induce ER proteostasis remodeling through IRE1/XBP1s activation. We employ transcriptional profiling to stringently confirm that our prioritized compounds selectively activate IRE1/XBP1s signaling without activating other cellular stress-responsive signaling pathways. Furthermore, we demonstrate that our compounds improve ER proteostasis of destabilized variants of amyloid precursor protein (APP) through an IRE1-dependent mechanism and reduce APP-associated mitochondrial toxicity in cellular models. These results establish highly-selective IRE1 activating compounds that can be widely employed to define the functional importance of IRE1/XBP1s activity for ER proteostasis regulation in the context of health and disease.

overall_design: Comparisons of the transcriptomes of HEK293T cells treated with DMSO or identified IRE1/XBP1s activating compounds IXA1, IXA4, IXA6, or global UPR activator thapsigargin (Tg)

first: Julia

middle: M

last: Grandjean

first: Evan

middle: T

last: Powers

first: R


title: Targeted determination of pri-miRNA processing

city: Aarhus

first: Ulf Andersson

middle: Vang

last: Ørom

address: C F Mollers Alle 3

country: Denmark

institute: Aarhus University

summary: Targeted sequencing at Microprocessor cleavage sites in pri-miRNAs to determine processing efficiency of several pri-miRNAs in vivo in one experiment

overall_design: Pri-miRNAs are enriched with targeted probes from Illumina RNA sequencing libraries and sequenced with low depth (high coverage) to focus sequencing reads on pri-miRNA processing

first: Ulf

middle: A

last: Vang Ørom


title: maternal microbiome modulation of fetal brain development and sensory behavior

city: Inglewood

first: Helen

last: Vuong

address: 10953 Penney Ave

institute: UCLA

summary: “Dysbiosis" of the maternal gut microbiome, in response to environmental challenges such as infection, altered diet and stress during pregnancy, has been increasingly associated with abnormalities in offspring brain function and behavior. However, whether the maternal gut microbiome regulates neurodevelopment in the absence of environmental challenge remains unclear. In addition, whether the maternal microbiome exerts such influences during critical periods of embryonic brain development is poorly understood. Here we investigate how depletion, and selective reconstitution, of the maternal gut microbiome influences fetal neurodevelopment in mice. Embryos from antibiotic-treated and germ-free dams exhibit widespread transcriptomic alterations in the fetal brain relative to conventionally-colonized controls, with reduced expression of several genes involved in axonogenesis. In addition, embryos from microbiome-depleted mothers exhibit deficient thalamocortical axons and impaired thalamic axon outgrowth in response to cell-extrinsic guidance cues and growth factors. Consistent with the importance of fetal thalamocortical axonogenesis for shaping neural circuits for sensory processing, restricted depletion of the maternal microbiome from pre-conception through mid-gestation yields offspring that exhibit tactile hyposensitivity in select sensorimotor behavioral tasks. Gnotobiotic colonization of antibiotic-treated dams with a limited consortium of spore-forming bacteria indigenous to the gut microbiome prevents abnormalities in fetal brain gene expression, fetal thalamocortical axonogenesis and adult tactile sensory behavior associated with maternal microbiome depletion. Metabolomic profiling reveals that the maternal microbiota regulates levels of numerous small molecules in the maternal serum as well as the brains of fetal offspring. Select microbiota-dependent metabolites – trimethylamine N-oxide, 5-aminovalerate, imidazole propionate, and hippurate – sufficiently promote axon outgrowth from fetal thalamic explants. Moreover, maternal supplementation with the metabolites during early gestation abrogates deficiencies in fetal thalamocortical axons and prevents abnormalities in tactile sensory behavior in offspring from microbiome-depleted dams. Altogether, these findings reveal that the maternal gut microbiome promotes fetal thalamocortical axonogenesis and select tactile sensory behaviors in mice, likely by signaling of microbially modulated metabolites to neurons in the developing brain.

overall_design: There are a total of 9 samples of embryonic day 14.5 fetal brains from three groups. The specific pathogen free (SPF) group is the control (N=3). The antibiotic-treated (ABX) group is the perturbation group (N=3). The Spore-former (Sp) colonization group is the restoration group (N=3).

first: Elaine

middle: Y


title: Formation of colorectal liver metastases induces musculoskeletal and metabolic abnormalities

city: Indianapolis

first: Andrea

last: Bonetto

state: IN

address: 980 W. Walnut Street, R3-C522

institute: Indiana University School of Medicine

summary: Advanced colorectal cancer (CRC), a leading cause of death worldwide, is often accompanied by the development of liver metastases (LM), as well as skeletal muscle (SkM) wasting, i.e. cachexia. Despite affecting the majority of CRC patients, cachexia remains understudied and uncured. Moreover, only a single model of LM associated with CRC has been developed for the study of cachexia. Here we examined differential gene expression of skeletal muscles deriving from subcutaneous and metastatic C26 tumor hosts. Tumor hosts displaying LM experienced exacerbated muscle wasting compared to tumor hosts without LM.

overall_design: 8-week old CD2F1 male mice were subcutaneously (C26) or intrasplenically injected with C26 tumor cells (mC26) to mimic hepatic dissemination of cancer cells, while sham-operated animals received saline. All animals were euthanized weeks following experimental intervention. mRNA was isolated from quadriceps muscle. For RNA seq.

first: Joshua

middle: R

last: Huot

first: Ashok

last: Narasimhan

first: Teresa

last: Zimmers


title: HRI activates ATF4 to promote BCL11A transcription and fetal hemoglobin silencing [ChIP-seq]

city: Guangzhou

first: Peng

last: Huang

state: Guangdong

address: Xinzao, Panyu District

department: GMU-GIBH Joint School of Life Sciences

institute: Guangzhou Medical University

summary: Through a CRISPR-Cas9 guided loss-of-function screen in human erythroid cells we identified transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin repressor. ATF4 binds to a BCL11A enhancer to augment promoter contacts, stimulates BCL11A transcription to repress γ-globin expression. Notably, mice deficient for HRI displayed normal Bcl11a levels, suggesting species selective regulation that we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. This illustrates potential limits of commonly used murine models of globin gene regulation.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: ATF4 ChIP-seq of HUDEP2, Human primary erythroblast and G1E-ER4 cell line


title: HRI activates ATF4 to promote BCL11A transcription and fetal hemoglobin silencing [Capture-C]

overall_design: We performed Capture-C using the BCL11A promoter as anchor to examine whether the +55 enhancer is in physical proximity with the BCL11A promoter.


title: HRI activates ATF4 to promote BCL11A transcription and fetal hemoglobin silencing

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

overall_design: Refer to individual Series


title: Vascular smooth muscle selective deletion of Srf

city: Rochester

first: Joseph

last: Miano

state: NY

address: 601 Elmwood Avenue

institute: University of Rochester

summary: Previous efforts to conditionally delete the Srf transcription factor in smooth muscle lineages were met with early demise of the mouse due to a gastrointestinal (GI) obstruction phenotype. We have developed a new, more VSMC selective Cre recombinase mouse (Itga8-CreERT2), that circumvents the GI phenotype thus affording the opportunity to evaluate, in an unambiguous manner, VSMC phenotypes where Srf is reduced.  The mice live for at least 6 months post-tamoxifen and here we report the first RNA-seq experiments in mouse aortic SMC where Srf is reduced.

overall_design: Mice homozygous or heterozygous for a floxed Srf allele and hemizygous for the Itga8-CreERT2 driver were treated with 5 daily doses of 33mg/kg Tamoxifen (i.p.).  Aortae from the experimental homozygous cKO (n=3) or the control heterozygous cKO (n=3) were harvested 10 days following the last tamoxifen injection.  Only 2 homozygous cKO replicates are reported here due to an outlier effect (or error in sampling) in one of the experimental samples.  Thus, the data herein reflect only 2 homoygous cKOs and 3 heterozygous cKO controls.

first: Qing

last: Lyu

first: Wei

last: Zhang

first: Orazio

last: Slivano

first: Christine

middle: K

last: Christie

first: Xiaochun

last: Long

first: Lin

last: Gan


title: Transcriptional analysis of transcription factor c-Maf as a molecular switch that controls immune suppression by programming macrophages in lung cancer

city: Louisville

first: Eric

middle: Christian

last: Rouchka

state: Kentucky

address: 522 East Gray Street

department: Biochemistry and Molecular Genetics

institute: University of Louisville

summary: To investigate c-Maf-controlled gene expression, M2-like bone marrow-derived macrophages (BMM) were transfected with c-Maf or control siRNA. Knockdown of c-Maf significantly decreased c-Maf protein and mRNA expression levels. In addition, IL-10 and arginase mRNA levels were significantly decreased while IL-12 was increased. In contrast, ectopic expression of c-Maf in the M1-like BMM significantly upregulated IL-10 and arginase mRNA expression levels while downregulated IL-12 expression level, suggesting an M2-like phenotype. These data indicate that c-Maf may be a critical controller in regulating M2-related gene expression.  To further determine which part of the M2 macrophage transcriptomic profile is controlled by c-Maf, we performed microarray analysis using polarized M2-like BMM from WT or c-Maf KO fetal liver chimeric mice. Notably, many M2 genes were differentially regulated by c-Maf. Further real-time PCR (qPCR) analysis confirmed that the mRNA expression levels of IL-12, IL-1β, IL-6, arginase, IL-10, VEGF, TGF-β, IRF-4, and CCR2 were significantly altered in c-Maf-deficient M2 BMM. Since M2-like macrophages have a potent immunosuppressive function on T cell activation, we next determined whether deficiency of c-Maf in M2 BMM would reverse such effect. M2 BMM from WT mice exhibited potent immunosuppressive activity as IFN-γ production from antigen (Ag)-specific CD4 and CD8 T cells was significantly diminished. In contrast, c-Maf deficiency in M2 BMM significantly increased IFN-γ production by CD4 and CD8 T cells compared to WT M2 BMM. These data suggest that c-Maf not only controls many M2-related gene expression but also is critical in regulating M2-like macrophage-mediated T cell immunosuppression.

overall_design: Gene expression of polarized M2-like BMM from WT or c-Maf KO fetal liver chimeric mice was compared using 3 replicates in each group.

first: Min

last: Liu

first: Zan

last: Tong

first: Chuanlin

last: Ding

first: Fengling

last: Luo

first: Shouzhen

last: Wu

first: Sabrin

last: Albeituni

first: Liqing

last: He

first: Xiaoling

last: Hu

first: David

last: Tieri

middle: C

first: Michito

last: Hamada

first: Satoru

last: Takahashi

first: Andrew

last: Gibb

first: Goetz

last: Kloecker

first: Hang-Ge

first: Michael

last: Bousamra II

first: Bradford

middle: G

last: Hill

first: Xiang

first: Jun

last: Yan


title: Transcriptomic analysis of human CD4+, CD8+ and DP T cells

city: Nantes

first: Delphine

last: Fradin

address: 8 quai Moncousu

institute: CRCI2NA, Inserm UMR1307, CNRS UMR6075

summary: Analysis of the transcriptome of human CD4+, CD8+ and DP T cells. The objective is to define the origin of the DP in melanoma

overall_design: Total RNA were isolated from T cells using Trizol and Rneasy micro kit (Qiagen) before or after stimulation during 6 hours by an anti-CD3

first: Nadine

last: Gervois


title: Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

city: Vilnius

first: Giedrius

last: Vilkaitis

address: Sauletekio av. 7

department: Department of Biological DNA Modification, Institute of Biotechnology

country: Lithuania

institute: Vilnius University

type: Methylation profiling by high throughput sequencing

summary: 5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single nucleotide resolution, named hmTOP-seq, which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct tethering chemistries suggested a putative model for the targeted non-homologous polymerase priming which permits theoretical evaluation of suitable tethers at the design stage. The robustness and resolution of hmTOP-seq was validated on a small model genome and mouse embryonic stem cells, which allowed construction of single nucleotide 5hmCG maps that reflect subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq expands the toolbox of valuable techniques for cost-effective identification of epigenetic changes and new disease biomarkers.

overall_design: 5-hydroxymethylcytosine analysis in mouse embryonic stemm cells. DNA modifications were analysed using various input DNA amounts (5, 50 and 500 ng) - two techinal replicates for each input. Control libraries were prepared using the same pipeline without the BGT labeling step.

first: Povilas

last: Gibas

first: Milda

last: Narmontė

first: Zdislav

last: Staševskij

first: Juozas

last: Gordevičius

first: Saulius

last: Klimašauskas

first: Edita

last: Kriukienė


title: Characterization of glyceollins as novel aryl hydrocarbon receptor ligands and their role in cell migration

city: Rennes

first: Frédéric

last: Chalmel

state: France

address: 9 avenue du Pr. Léon Bernard

department: Physiology and physiopathology of the urogenital tract

institute: Inserm U1085-Irset

summary:
This study is to investigate the effects of glycellin I (GI) and glyceollin II (GII), a aryl hydorcarbon (AhR) ligand  in genome. Cells were treated during 24h with glyceollins alone or in combination with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Total RNA was prepared and controlled the quality. Total RNA was prepared and controlled the quality. Then, a 3’Digital Gene Expression (DGE) library was constructed using a modified SCRB-Seq protocol [55] with barcoded poly-dT RT primers and a hybrid Nextera/TruSeq sequencing strategy. Data quality control and data preprocessing were performed by the GenoBiRD Platform in Nantes, France. The results showed that there are numerous gene regulated by TCDD are also modified by GI or GII. GI et GII can have antagonistic or synergistic effects together with TCDD. This is the first study elucidating the effect of GI and GII in three cell types.
Method: Cells were treated during 24h with glyceollins alone or in combination with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Total RNA was prepared and controlled the quality. Total RNA was prepared and controlled the quality. Then, a high-Throughput mRNASeq library for 3’Digital Gene Expression (DGE) was constructed using a modified SCRB-Seq protocol [55] with barcoded poly-dT RT primers and a hybrid Nextera/TruSeq sequencing strategy. Data quality control and primary analysis were performed by the GenoBiRD Platform in Nantes, France.

overall_design: Examination the effect of glycellins in three cell types: MDA-MB-231, HepG2 and MCF-7

first: Bertrand

last: Evrard

first: Aurelie

last: Lardenois

first: Thu

middle: H

last: Pham

first: Farzad

last: Pakdel


title: An Exome-Wide Association Study Identifies New Susceptibility Loci for the Risk of Nicotine Dependence in European-American Populations

city: Hangzhou

first: Yi

last: Xu

state: Zhejiang

address: No. 17, Dazhi Road, old Zhejiang, Shangcheng District

department: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases

institute: The First Affiliated Hospital, Zhejiang University School of Medicine

type: Genome variation profiling by genome tiling array

summary: Cigarette smoking is one of the largest causes of preventable death worldwide. Smoking behaviors, including age at smoking initiation (ASI), smoking dependence (SD), and smoking cessation (SC), are all complex phenotypes determined by both genetic and environmental factors as well as their interactions. To identify susceptibility loci for each smoking phenotype, numerous studies have been conducted, with approaches including genome-wide linkage scans, candidate gene-based association analysis, and genome-wide association study (GWAS). Therefore, we conducted an exome-wide association study to identify new susceptibility loci for the risk of nicotine dependence in European-American populations.

overall_design: To reveal the molecular mechanism underling each smoking phenotype, we used high-throughput approaches such as exome-based association study to identify genetic variants that contribute to nicotine dependence and other smoking-related phenotypes. First, we evaluated each common variant individually with a univariate statistic; i.e., logistic and linear regression models. Second, rare variants were grouped by genomic regions and analysed using burden tests, i.e., the Weighted Sum Statistic (WSS). Third, we tested for combined effects of rare and common variants with a unified statistical test that allows both types of variants to contribute fully to the overall test statistic.

first: Ming

middle: D

last: Li


title: Tumor suppressive long non-coding RNA AFDN-DT inhibits gastric cancer invasion via transcriptional regulation

first: Ping

address: Songjiang Hospital Affiliated to Shanghai Jiaotong University School of Medicine

summary: We identifed genes differentially expression in HGC27 cells withor without AFDN-DT overexpression

overall_design: RNA-seq in HGC27 cells transduced with AFDN-DT or vector

first: Yuexing

last: Lai


title: Effects of newly developed pulp-capping materials on early response of stem cells from exfoliated deciduous teeth: cell differentiation, migration, and proliferation based on next gene sequencing analysis

city: Dongdaemun-gu

first: Hosun

last: Lee

state: Seoul

address: 26, Kyungheedae-ro

country: South Korea

institute: School of Dentistry, Kyung Hee University

summary:
Aim : This study aimed to identify early effect of newly developed pulp capping materials on differentiation, migration, and proliferation of stem cells from exfoliated deciduous teeth (SHED) using next gen sequencing analysis.
Methodology : SHED were cultured and exposed to MTA, Biodentine (BD), and Theracal (TC) for 24 and 72 h. WST assay was performed to evaluate cell viability. After total RNA was isolated, QuantSeq 3’ mRNA-Sequencing was performed to identify differential gene expression in MTA-, BD-, TC-exposed SHED. Bioinformatics analysis was also performed.
Results : WST assay showed no significant difference in SHED viability among materials (p>0.05). In gene set enrichment analysis, hallmark gene set enrichment differed. Gene sets associated with epithelial mesenchymal transition and hedgehog signaling were enriched in all materials. In BD, gene sets with inflammatory response and Wnt/β-catenin signaling were enriched, whereas enrichment of gene sets with TNF-α signaling and apoptosis were identified in TC.
Conclusions : Despite cell viability of SHED exhibited similar results among MTA, BD, and TC, patterns of gene networks differed, suggesting that diverse gene functional difference may be involved in these materials.

first: Ho

middle: S

first: Jae-Hwan

last: Kim

first: Yong

last: Chae

first: Hyo-Seol

first: Sung

last: Choi

first: Ok

last: Nam


title: Dynamics of the transcriptome response to heat in the moss, Physcomitrella Patens

city: Aubière

first: Said

last: Mouzeyar

address: 1 Impasse Amélie Murat

department: Biology

institute: Université Clermont Auvergne

summary: The moss Physcomitrella patens was employed to study abiotic stress responses in order to understand the effects of mild (30°C) and strong (37°C) continuous heat stresses from 1h to 24h at a transcriptomic level.

overall_design: Identification of differentially expressed genes in heat treated samples (30 °C or 37°C) compared to controls (24 °C)

first: Osama

last: Elzanati

first: Mohamed

middle: F

last: Bouzidi

first: Jane

last: Roche


title: Transcriptomic profiling of microglia and astrocytes throughout aging

city: shenzhen

first: jie

last: pan

address: Lian Hua Lu 1120, FuTian District  Guangdong

institute: Shenzhen	Peking	University	– The	Hong	Kong	University	of	Science	and	Technology	Medical	Center

summary: Purpose:The goal of this study are to understand the role of miceoglia and astrocyte in both physiological and pathological conditions. Methods: Microglia and astrocyte were isolated from 5 time points WT/APP-PS1 mice in triplicate. mRNA were generated by SMART seq2 using Illumina Novaseq platform. Conclusions:  Using DESeq2 to sequence polyA-selected mRNAs from microglia and astrocytes isolated from whole brain. We mapped >85% of reads for all of our sample. We found good reproducibility between the replicates

overall_design: We performed RNA-seq in microglia and astrocytes freshly isolated from Wild-type(WT)/APP-PS1(AD) mice brains across the lifespan to analyze the longitudinal genes expression profile.

first: J

last: Pan

last: Wan


title: Aspergillus flavus gene expression profile under different conditions of ustiloxin production

city: Tsukuba

first: Myco

last: Umemura

state: Ibaraki

address: Higashi 1-1-1

department: Bioproduction Research Institute

institute: National Institute of Advanced Industrial Science and Technology (AIST)

summary: Transcriptome data of A. flavus was assembled under four different conditions (two strains and two media) for omics data analyses of genes related to ustiloxin production. The used two strains were the over-expressed strain (ustR-oe) of the transcription factor in ustiloxin gene cluster, ustR, and the pyrG marker complemented strain (control). The two media were V8 juice liquid and potato dextrose broth (PDB) media. The ustR-oe strain produced more ustiloxin B than the control strain, and the production is much higher in V8 than in PDB. Therefore, four different state of transcriptome could be obtained under these four conditions. The obtained transcriptome data was combined with the GEO data acc. no. GSE15435 and uesd for omics data analyses.

overall_design: 1E5 spores of ustR-oe and control strains were inoculated to 30 mL of V8 and PDB media in 100-mL flask with buffle and cultured for 2 days at 30C, 165 rpm. The mRNA was extracted and labeled according to the manufacturer's protocol. Two biological replicates were taken for each sample. The raw data was normalized (Z-scored) using Agilent GeneSpring software.

first: Maiko


title: Hedgehog signaling pathway regulates gene expression profiling of epididymal principal cells through the primary cilium

city: Quebec

first: Ezequiel

middle: L

last: Calvo

state: Quebec

address: 2705 Boul. Laurier

department: Molecular Endocrinilogy

institute: CRCHUL

summary: Background. Primary cilia (PC) are solitary antennae present at the cell surface. These non-motile cilia play an important role in organ development and tissue homeostasis through the transduction of the Hedgehog (Hh) signaling pathway. We recently revealed the presence of PC in the epithelium of the developing epididymis, an organ of the male reproductive system whose dysfunction triggers male infertility. Acknowledging that systemic blockade of the Hh pathway trigger epididymal dysfunctions in vivo, our main goals were 1) to portray the epididymal Hh environment, 2) to determine the direct responsiveness of epididymal epithelial cells to Hh, and 3) to define the contribution of PC to the transduction of this pathway.  Results. The Hh ligands Indian and Sonic hedgehog (Ihh and Shh) were respectively located in principal and clear cells of the mouse epididymis by immunofluorescent staining. The propensity of epididymal principal cells to respond to Hh signaling was assessed on immortalized epididymal DC2 cells by western-blot, confocal imaging and 3D-reconstruction. Our results indicate that epididymal principal cells secrete Ihh and expose PC that co-localize with the conventional acetylated tubulin/Arl13b ciliary markers, as well as with GLI3 Hh signaling factor. Gene expression microarray profiling indicated that the expression of 43 and 248 genes was respectively and significantly modified following pharmacological treatment of DC2 cells with the Hh agonist SAG (250 nM) or the Hh antagonist cyclopamine (20 µM) compared with the control. Among Hh target genes identified, 6.7 % presented perfect matches for GLI-transcription factor consensus sequences, and the majority belonged to interferon-dependent immune response and lipocalin 2 pathways. Finally, the contribution of epididymal PC to the transduction of canonical Hh pathway was validated by ciliobrevinD treatment, which induced a significant decrease of PC length and the expressional reduction of Hh signalling targets. Conclusions. All together our data indicate that PC from epithelial principal cells regulate gene expression profile through a possible autocrine Hh signaling. This provides new hypotheses regarding the potential contribution of PC and Hh signaling in intercellular cross-talk and immunological regulation of the epididymis.

overall_design: Immortalized DC2 cells were treated for 24 hrs with 250 nM of Smoothened agonist (SAG, n=3, SAG 1, SAG 2 and SAG 3), 20 uM of Cyclopamine (Cyclo, n=3, Cyclo 1, Cyclo 2 and Cyclo 3) and control medium condition (Ctrl, n=3, Ctrl 1, Ctrl 2 and Ctrl 3). Total RNA extraction was performed with the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol, and potential genomic DNA contamination was eliminated with the RNase-free DNase set (Qiagen). Total RNA was eluted in RNAse-free water and its concentration was quantified with a NanoDrop 1000 microvolume spectrophotometer (Thermo Scientific). Ribonucleic acid quality was assessed with the Eukaryote Total RNA Pico Series II kit (Agilent) on a 2100 Bioanalyzer by the transcriptomic core facility of the CHUQ. The minimal accepted number for RNA integrity was 7. DNA microarray analyses were carried out with Affymetrix Mouse Clariom S arrays (Thermofisher) according to the Affymetrix standard protocol. Briefly, total RNA (100 ng per sample) was labeled using the GeneChip® WT Plus Reagent Kit protocol and hybridized to the arrays as described by the manufacturer (Affymetrix, Thermofisher). The cRNA hybridization cocktail was incubated overnight at 45°C while rotating in a hybridization oven. After 16 h of hybridization, the cocktail was removed and the arrays were washed and stained in an Affymetrix GeneChip fluidics station 450, according to the Affymetrix-recommended protocol. The arrays were scanned using the Affymetrix GCS 3000 7G and the Gene-Chip Command Console Software (AGCC) (Affymetrix, Thermofisher) to produce the probe cell intensity data (CEL). The image data were analyzed by using the Affymetrix Expression Console Software to perform the quality control, the background subtraction and the normalization of probe set intensities with the method of Robust Multiarray Analysis (RMA). Microarray analyses were performed by the CHU de Québec Research Center (CHUL) Gene Expression Platform, Quebec, Canada.

first: Clémence

last: Belleanée

first: Laura

last: Girardet

first: Agathe

last: Bernet

first: Denis

last: Soule

first: Charles

middle: J

last: Beauparlant

first: Arnaud

last: Droit

first: Daniel

last: Cyr


title: Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency

city: Duesseldorf

first: Birgit

last: Knebel

address: Auf'm Hennekamp 65

department: Cinical Biochemistry and Pathobiochemistry

country: Germany

institute: German Diabetes Center

summary:
Rationale: Myocardial fibrosis manifests progressively in several forms of cardiomyopathies. Although fibrosis depicts a reparative mechanism, maladaptation of the heart due to excessive production of extracellular matrix accelerates cardiac dysfunction.
Objective: The anthraquinone Rhein, a compound from rhubarb, was examined for its anti-fibrotic potency to mitigate cardiac fibroblast-to-myofibroblast transition (FMT).
Methods and Results: Primary human ventricular cardiac fibroblasts were subjected to hypoxia and characterized with proteomics, transcriptomics and cell functional techniques. Knowledge based analyses of the omics data revealed a modulation of fibrosis-associated pathways and cell cycle due to Rhein administration, whereas p53 and p21 were identified as upstream regulators involved in the manifestation of cardiac fibroblast phenotypes. Mechanistically, Rhein-mediated cellular effects were linked to the histone deacetylase (HDAC)-dependent acetylation status of p53 a posttranslational modification that acts protein stabilizing. Direct enzymatic testing revealed an inhibitory potency of Rhein for HDAC classes I/II. Functionally, Rhein inhibited collagen contraction in response to protein abundance of SMAD7, endogenous inhibitor of TGFβ1 action, thus demonstrating its anti-fibrotic property in cardiac remodeling.
Conclusion: In conclusion, this study identifies Rhein as a novel potent HDAC inhibitor and provides evidence that Rhein may contribute to the treatment of cardiac fibrosis as anti-fibrotic agent. As readily available drug with approved safety, Rhein constitutes a promising potential therapeutic approach in the supplemental and protective intervention of cardiac fibrosis.

overall_design: HCF-v culture and chronic hypoxia treatment  Primary HCF-v of 5 different donors were purchased (Lonza, Basel, Switzerland; Promocell, Heidelberg, Germany) and cultured in FGMTM-3 medium (Lonza, Basel, Switzerland) (37°C, 5% CO2). Prior treatment HCF-v were seeded in 145mm dishes. After 16h, cells were subjected to the hypoxic workstation (Xvivo System (Biospherix, Parish, NY, USA) set to 0.5% O2, 5% CO2, 94.5% N2 and 37°C) with N2-pre-gassed DMEM (10% FCS, 1% glutamine, 1% antibiotic/antimycotic). After 60h, sub-confluent cells were washed (1x PBS) and serum-starved nitrogenated serum-free media (DMEM, 1% glutamine, 1% antibiotic/antimycotic) for 36h. Normoxic control cells were treated in parallel in a second incubator of the hypoxic workstation (21% O2, 74% N2, 5% CO2 and 37°C). For Rhein treatment, 35µM Rhein was added during normoxic or hypoxic treatment for a total incubation time of 96h.

first: David

last: Barbosa

first: Jorg

last: Kotzka


title: Transcriptome analysis of CA1 rat hippocampus 1 week after kainate seizure at P7

city: Aurora

first: Tim

last: Benke

state: CO

department: Pediatrics

institute: University of Colorado School of Medicine

summary: Seizures that occur during early development are associated with adverse neurodevelopmental outcomes.  Causation and mechanisms are currently under investigation.  Induction of an early life seizure by kainic acid (KA) in immature rats on post-natal day (P) 7 results in behavioral changes in the adult rat that reflect social and intellectual deficits without overt cellular damage.  Our previous work also demonstrated increased expression of CA1 hippocampal long-term potentiation (LTP) and reduced desensitization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic glutamate receptors (AMPA-R) one week following a kainic acid induced seizure (KA-ELS).  Here we used RNA sequencing (RNAseq) of mRNA from dorsal hippocampal CA1 to probe changes in mRNA levels one week following KA-ELS as a means to investigate the mechanisms for these functional changes.  Ingenuity pathway analysis (IPA) confirmed our previous results by predicting an up-regulation of the synaptic LTP pathway.  Differential gene expression results revealed significant differences in 7 gene isoforms.  Additional assessments included AMPA-R splice variants and adenosine deaminase acting on RNA 2 (ADAR2) editing sites as a means to determine the mechanism for reduced AMPA-R desensitization.  Splice variant analysis demonstrated that KA-ELS result in a small, but significant decrease in the "flop" isoform of Gria3, and editing site analysis revealed significant changes in the editing of a kainate receptor subunit, Grik2, and a serotonin receptor, Htr2c.  While these specific changes may not account for altered AMPA-R desensitization, the differences indicate that KA-ELS alters gene expression in the hippocampal CA1 one week after the insult.

overall_design: 8 ka-els, 8 control, no replicates

first: Heather

last: O'Leary

last: Saba


title: Reliability of a Single-region Tumor Sample for Evaluating the Immune Tumor Microenvironment of Hepatocellular Carcinoma Tumors

city: Taipei

first: Chia-Lang

last: Hsu

address: No. 7, Zhongshan S. Rd.

department: Department of Medical Research

institute: National Taiwan University Hospital

summary: Heterogeneity in immune microenvironment within a tumor may pose a barrier to evaluate immune tumor microenvironment by using a single-region tumor sample, such as a single tumor biopsy. Our study using both immunohistochemical and transcriptomic analyses showed that a single-region sample is reliable for evaluation of immune tumor microenvironment in 60-70% of HCC patients.

overall_design: Multiregion sampling was performed from newly resected tumors. The immune tumor microenvironment was evaluated by RNA-seq in selected samples.

first: Y

last: Shen

first: C


title: The Arabidopsis YDL1 suppresses singlet oxygen-induced gene expression changes

city: Planegg

first: Tatjana

last: Kleine

address: Großhaderner Straße 2

department: Biologie I, Botanik

institute: Ludwig-Maximilians-Universität München

summary: Singlet oxygen (1O2), the main ROS produced in chloroplasts, has long been recognized as a cytotoxin that inhibits photosynthesis and damages the cell. However, more recent findings have proposed 1O2 as a highly versatile signal that induces various stress responses. A signaling role for 1O2 was first documented in the conditional fluorescent (flu) mutant of Arabidopsis. Release of 1O2 in flu mutant induces bleach/die of young seedlings and growth inhibition of mature plants. However, all these drastic phenotypic changes can be suppressed by mutation of EXCUTER1 (EX1) gene. The Arabidopsis YDL1 was identified during a screen of revertant mutant from EMS mutagenized flu/ex1. Loss of function of SAFE1 leads to highly expression of 1O2-indluced genes in flu/ex1 mutant, mimicking the flu mutant. We found that YDL1 localized in the stroma of chloroplast, and suppressed 1O2-mediated stress responses via inhibiting 1O2-induced damage on the grana margins.

overall_design: Examination of differential gene expression in the Arabidopsis flu/ex1 and flu/ex1/ydl1 mutant before and after the release of 1O2

first: Wang

last: Liangsheng


title: Cis-regulatory analysis of Onecut1 in fate-restricted retinal progenitor cells

city: New York

first: Sruti

last: Patoori

address: 85 Saint Nicholas Terrace

institute: CUNY The City College of New York

summary: Background: The vertebrate retina consists of six major classes of neuronal cells. During development, these cells are generated from a pool of multipotent retinal progenitor cells (RPCs) that express the gene Vsx2. Fate-restricted RPCs have recently been identified, with limited mitotic potential and cell fate possibilities compared to multipotent RPCs. One population of fate-restricted RPCs, marked by activity of the regulatory element ThrbCRM1, gives rise to both cone photoreceptors and horizontal cells. These cells do not express Vsx2, but co-express the transcription factors (TFs) Onecut1 and Otx2, which bind to ThrbCRM1. The components of the gene regulatory networks that control the transition from multipotent to fate-restricted gene expression are not known.  This work aims to identify and evaluate cis-regulatory elements proximal to Onecut1 to identify the gene regulatory networks involved in RPC fate-restriction. Method: We identified regulatory elements through ATAC-seq and conservation, followed by reporter assays to screen for activity based on temporal and spatial criteria. The regulatory elements of interest were subject to deletion and mutation analysis to identify functional sequences and evaluated by quantitative flow cytometry assays. Finally, we combined the enhancer::reporter assays with candidate TF overexpression to evaluate the relationship between the TFs, the enhancers, and early vertebrate retinal development. Statistical tests included ANOVA, Kruskal-Wallis, or unpaired t-tests.Results: Two regulatory elements, ECR9 and ECR65, were identified to be active in ThrbCRM1(+) restricted RPCs. Candidate bHLH binding sites were identified as critical sequences in both elements. Overexpression of candidate bHLH TFs revealed specific enhancer-bHLH interactions. Nhlh1 overexpression expanded ECR65 activity into the Vsx2(+) RPC population, and overexpression of NeuroD1/NeuroG2/NeuroD4 had a similar effect on ECR9. Furthermore, bHLHs that were able to activate ectopic ECR9 reporter were able to induce endogenous Otx2 expression. Conclusions: This work reports a large-scale screen to identify spatiotemporally specific regulatory elements near the Onecut1 locus. These elements were used to identify distinct populations in the developing retina. In addition, fate-restricted regulatory elements responded differentially to bHLH factors, and suggest a role for retinal bHLHs upstream of the Otx2 and Onecut1 genes during the formation of restricted RPCs from multipotent RPCs.

overall_design: 2 samples, one replicate each. ThrbCRM1(+) and ThrbCRM1(-) were sorted and collected through FACS after electroporation with ThrbCRM1::GFP and UbiqC::TdT. ThrbCRM1(-) served as a comparison to ThrbCRM1(+)

first: Mark

last: Emerson


title: Next Generation Sequencing Facilitates Quantitative Analysis of transcriptomes in Human PBMC-derived MΦs and TAMs

city: Houston

first: Pan

last: Su

state: TX

address: 6550 FANNIN STREET

institute: Houston Methodist Research Institute

summary: The goals of NGS-derived macrophage transcriptome profiling (RNA-seq) is to identify the differences of signaling pathway between normal MΦs and tumor-associated macrophages (TAMs). Human PBMC-derived MΦs were generated in vitro, and TAMs were generated by using coculture with human myeloma cell lines U266 or ARP1 for 72 hours. Total RNA was extracted using RNeasy Mini Kit (Qiagen). 5-10 µg RNA samples were sent to BGI genomics for RNA-seq followed by data analysis.

overall_design: Examination of gene-profiling data in human PBMC-derived MΦs and TAMs.


title: Transcriptome analysis of isolated glomeruli from obese type 2 diabetic mice treated with enarodustat

city: Bunkyo-ku

first: Mai

last: Sugahara

state: Tokyo

address: 7-3-1 Hongo

department: Nephrology and Endocrinology

institute: The University of Tokyo Graduate School of Medicine

summary: We found that 18-week administration of a prolyl hydroxylase inhibitor, enarodustat, improved glucose/lipid metabolism of BTBR ob/ob mice, which is a model of obesity and type 2 diabetes mellitus. Enarodustat-treated mice also exhibited reduced albuminuria along with ameliorated glomerular epithelial and endothelial damage. In order to elucidate the mechanism of renoprotection, we performed microarray gene expression analysis of isolated glomeruli. The initial screening process revealed 8965 probes whose absolute values of log2 (BTBR ob/ob mice treated with enarodustat/BTBR ob/ob mice treated with vehicle-only) exceeded 0.5. We then compared the expression levels of those 8965 probes between BTBR ob/ob and wild-type mice to identify molecules that were likely to be involved in the pathogenesis of glomerular injury. Such analysis revealed 71 genes which were significantly up-regulated and 47 genes which were significantly down-regulated in BTBR ob/ob mice compared to wild-type mice. The genes were ranked according to their fold-change values, and the analysis presented Ccl2/Mcp1 as the second-most up-regulated gene in BTBR ob/ob mice. The expression level of Ccl2/Mcp1 increased by 24.42-fold in BTBR ob/ob compared to wild-type mice, and its expression in enarodustat-treated BTBR ob/ob mice was decreased to 0.62 of the vehicle-only treated BTBR ob/ob mice. Urinary CCL2/MCP-1 was indeed decreased in enarodustat-treated BTBR ob/ob mice. In vitro experiments also showed that enarodustat suppressed palmitate-induced CCL2/MCP-1 production in murine mesangial cells. Taken together, enarodustat is likely to confer renoprotection through regulating the expression of CCL2/MCP-1 in the glomerulus.

overall_design: Glomeruli were isolated from three groups of mice; BTBR wild type and BTBR ob/ob mice treated with vehicle-only, and BTBR ob/ob mice treated with enarodustat. n = 3 for each group.

first: Shinji

last: Tanaka

first: Tetsuhiro

first: Hisako

last: Saito

first: Yu

last: Ishimoto

first: Takeshi

last: Wakashima

first: Masatoshi

last: Ueda

first: Kenji

last: Fukui

first: Akira

last: Shimizu

first: Reiko

last: Inagi

first: Toshimasa

last: Yamauchi

first: Takashi

last: Kadowaki

first: Masaomi

last: Nangaku


title: Reprogrammed alveolar macrophages after pneumonia recovery

city: Boston

first: Boston University

last: Microarray and Sequencing Resource

state: MA

address: 72 East Concord Street, E631

department: Microarray and Sequencing Resource

institute: Boston University

summary:
Community-acquired pneumonia is a widespread disease with significant morbidity and mortality.  Alveolar macrophages are tissue-resident lung cells that play a crucial role in innate immunity against bacteria causing pneumonia.  We hypothesized that alveolar macrophages display adaptive characteristics after resolution of bacterial pneumonia.  We studied mice one to six months after self-limiting lung infection due to Streptococcus pneumoniae, the most common cause of bacterial pneumonia.  Among the myeloid cells recovered from the lung, only alveolar macrophages showed long-term modifications of their surface marker phenotype.  The remodeling of alveolar macrophages was: (i) long-lasting (still observed 6 months post infection), (ii) regionally localized (only observed in the affected lobe after lobar pneumonia), and (iii) associated with a macrophage-dependent enhanced lung protection to another pneumococcal serotype.  Metabolomic and transcriptomic profiling revealed that alveolar macrophages of mice which recovered from pneumonia had new baseline activities and altered responses to infection.  Thus, the enhanced lung protection after mild and self-limiting respiratory infection includes a profound remodeling of alveolar macrophages that is long-lasting, compartmentalized, and manifest across surface receptors, metabolites, and both resting and stimulated transcriptomes.
We used microarrays to detail the global program of gene expression for mouse alveolar macrophages sorted from lungs that were naïve or infected more than a month previously, at rest and during an acute (4-hour) infection.

overall_design: Mouse lungs were selected  with varying types of infectious history for alveolar macrophage sorting and RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of cells at each infection history stage in order to increase the resolution of how infection history influences expression profiles. To that end, we selected mice according to infectious criteria at four settings: not previously infected (Naive mice who receieved saline instead of SP19 a month previously) or previously infected twice with pneumococcus serotype 19F over a one-week interval more than a month previously (Experienced), either at rest (Control) or 4 hours into a current heterotypic infection with pneumococcus serotype 3 (SP3).

first: Antoine

last: Guillon

first: Joseph

middle: P

last: Mizgerd


title: Alveolar macrophage immunometabolism and lung function impairment in smoking and chronic obstructive pulmonary disease

first: Yael

last: Strulovici-Barel

address: 1300 York Avenue

department: Department of Genetic Medicine

institute: Weill Cornell Medical College

summary: Background: Metabolic plasticity involving shifts between mitochondrial respiration and glycolysis is emerging as a crucial component of efficient innate immune cell responses. Alveolar macrophages (AMs), the most abundant antigen-presenting cells in the lung, are dramatically increased in the lungs of patients with chronic obstructive pulmonary disease (COPD). However, COPD AMs exhibit dysfunctional responses to infection with lower phagocytic ability and impairment of mitochondrial reactive oxygen species (ROS) generation. Little is known about the mitochondrial function or respiration of these cells and whether alterations in their mitochondrial or glycolytic activities may contribute to the pathogenesis of COPD.

overall_design:
In this study, utilizing AMs isolated from the bronchoalveolar lavage fluid (BALF) of subjects with COPD, smokers and healthy non-smoker controls, we demonstrate that BAL macrophages from individuals with COPD have altered expression of mitochondrial-related genes from MitoCarta 2.0 and severely blunted maximal respiration, spare respiratory capacities and compensatory glycolytic rates when compared to smokers and healthy controls. We demonstrate for the first time that such impaired immunometabolic activities associate with the degree of lung function impairment, as defined by %FEV1 in these patients.
Comparison of gene expression in alveolar macrophages of normal non-smokers and normal smokers and smokers with COPD
The Mito Carta 2.0 website can be found at https://www.broadinstitute.org/scientific-community/science/programs/metabolic-disease-program/publications/mitocarta/mitocarta-in-0/

first: Sarah

last: O'Beirne

last: Kikkers

first: Clara

last: Oromendia

first: Jacqueline

last: Salit

first: Mahboubeh

last: Rostmai

first: Karla

middle: V

last: Ballman

first: Robert

last: Kaner

first: Ronald

last: Crystal

first: Suzanne

middle: M

last: Cloonan


title: Expression data from regulatory T cells in mice

city: Shanghai

first: Hui

last: Yang

address: Ruijin Second Road No.197

institute: Shanghai Jiaotong University, School of Medcine

summary:
It is well accepted that the generation and functional stability of Treg cells stand as one dominant force to maintain immunological homeostasis during steady-state or under inflammatory conditions, as the primary Treg cell deficiency causes numerous types of  autoimmunopathy, hypersensitivity or inflammation-induced carcinogenesis.
We used microarrays to indentify the global programme of gene expression underlying regulatory mechanism and explore the differentially expressed genes during the process of immunology regulation.

overall_design: WT or KO naïve T cells were freshly isolated from spleen and stimulated under Treg-induction conditions for 3 days. Cells were collected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain differential genes between WT and KO Treg cells.

first: He-Zhou

last: Guo

first: Xian-Yang

last: Li

first: Jiang

last: Zhu