title: The Power of Resolution: Contextualized Understanding of Chemical-biological Interactions

status: Public on Mar 06 2020

summary: Prediction of human response to chemical exposures is a major challenge in both pharmaceutical and toxicological research. Transcriptomics has been a powerful tool to explore chemical-biological interactions. However, limited throughput, high-costs and complexity of transcriptomic interpretations have yielded numerous studies lacking sufficient experimental context for predictive application. We utilized a novel high-throughput transcriptomics platform to explore a broad range of exposures to 24 reference compounds in both differentiated and undifferentiated human HepaRG cultures. Our goals were to 1) explore transcriptomic characteristics distinguishing liver injury compounds, 2) assess impacts of differentiation state on baseline and compound-induced responses (e.g., metabolically-activated), and 3) identify and resolve reference biological-response pathways and their quantitative translation to human exposures. Study data revealed the predictive utility of transcriptomic concentration-response modeling to quantitatively identify human liver injury compounds by their respective benchmark concentrations (BMCs), and model hepatic responses to classical reference compounds yielding plausibly-relevant estimations of human potency.

overall_design: BioProject accession (PRJNA497448) HepaRG cells were seeded at a density of ~2000 cells per well onto collagen(I)-coated 384-well plates and kept in culture only 3 days prior to compound exposures for maintain proliferation and the undifferentiated state. Cells were seeded using Williams E medium (ThermoFisher) supplemented with plating additive (Lonza, Catalog), and 24 hours later exchanged with William’s E medium supplemented with maintenance additive (Lonza) for the duration of cultures. After the initial 3/10 days, 50 μL of compound mixtures was transferred to the plates containing cells. Media was exchanged after the initial 48h of treatment. At the end of the 96-hour compound exposure period, exposure media was removed, and cells were washed once with 50 μL Phosphate-buffered saline (ThermoFisher) followed by addition of 20 μL of 1X TempO-Seq lysis buffer (Biospyder) to culture plates. Plates were then incubated for 15 minutes at room temperature and frozen at -80° C. TempO-Seq analysis was performed as described previously, at a targeted sequencing depth of 500 mapped read counts per transcript (Yeakley, et al., 2017).

first: Sreenivasa

last: Ramaiahgari

first: Trey

middle: O

last: Saddler

first: Julie

last: Rice

first: Paul

last: Dunlap

first: Nisha

last: Sipes

first: Michael

last: DeVito

first: Ruchir

last: Shah

first: Pierre

last: Bushel

first: Bruce

middle: A

last: Merrick

first: Richard

last: Paules

first: Stephen

last: Ferguson


title: Gene exression in single T cells across division states.

status: Public on Dec 01 2018

city: Stanford

first: Zinaida

last: Good

state: California

address: 265 Campus Drive, Room G2105

department: Stanford Cancer Institute, Department of Biomedical Data Science

country: USA

institute: Stanford University School of Medicine

type: Expression profiling by high throughput sequencing

summary:
Purpose: To compare diversity of primary human CD8+ T cells that have divided 0, 1, or 2 times on day 3 of ex vivo expansion from naïve resting state.
Methods: Naïve T cells were enriched from human peripheral blood monoluclear cells (PBMCs), labeled with CFSE dye, and expanded for 3 days using rapid expansion protocol (Li, Y. & Kurlander, R.J. Journal of Translational Medicine, 2010). On day 3, 10,000 single live CFSE+ CD8+ T cells from each of divisions 0, 1, and 2 were sorted and immediately processed using 10X Genomics single-cell RNA-sequencing platform.
Results: We found that undivided cells display the highest gene expression diversity. Using 1,000 most variably expressed genes, we created a force-directed layout, representing a phenotypic map of cellular differentiation across division states. To understand the basis of T-cell diversity, we defined and quantified regions of interest on this map based on diffusion pseudo-time (DPT), a metric of cell differentiation state. Finally, we examined gene expression in cells from each region and found that undivided cells acquire gene expression associated with effector cell function, while remaining cells go on to grow and differentiate.
Conclusions: Our study provides insights into T-cell differentiation within an ex vivo expansion system for cancer immunotherapy applications.

overall_design: A total of 4,060 cells (division 0: n = 552 cells, division 1: n = 1,777 cells, division23: n = 1,731 cells) were sequenced to an average of 52,040 post-normalization reads per cell capturing a median of 18,770 unique molecular identifier (UMI) counts per cell mapping to 3,544 unique genes per cell.

first: Sean

middle: C

last: Bendall


title: Triphenyl Phosphate (CAS No. 115-86-6): 5-Day Rat Gavage Toxicogenomic Study

status: Public on Oct 23 2018

city: Durham

first: Scott

last: Auerbach

state: North Carolina

address: 111 TW Alexander Drive

institute: Division of the National Toxicology Program at NIEHS

summary: Triphenyl phosphate (TPHP) is an organophosphate flame retardant currently on the market and used as a replacement for phased-out polybrominated diphenyl ethers. Toxicity information on this class of chemicals is inadequate. Herein, the NTP use short-term, in vivo transcriptomic studies to inform potential points of departure for TPHP.

overall_design: The National Toxicity Program (NTP) conducted a transcriptomic study on TPHP, in male Harlan Sprague Dawley rats. TPHP was dissolved in corn oil. Exposure was once daily for 4 days by oral gavage. TPHP (>99%) was tested at six doses (0, 55, 110, 220, 441, and 881 mg/kg body weight). On Day 5, animals were sacrificed, standard toxicological measures assessed, and the liver taken for gene expression studies using Affymetrix microarrays.


title: Contractile activity-specific transcriptome response to acute endurance exercise and training in human skeletal muscle

status: Public on Feb 16 2019

city: Moscow

first: Pavel

last: Makhnovsky

address: 76A Khoroshevskoye shosse

country: Russia

institute: Institute of Biomedical Problems of the Russian Academy of Sciences

summary: In our study, we investigated for contractile activity-specific changes in the transcriptome in untrained and trained (after an aerobic training programme) human skeletal muscle. The second goal was to examine effect of aerobic training on gene expression in muscle at baseline (after long term training). Seven untrained males performed the one-legged knee extension exercise (for 60 min) with the same relative intensity before and after a 2 month aerobic training programme (1 h/day, 5/week). Biopsy samples were taken at rest (baseline condition, 48 h after exercise), 1 and 4 h after the one-legged exercise from m. vastus lateralis of either leg. Comparison of gene expression in exercised leg with that in non-exercised [control] leg allowed us to identify contractile activity-specific genes in both untrained and trained skeletal muscle, i.e., genes that play a key role in adapting to acute exercise, regardless of the level of fitness. RNA-sequencing (84 samples in total; ~47 million reads/sample) was performed by NextSeq 500 and HiSeq 2500 (Illumina). Two months aerobic training increased the aerobic capacity of the knee-extensor muscles (power at anaerobic threshold in the incremental one-legged and cycling tests), the maximum rate of ADP-stimulated mitochondrial respiration in permeabilized muscle fibres and amounts of oxidative phosphorylation proteins. Contractile activity-specific changes in the transcriptome in untrained and trained human skeletal muscle were revealed for the first time. After 2 month aerobic training, transcriptome responses specific for contractile activity in trained muscle substantially decreased relative to those in untrained muscle. We found out that adaptation of skeletal muscle to regular exercise is associated not only with a transient change in the transcriptome after each stress (acute exercise), but also with a marked change in baseline expression of many genes after repeated stress (e.g., long term training).

overall_design:
Seven untrained males performed the one-legged knee extension exercise (for 60 min) with the same relative intensity (at ~65% of the maximal power determined in the one-legged knee extension ramp test) before and after a 2 month aerobic training programme (1 h/day, 5/week). Biopsy samples were taken at rest (baseline condition, 48 h after exercise), 1 and 4 h after the one-legged exercise from m. vastus lateralis of either leg. Comparison of gene expression in exercised leg with that in non-exercised leg allowed us to identify genes that are directly related to contractile activity in human skeletal muscle, regardless of the level of fitness as well as a change in baseline transcriptome after long term training. RNA-sequencing (84 samples in total; ~47 million reads/sample) was performed by NextSeq 500 and HiSeq 2500 (Illumina).
Symbols in file names: ‘Ex’ – exercised leg; ‘NE’ – non-exercised leg; ‘baseline’ – prior to the one-legged exercise; ‘+1 h’ – 1 h after the one-legged exercise; ‘+4 h’ – 4 h after the one-legged exercise; ‘untrained’ – before a 2 month aerobic training programme; ‘trained’ – after a 2 month aerobic training programme; the last symbol in file name – subject’s ID.

first: Daniil

middle: V

last: Popov

last: Makhnovskii


title: Transcriptional control of macrophage inducible gene expression by cohesin [ChIP-Seq II]

status: Public on Jul 31 2018

city: London

first: Gopuraja

last: Dharmalingam

address: Faculty of Medicine, Imperial College, Hammersmith Hospital Campus

department: Epigenetics section

country: United Kingdom

institute: MRC London Institute of Medical Sciences

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Although cohesin and CTCF are major determinants of interphase 3D genome organisation, their impact on the maintenance of transcriptional control, chromatin marks, and enhancer states appears surprisingly limited. Here we study how cohesin promotes the dynamics of eRNA transcription and enhancer states and facilitates the regulation of inducible inflammatory genes in macrophages.

overall_design: RNA-seq, GRO-seq, ChIP-seq and ATAC-seq profiling were performed in Rad21-/- macrophages treated with LPS at different time points as indicated.

first: Matthias

last: Merkenschlager

first: Sergi

last: Cuartero


title: Transcriptional control of macrophage inducible gene expression by cohesin [ATAC-Seq]


title: Transcriptional control of macrophage inducible gene expression by cohesin [RNA-Seq I]


title: Transcriptional control of macrophage inducible gene expression by cohesin [ChIP-Seq I]


title: Single-site CpG Profiling for KMT2D and HNF4A genomic region in pancreatic cancer cells.

status: Public on Aug 20 2018

city: LOS ANGELES

first: MARINA

last: KOUTSIOUMPA

state: CA

address: 650 Charles E. Young Dr South, CHS 33-315

department: MEDICINE

institute: UCLA

type: Methylation profiling by high throughput sequencing

summary: Differential CpG methylation profiling for KMT2D and HNF4A genomic region in pancreatic cancer cells was performed using Illumina MiSeq.

overall_design: Triplicates of 5-AZA-CdR treated or untreated cells were used for genomic DNA isolation and subjected to Targeted 5-mC Bisulfite Sequencing for the selected regions of interest i) chr12: 49448986-49449286 for KMT2D and ii) chr20: 43029895, chr20: 42984320, chr20: 42984324, chr20: 43029732, chr20: 43036036, chr20: 42984099, chr20: 42984209, chr20: 42984394, chr20: 42984878, chr20: 42984338, chr20: 42984579, chr20: 42984347, chr20: 42984453, chr20: 42983920, chr20: 42984276, chr20: 43029777 for HNF4A. Primers used were designed and validated by Zymo Research Corporation.

first: Dimitrios

last: Iliopoulos

first: Marina

last: Koutsioumpa

first: Maria

last: Hatziapostolou


title: Transcriptional control of macrophage inducible gene expression by cohesin

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

overall_design: Refer to individual Series


title: Transcriptional control of macrophage inducible gene expression by cohesin [RNA-Seq II]

first: Felix

middle: D

last: Weiss


title: SMRT-Cappable-seq reveals complex operon variants in bacteria

status: Public on Jul 22 2018

city: Ipswich

first: Bo

last: Yan

state: MA

address: County Road

institute: New England Biolabs

summary:
We develop a method SMRT-Cappable-seq that combines the isolation of unfragmented bacterial primary transcripts with the longread sequencing using PacBio. This method allows the identification and phasing of the transcrription start sites and the termination sites, thereby revealing the operon structure and the regulation of gene experession in bacteria.
Applied to E.coli, our method results in an unprecedented definition of the transcriptome with 34% of the known operons from RegulonDB database being extended by at least one gene, and identifies a total of 2300 operons from which around 900 are novel.

overall_design: Total RNAs were isolated from E. coli MG1655 cells grown under both M9 minimal and Rich condition. Primary transcripts were capped with desthiobiotin and extracted using streptavidin beads to form Enrich Library. While total RNAs without streptavidin enrichment were used to form Control Library. The RNAs were converted to cDNAs, then the cDNAs were amplified and sequenced using PacBio RSII and Sequel.

first: Laurence

last: Ettwiller


title: Transcriptome analysis of SH-SY5Y and SK-N-AS neuroblastoma cells with KIT gene knockdown

status: Public on Jun 23 2018

middle: Vladimirovna

last: Suntsova

address: Miklukho-Maklaya, 16/10

department: Department of Genetics and Postgenomic Technologies

institute: M.M. Shemyakin and Yu.A. Ovchinnikov  Institute of  bioorganic chemistry of the Russian Academy of Sciences

summary: The goal of this study was to identify dynamic changes in signaling pathway activities after KIT knockdown by RNAi in neuroblastoma with relatively high KIT expression (SH-SY5Y) and low expression (SK-N-AS).

overall_design: In two neuroblastoma cell line KIT expression was downregulated by shRNA lentiviral vector and transcriptome profiling was performed 3, 6 and 30 days after lentiviral transduction. Gene expression was normalized on neuroblastoma cell lines, transduced with control shRNA lentiviral vector.

last: Spirin

first: Ivan

last: Petrov

first: Anton

last: Buzdin


title: Transcriptional control of macrophage inducible gene expression by cohesin [GRO-Seq]


title: RNA-seq in KPC mice treated with a hypomethylating drug (decitabine) and in untreated KPC mice

status: Public on Jul 01 2018

city: New York

first: Tamas

last: Gonda

state: NY

address: 630 West 168th Street

department: Department of Medicine, Division of Digestive and Liver Diseases

institute: Columbia University

summary: Background: Treatment with single-agent decitabine (5aza-dC; DAC), a well-tolerated DNA hypomethylating drug, in a mouse model of pancreatic ductal adenocarcinoma (PDAC), KPC-Brca1, extended the survival of the animals and upregulated immune-related pathways. Here we extend these findings to combination therapy, using DAC followed by the immune checkpoint inhibitor anti-PD-1H in the original more widely utilized KPC (Pdx-Cre Kras/p53) model.   Methods: We treated tumor-bearing KPC mice with DAC, and with anti-PD-1H, separately and in combination, and assessed tumor growth, mouse survival, immune cell infiltration of the tumors, and gene expression, compared to historical and mock-treated control mice.  Results: Treatment with single-agent DAC led to increased Cd8+ tumor-infiltrating T cells (TILs), increased tumor necrosis, and slower tumor growth. RNA-seq analysis revealed increased expression of a group of myeloid-lineage markers, including Chi3l3 (Ym1), which proved to reflect recruitment or expansion of a unique population of Chi3l3/Arginase-1 double-positive “M2-polarized” tumor-infiltrating myeloid cells. Anti-PD-1H alone had only modest effects on tumor growth and number of Cd8+ TILs. However, PD-1H-expressing TILs were significantly increased by single agent DAC, and DAC treatment followed by anti-PD-1H produced the strongest increase in Cd8+ TILS, inhibition of tumor growth, and prolongation of survival.   Conclusions: Treatment with DAC alone, and DAC plus anti-PD-1H, inhibits PDAC tumor growth in the KPC model and produces changes in tumor-infiltrating lymphoid and myeloid cell populations, with an additive therapeutic benefit from combining the two agents. Since the influx of M2-polarized macrophages induced by DAC is predicted to antagonize the anti-tumor effects, future work should investigate eliminating or reprogramming these cells.

overall_design: Paradigm I design, explanted KPC tumor epithelial cells treated with the hypomethylating drug (DAC) in tissue culture; paradigm II, graft tumors  from DAC pre-treated and untreated cultured KPC tumor cells  injected subcutaneously in syngeneic host mice; paradigm III, whole tumors from the fully in vivo KPC model, following three cycles of DAC treatment or PBS

first: Jarwei

last: Fang

first: Martha

last: Salas

first: Emily

last: Hsu

first: Catherine

last: Do

first: Anna

last: Zhukovskaya

first: Ariel

last: Siegel

first: Timothy

last: Wang

first: Kenneth

middle: P

last: Olive

first: Benjamin

last: Tycko


title: Transcriptome Alterations Following Developmental Atrazine Exposure in Zebrafish Are Associated with Disruption of Neuroendocrine and Reproductive System Function, Cell Cycle, and Carcinogenesis

status: Public on Jul 20 2018

city: West Lafayette

first: Jennifer

last: Freeman

state: IN

address: 550 Stadium Mall Drive

department: School of Health Sciences

institute: Purdue University

summary: Atrazine, a herbicide commonly applied to agricultural areas and a common contaminant of potable water supplies, is implicated as an endocrine-disrupting chemical (EDC) and potential carcinogen. Studies show that EDCs can cause irreversible changes in tissue formation, decreased reproductive potential, obesity, and cancer. The U.S. Environmental Protection Agency considers an atrazine concentration of ≤ 3 ppb in drinking water safe for consumption. The specific adverse human health effects associated with a developmental atrazine exposure and the underlying genetic mechanisms of these effects are not well defined. In this study, zebrafish embryos were exposed to a range of atrazine concentrations to establish toxicity. Morphological, transcriptomic, and protein alterations were then assessed at 72 h postfertilization following developmental atrazine exposure at 0, 0.3, 3, or  30 ppb. A significant increase in head length was observed in all three atrazine treatments. Transcriptomic profiles revealed 21, 62, and 64 genes with altered expression in the 0.3, 3, and 30 ppb atrazine treatments, respectively. Altered genes were associated with neuroendocrine and reproductive system development, function, and disease; cell cycle control; and carcinogenesis. There was a significant overlap (42 genes) between the 3 and 30 ppb differentially expressed gene lists, with two of these genes (CYP17A1 and SAMHD1) present in all three atrazine treatments. Increased transcript levels were translated to significant upregulation in protein expression. Overall, this study identifies genetic and molecular targets altered in response to a developmental atrazine  exposure to further define the biological pathways and mechanisms of toxicity.

overall_design:
We treated zebrafish embryos from approximately 1 hour post fertilization through 72 hours post fertilization (embryogenesis) to 0, 0.3, 3, or 30 ppb atrazine. Following the 72 hour exposure period, larvae were collected and processed for transcriptomic analysis (n=3 pools of ~50 larvae).
Tables of probes altered in the 0.3, 3, or 30 ppb treatments

middle: L


title: Developmental origins of neurotransmitter and transcriptome alterations in adult female zebrafish exposed to atrazine during embryogenesis

status: Public on Jul 18 2018

summary: Atrazine is an herbicide applied to agricultural crops and is indicated to be an endocrine disruptor. Atrazine is frequently found to contaminate potable water supplies above the maximum contaminant level of 3 mg/L as defined by the U.S. Environmental Protection Agency. The developmental origin of adult disease hypothesis suggests that toxicant exposure during development can increase the risk of certain diseases during adulthood. However, the molecular mechanisms underlying disease progression are still unknown. In this study, zebrafish embryos were exposed to 0, 0.3, 3, or 30 mg/L atrazine throughout embryogenesis. Larvae were then allowed to mature under normal laboratory conditions with no further chemical treatment until 7 days post fertilization (dpf) or adulthood and neurotransmitter analysis completed. No significant alterations in neurotransmitter levels was observed at 7 dpf or in adult males, but a significant decrease in 5-hydroxyindoleacetic acid (5-HIAA) and serotonin turnover was seen in adult female brain tissue. Transcriptomic analysis was completed on adult female brain tissue to identify molecular pathways underlying the observed neurological alterations. Altered expression of 1928, 89,  and 435 genes in the females exposed to 0.3, 3, or 30 mg/L atrazine during embryogenesis were identified, respectively. There was a high level of overlap between the biological processes and molecular pathways in which the altered genes were associated. Moreover, a subset of genes was down regulated throughout the serotonergic pathway. These results provide support of the developmental origins of neurological alterations observed in adult female zebrafish exposed to atrazine during embryogenesis.

overall_design:
Global gene expression changes induced by a developmental exposure to 0.3, 3, or 30 ppb atrazine were analyzed in 5-6  months aged adult female (1 fish brain per sample; N=3 per treatment group) zebarfish brains. A developmental atrazine exposure was performed shortly after fertilization until 72 hours after fertilization.
Probes altered in aged adult female zebrafish developmentally exposed to 0.3, 3, or 30 ppb atrazine

first: Sara

middle: E

last: Wirbisky


title: Global Gene Expression Analysis Reveals Dynamic and Developmental Stage–Dependent Enrichment of Lead-Induced Neurological Gene Alterations

summary: Background: The underlying genetic mechanisms specific to subtle neurological alterations associated with environmental lead (Pb) exposures have not been clearly elucidated.   Objectives: The goal of this study was to identify novel gene targets and the underlying genetic mechanisms associated with developmental Pb neurotoxicity.  Methods: We first exposed zebrafish embryos to a range of Pb concentrations throughout early development to establish relative toxicity. Using the data from that experiment, we exposed another group of zebrafish embryos to a sublethal dose of Pb (100 ppb) immediately after fertilization through 72 or 120 hr postfertilization (hpf). Global gene expression was then analyzed for molecular pathways and gene ontology enrichment, and Western blot analysis was performed to investigate the translation of gene expression changes to protein levels.  Results: After 72 hpf, we identified 231 probes representing 90 nonredundant genes with well-established function or orthology to human genes as being altered by Pb exposure. This gene set was both confirmatory and novel in nature and was highly enriched for neurological development, function, and disease. Moreover, gene changes at this time point were correlated to altered protein levels. Alternatively, the gene set at 120 hpf did not share association with neurological development.  Conclusions: Global gene expression alterations associated with developmental Pb exposure were dynamic and dependent on developmental stage. Gene expression alterations at the 72‑hpf time point were highly enriched with genes and molecular pathways associated with neurological development and disease. Moreover, we identified a number of novel targets for future exploration

overall_design: We treated zebrafish embryos from approximately 1 hour post fertilization through 72 hours post fertilization (embryogenesis) or 120 hours post fertilization to 0 or 100 ppb Pb (lead acetate). Following the chemical exposure period, larvae were collected and processed for transcriptomic analysis to determine differences between control and Pb treatment at each time point (n=3 pools of ~50 larvae per time point).


title: Analysis of gene expression in SKOV3 ovarian cancer cells after knockdown of the long non-coding RNA DNM3OS

status: Public on Feb 13 2018

city: Philadelphia

first: Ramkrishna

last: Mitra

state: Pennsylvania

address: 233 S 10th street

institute: Thomas Jefferson University

summary: RNA-sequencing was performed to gain insight into the mechanism responsible for the mesenchymal-to-epithelial transition (MET) induced by loss of long non-coding RNA (lncRNA) DNM3OS in SKOV3 ovarian cancer cells. Following siRNA-mediated knockdown of DNM3OS or non-targeting control,  RNA-sequencing was performed. This high-throughput data revealed knockdown of DNM3OS down-regulated the expression of genes and pathways known to induce EMT in ovarian cancer.

overall_design: DNM3OS was knockdown in SKOV3 ovarian cancer cells and gene expression profiles were compared with controls using RNA-sequencing.

first: Christine

middle: M

last: Eischen


title: Embryonic ionizing radiation exposure results in expression alterations of genes associated with cardiovascular and neurological development, function, and disease and modified cardiovascular function in zebrafish

summary: The relationship between ionizing radiation (IR) and carcinogenesis is long established, but recently the association between IR and other diseases is starting to be recognized. Currently, there is limited information on the genetic mechanisms governing the role of IR in non-cancer related adverse health effects and in regards to an early developmental exposure. In this study, zebrafish embryos were exposed to a range of IR doses (0, 1, 2, 5, 10 Gy) at 26 hours post fertilization (hpf). No significant increase in mortality or hatching rate was observed, but a significant decrease in total larval length, head length, and eye diameter was observed in the 10 Gy dose. Transcriptomic analysis was conducted at 120 hpf to compare gene expression profiles between the control and highest IR dose at which no significant differences were observed in morphological measurements (5 Gy). 253 genes with well-established function or orthology to human genes were significantly altered. Gene ontology and molecular network analysis revealed enrichment of genes associated with cardiovascular and neurological development, function, and disease. Expression of a subset of genetic targets with an emphasis on those associated with the cardiovascular system was assessed using qPCR to confirm altered expression at 5 Gy and then to investigate alterations at lower doses (1 and 2 Gy). Strong correlation between microarray and qPCR expression values was observed, but zebrafish exposed to 1 or 2 Gy resulted in a significant expression alteration in only one of these genes (LIN7B). Moreover, heart rate was analyzed through 120 hpf following IR dosing at 26 hpf. A significant decrease in heart rate was observed at 10 Gy, while a significant increase in heart rate was observed at 1, 2, and 5 Gy. Overall these findings indicate IR exposure at doses below those that induce gross morphological changes alters heart rate and expression of genes associated with cardiovascular and neurological functions.

overall_design: Zebrafish embryos were irradiated at 5 Gy at 26 hours post fertilization in a Co-60 irradiator. At 120 hours post fertilization larvae were collected and processed for transcriptomic analysis to determine differences between control and radiation treatment (n=3 pools of ~50 larvae).


title: Distinct molecular programs regulate synapse specificity in cortical inhibitory circuits

status: Public on Jan 25 2019

city: Boston

first: Emilia

last: Favuzzi

address: 220 Longwood Avenue, Armenise 217

department: Neurobiology

institute: Harvard Medical School

summary: How neuronal connections are established and organized in functional networks determines brain function. In the mouse cerebral cortex, different classes of GABAergic interneurons exhibit specific connectivity patterns that underlie their ability to shape temporal dynamics and information processing. Much progress has been made parsing interneuron diversity, yet the molecular mechanisms by which interneuron subtype-specific connectivity motifs emerge remain unclear. Here we investigate transcriptional dynamics in different classes of interneurons during the formation of cortical inhibitory circuits. We found that whether the interneurons synapse with pyramidal neurons on their dendrites, soma, or axon initial segment is determined by synaptic molecules that are expressed in a subtype-specific manner. Thus cell-specific molecular programs that unfold during early postnatal development underlie the connectivity patterns of cortical interneurons.

overall_design: RNAseq of three different subtypes of GABAergic inhibitory neurons (SST+, PV+ and Chandelier cells) at two developmental stages (P5 and P10) from mouse cortical regions as well as three control populations: interneurons at P0, pyramidal excitatory neurons at P12 and PLP+ oligodendrocytes at P10.

first: Rubén

last: Deogracias

first: Patricia

last: Maeso

first: Beatriz

last: Rico


title: Genomic signatures of platinum re-sensitization in ovarian cancer [RNA-Seq]

status: Public on Jun 05 2018

city: Bloomington

last: Nephew

institute: Indiana University

summary: Epigenetic changes, particularly DNA methylation aberrations have been implicated in acquired resistance to platinum in ovarian cancer. A multi-institutional randomized clinical trial compared a regimen of a DNA methyl transferase (DNMT) inhibitor guadecitabine and carboplatin to physician’s choice chemotherapy for patients with recurrent platinum resistant ovarian cancer. Tumor biopsies or malignant ascites were collected at day 1 of cycle 1 (pre-guadecitabine) and after two cycles of treatment (post-decitabine). The goal of the current study was to analyze guadecitabine-induced DNA methylation and gene expression changes and correlate pretreatment levels with clinical outcomes. Epigenomic and transcriptomic profiling using the Infinium HumanMethylation450 BeadChip (HM450) and RNA sequencing revealed extensive methylation and gene expression changes induced by guadecitabine in ovarian tumors. Ninety-four gene promoters were significantly hypomethylated after treatment with guadecitabine and 949 genes were differentially expressed in pre vs. post-treatment tumors. Pathways associated with immune reactivation and DNA repair were significantly altered by guadecitabine treatment.  Expression levels of 1155 genes involved in 25 networks on day 1 of cycle 1 correlated with progression free survival. Increased expression of selected genes (e.g. DOK2, miR193a) silenced through promoter methylation restored platinum sensitivity in ovarian cancer cells.  Together, these results support that guadecitabine altered DNA methylation and expression of genes and gene networks correlate with re-sensitization to carboplatin in ovarian cancer patients.

overall_design: 93 samples are analyzed, including tumor samples collected from patients with platinum resistant ovarian cancer before treated by guadecitabine and after treated by guadecitabine.

first: Fang

last: Fang

first: Daniela

last: Matei


title: Genomic and Epigenomic Signatures of Platinum Re-sensitization in ovarian cancer

type: Methylation profiling by genome tiling array


title: Clearance of a persistent picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses

status: Public on Dec 19 2017

city: Orient

first: James

middle: J.

last: Zhu

department: Foreign Animal Disease Research Unit

institute: USDA-ARS

summary: Immunomodulatory mechanisms associated with clearance versus persistence of foot-and-mouth disease virus (FMDV) in micro-dissected compartments of the bovine nasopharynx were investigated using qRT-PCR and whole transcriptome microarray. Analysis of tissue samples obtained by laser capture microdissection (LCM) during transitional and persistent phases of infection demonstrated significant differences in transcriptome profiles of animals that cleared infection versus those that became persistently infected carriers. The combined output of the analyses suggested that clearance of FMDV from the nasopharyngeal mucosa is associated with an activated cellular immune response. This was supported by induction of Th1-associated mediators and upregulation of multiple targets associated with activation of T cell-mediated cytotoxicity in tissues from animals that cleared infection. Contrastingly, the pattern of gene regulation in FMDV carriers during transitional and persistent phases of infection suggested inhibition of T cell activation and promotion of Th2 polarization. Regulation of genes associated with apoptosis or cellular proliferation suggested inhibition of apoptotic pathways and promotion of cellular proliferation associated with FMDV persistence while the opposite patterns were found in animals that cleared infection. The findings presented herein emphasize the critical importance of Th1-mediated cellular immunity for clearance of persistent FMDV infection. Additionally, the strong antibody-mediated immune response that is induced during acute infection may impair efficient clearance of intra-cellular virus, thereby promoting FMDV persistence. Thus, a critical balance between Th1 and Th2 -mediated immunity is essential for successful clearance of FMDV infection and should be considered for development of next-generation vaccines and antiviral products.

platform_organism:
Bos taurus
Foot-and-mouth disease virus
Human adenovirus 5

overall_design: Epithelial tissue associated with lymphoid follicles in the dorsal nasopharynx or the dorsal soft palate of 15 Holstein cattle was obtained by laser-capture microdissection. RNA extracted from the tissues was amplified by REPLI-g whole-transcriptome amplification, heat-fragmented, labeled with Cy3 and Cy5 and hybridized to paired 4x45K arrays in a dye-swap arrangement.  The numbers of FMDV carriers, non-carriers and controls were unbalanced, precluding a fully connected dye-swap design, and a separate-channel approach using a common correlation model (Smyth and Altman, 2013,  BMC Bioinformatics 14:165, doi: 10.1186/1471-2105-14-165) was used instead.

first: Carolina

last: Stenfeldt

first: Michael

last: Eschbaumer

first: George

middle: R

last: Smoliga

first: Luis

last: Rodriguez

first: Jonathan

last: Arzt


title: Embryonic exposure to an aqueous coal dust extract results in gene expression alterations associated with development and function of connective tissue and the hematological system in zebrafish

status: Public on Nov 30 2017

last: Freeman

summary: Coal is a major energy source that generates diverse environmental impacts through its production, primarily by the release of coal dust particles. An aqueous coal dust extract was obtained from a mineral sample taken from one of the largest coal mines in Colombia (La Loma, Cesar), trace elements by ICP/MS were measured, and its toxicity evaluated using the zebrafish (Danio rerio) vertebrate model. In this study, zebrafish embryos were exposed to different concentrations of aqueous coal extract (0, 0.1, 1, 10, 100 and 1000 parts per million (ppm; μg/mL) to establish acute toxicity, as well as morphological and transcriptome alterations. Trace elements within the coal extract yielding the highest concentrations included Sr, Zn, Ba, As, Cu, Se, Li, Ni, Sb, Rb, Co, and Cr. In addition, Cd and Pb were found in lower concentrations. No significant difference in mortality was observed with survival near 90% in all treatments. A significant decrease in rate of hatching was observed in the 0.1 and 1000 ppm treatment groups at 72 hpf. Furthermore, no significant differences in total body length, head length, or head diameter was observed in any of the treatment groups. Transcriptomic results of zebrafish larvae revealed alterations in 77, 61, and 1,376 genes in the 1, 10, and 100 ppm treatments, respectively. Gene ontology analysis revealed gene alterations associated with hematological system development and function, tissue morphology and development, connective tissue development and function, and embryonic development. Overall, these findings are the first to identify gene expression alterations in response to a developmental aqueous coal dust residue from coal mining.

overall_design: We treated zebrafish embryos from approximately 1 hour post fertilization through 72 hours post fertilization (embryogenesis) to 0, 1, 10, or 100 ppm aqueous coal extract mixture. Following the 72 hour exposure period, larvae were collected and processed for transcriptomic analysis.

first: Karina

last: Caballero-Gallardo

first: Jesus

last: Olivero-Verbel


title: Embryonic exposure to 100 µg/L lead results in sex-specific expression changes in genes associated with neurological system and cancer in the aged adult zebrafish brain

first: Jennifer


title: Analysis of gene expression in p53-null cell types following deletion of Mdm2

status: Public on Sep 06 2017

first: Ramkrishna

last: Mitra

summary: RNA-sequencing was performed to gain insight into the mechanism responsible for the apoptosis and cell cycle arrest induced by loss of Mdm2 in p53-null cells. Following CreERT2-mediated deletion of Mdm2 in three p53-null cell types (T cell lymphoma, sarcoma, and fibroblasts), RNA-sequencing was performed. This high-throughput data revealed Mdm2 deletion in p53-null cells upregulated p53/p73 transcriptional target genes known to induce apoptosis and cell cycle arrest.

overall_design: Mdm2 was deleted in p53-null cells (T cell lymphoma and sarcoma and non-transformed fibroblasts) and gene expression profiles were compared using RNA-sequencing.

last: Eischen


title: Uncovering a Predictive Molecular Signature for the Onset of  NASH-Related Fibrosis in a Translational NASH Mouse Model

status: Public on Jan 19 2018

city: Zeist

first: Lars

last: Verschuren

address: Utrechtseweg 48

department: Microbiology and Systems Biology

country: Netherlands

institute: TNO

summary:
SUMMARY: This article presents a predictive molecular signature that marks the early onset of fibrosis in a translational nonalcoholic steatohepatitis mouse model. Overlap of genes and processes with human nonalcoholic steatohepatitis and a list of top candidate biomarkers for early fibrosis are described.

BACKGROUND & AIMS: The incidence of nonalcoholic steatohepatitis (NASH) is increasing. The pathophysiological mechanisms of NASH and the sequence of events leading to hepatic fibrosis are incompletely understood. The aim of this study was to gain insight into the dynamics of key molecular processes involved in NASH and to rank early markers for hepatic fibrosis.

METHODS: A time-course study in low-density lipoprotein–receptor knockout. Leiden mice on a high-fat diet was performed to identify the temporal dynamics of key processes contributing to NASH and fibrosis. An integrative systems biology approach was used to elucidate candidate markers linked to the active fibrosis process by combining transcriptomics, dynamic proteomics, and histopathology. The translational value of these findings were confirmed using human NASH data sets.

RESULTS: High-fat-diet feeding resulted in obesity, hyperlipidemia, insulin resistance, and NASH with fibrosis in a time-dependent manner. Temporal dynamics of key molecular processes involved in the development of NASH were identified, including lipid metabolism, inflammation, oxidative stress, and fibrosis. A data-integrative approach enabled identification of the active fibrotic process preceding histopathologic detection using a novel molecular fibrosis signature. Human studies were used to identify overlap of genes and processes and to perform a network biology-based prioritization to rank top candidate markers representing the early manifestation of fibrosis.

CONCLUSIONS: An early predictive molecular signature was identified that marked the active profibrotic process before histopathologic fibrosis becomes manifest. Early detection of the onset of NASH and fibrosis enables identification of novel blood-based biomarkers to stratify patients at risk, development of new therapeutics, and help shorten (pre)clinical experimental time frames.
Keywords: Systems Biology; Metabolic Syndrome; Liver Disease; Diagnosis.

overall_design: In total 9 treatment groups: 5 Control groups (chow = standard diet; t=0, 6, 12, 18, 24 weeks), 4 Treatment groups (HFD = High Fat diet; 6, 12, 18, 24 weeks).

first: Arianne

last: van Koppen

first: Anita

last: van den Hoek

first: Joanne

last: Verheij

first: Martine

last: Morrison

first: Kelvin

last: Li

first: Hiroshi

last: Nagabukuro

first: Adalberto

last: Costessi

first: Martien

last: Caspers

first: Tim

middle: J

last: van den Broek

first: John

last: Sagartz

first: Cornelis

last: Kluft

first: Carine

last: Beysen

first: Claire

last: Emson

first: Alain

last: van Gool

first: Roel

last: Goldschmeding

first: Reinout

last: Stoop

first: Ivana

last: Bobeldijk-Pastorova

first: Scott

last: Turner

first: Guido

last: Hanauer

first: Roeland

last: Hanemaaijer


title: Embryonic exposure to 100 µg/L lead results in sex-specific expression changes in genes associated with neurological system and cancer in the aged adult zebrafish brain [male]

first: Jennifer

last: Freeman

summary: Developmental lead (Pb) exposure has been linked to neurological health issues appearing in children. Results from non-human primate studies also suggest detrimental effects of an early life Pb exposure on neurological health, showing a pathological evidence of Alzheimer’s disease in the aged animal brain. To elucidate the impacts of a developmental Pb exposure on neurological health-related molecular changes occurring in the aged brain, aged zebrafish with a control treatment or an embryonic exposure to 100 µg/L Pb were subjected to a zebrafish-specific microarray analysis by sex. Gene ontology analysis showed alterations in distinct sets of genes associated with nervous system development and function in each sex. In aged adult female zebrafish, significant changes in expression profiles occurred in a number of genes involved in neurological disease and nervous system development and function. On the other hand, in aged adult males, a number of genes with significant expression alterations were found to be associated with cancer and tumors.

overall_design: Global gene expression changes induced by a developmental exposure to 100ppb lead (Pb) were analyzed in 12 months aged adult male (1 fish brain per sample; N=3 for control group, N=3 for Pb exposed group) and female (1 fish brain per sample; N=3 for control group, N=3 for Pb exposed group) zebarfish brains. A developmental Pb exposure was performed shortly after fertilization until 72 hours after fertilization.

first: Jinyoung

last: Lee


title: Transcriptomic analysis of third instar larva wing imaginal discs deregulating Cyclin G in Drosophila melanogaster

status: Public on Jul 01 2017

city: Paris

first: Frédérique

last: Peronnet

address: 9, quai saint-Bernard

department: UMR7622-Developmental biology

country: France

institute: CNRS-SU

summary: We report here the transcriptomic analysis of Drosophila melanogaster wing imaginal discs from third instar female larvae expressing Cyclin G deleted of the PEST domain (the 25 COOH-terminal amin-acids) under the control of the daugterless-Gal4 ubiquitous driver. The negative control was transgenic flies wearing only the daugterless-Gal4  driver.

overall_design: Imaginal wing disc mRNA profiles of female  UAS-CycGΔP, da-Gal4/+ or da-Gal4/+  Drosophila melanogaster third instar larvae were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.

first: Delphine

last: Dardalhon-Cuménal

first: Jérôme

last: Deraze


title: Promoter-bound METTL3 maintains myeloid leukemia by m6A-dependent regulation of protein translation

status: Public on Nov 28 2017

city: Portsmouth

first: Samuel

last: Robson

address: St Michael's Building

department: School of Pharmacy & Biomedical Science

institute: University of Portsmouth

summary: N6-methyladenosine (m6A) is an abundant internal RNA modification, in both coding and non-coding RNAs, catalyzed by the METTL3/METTL14 methyltransferase complex. We identified METTL3 as an essential gene for acute myeloid leukemia (AML) cell growth in two distinct genetic screens. Down-regulation of METTL3 results in cell cycle arrest, differentiation of leukemic cells and failure to establish leukemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start site (TSS) of 83 active genes. The vast majority of these genes have a CAATT-box motif at their TSS, are occupied by a specific set of transcription factors including NFY, WDR5, KLF9 and they harbor specific histone modifications (e.g. H3R2me2s). Promoter bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript and it enhances translation due to relief of ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for AML-leukemia. Together, these data define a new mechanism of gene regulation by METTL3 and identify this enzyme as a novel therapeutic target for AML.

overall_design: This experiment contains data for 3 high-throughput sequencing experiments; ChIP seq, m6A RNA IP seq and Riboprofiling. ChIP seq of METTL3 and METTL14 was conducted with 5 biological replicates each, with IgG and Input controls in triplicate and H3K4me3 in duplicate. All biological replicates were run across 2 lanes on an Illumina HiSeq 1500 providing technical replicates and increased coverage. m6A RNA IP was run in duplicate for two METTL3 shRNA inducible knockdown cell lines (KD1 and KD2) and a scramble shRNA control at day 8 following doxycyclin induction. In addition, matching non-immuno-precipitated Input controls were also sequenced. All biological replicates were run across 2 lanes on an Illumina HiSeq 4000 providing technical replicates and increased coverage. Riboprofiling was performed on two METTL3 shRNA inducible knockdown (KD) cell lines and a scramble shRNA control at day 5 and day 8 following doxycyclin induction. Matching Input controls were sequenced for each condition for direct comparison. All samples were run across 6 lanes on an Illumina HiSeq 1500 as technical replicates and to provide increased coverage. Files are named with the following structure: [Experiment Type]_[Sample Name]_[Biological Replicate Num]_[Technical Replicate Num].[File Extension].

first: Isaia

last: Barbieri

first: Konstantinos

last: Tzelepis

first: Luca

last: Pandolfini

first: Junwei

last: Shi

first: Valentina

last: Migliori

first: Andrew

last: Bannister

first: Namshik

last: Han

first: Etienne

last: De Braekeleer

first: Hannes

last: Ponstingl

first: Christopher

last: Vakoc

first: George

last: Vassiliou

first: Tony

last: Kouzarides


title: Lack of Repressive Capacity of Human Promoter DNA Methylation identified through Genome-Wide Epigenomic Manipulation

status: Public on Aug 10 2017

city: Crawley

first: Ethan

middle: Edward

last: Ford

state: WA

address: 35 Stirling Highway

department: Plant Energy Biology

country: Australia

institute: University of Western Australia

summary: It is widely assumed that the addition of DNA methylation at gene promoters silences gene transcription. However, this conclusion is largely drawn from the observation that promoter DNA methylation inversely correlates with gene expression. The effect of forced DNA methylation on endogenous promoters has yet to be comprehensively assessed. Here, we conducted artificial methylation of thousands of human promoters in human cells using an artificial zinc finger-DNMT3A fusion protein, enabling assessment of the effect of forced DNA methylation upon transcription and histone modifications, and the durability of DNA methylation after the removal of the fusion protein. We find that DNA methylation is insufficient to transcriptionally repress most promoters. Furthermore, DNA methylation deposited at promoter regions associated with H3K4me3 is rapidly erased after removal of the zinc finger-DNMT3A fusion protein. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3. These findings suggest that promoter DNA methylation is not generally sufficient for transcriptional inactivation suggesting important implications for the emerging field of epigenome engineering.

overall_design: The response of MCF-7 cells to enforced DNA methylation by ZF-DNMT3A as measured by MethylC-seq, RNA-seq, ChIP-seq and ChIP-BS-seq. ZF-DNMT3A expression was induced the addition of doxycycline to the growth media for 3 days. Doxycycline was then withdrawn for 9 days. DNA, RNA and/or chrmatin was extracted from  the cells before and after doxycycline induction as well as after doxycycline withdrawl. WGBS, RNA-seq, ChIP-seq and ChIP-BS-seq libraries were constructed for sequencing on the Illumina HiSeq1500.

first: Matthew

last: Grimmer

first: Sabine

last: Stolzenburg

first: Ozren

last: Bogdanovic

first: Alex

last: de Mendoza

first: Peggy

last: Farnham

first: Pilar

last: Blancafort

first: Ryan

last: Lister


title: Embryonic exposure to 100 µg/L lead results in sex-specific expression changes in genes associated with neurological system and cancer in the aged adult zebrafish brain [female]

first: Jennifer

last: Freeman

first: Jinyoung

last: Lee


title: Jasper Ridge Global Change Experiment

status: Public on Nov 21 2017

city: Beijing

first: Yunfeng

last: Yang

address: No.1 Qinghuayuan, Haidian District

department: School of Environment

country: China

institute: Tsinghua University

summary: Anthropogenic activities have dramatically increased the inputs of reactive nitrogen (N) into terrestrial ecosystems, with potentially important effects on the soil microbial community and consequently soil C and N dynamics. Our analysis of microbial communities in soils subjected to 14 years of 7 g N m-2 year-1 Ca(NO3)2 amendment in a Californian grassland showed that the taxonomic composition of bacterial communities, examined by 16S rRNA gene amplicon sequencing, was significantly altered by nitrate amendment, supporting the hypothesis that N amendment- induced increased nutrient availability, yielded more fast-growing bacterial taxa while reduced slow-growing bacterial taxa. Nitrate amendment significantly increased genes associated with labile C degradation (e.g. amyA and xylA) but had no effect or decreased the relative abundances of genes associated with degradation of more recalcitrant C (e.g. mannanase and chitinase), as shown by data from GeoChip targeting a wide variety of functional genes. The abundances of most N cycling genes remained unchanged or decreased except for increases in both the nifH gene (associated with N fixation), and the amoA gene (associated with nitrification) concurrent with increases of ammonia-oxidizing bacteria. Based on those observations, we propose a conceptual model to illustrate how changes of functional microbial communities may correspond to soil C and N accumulation.

overall_design: The JRGCE was designed to assess grassland responses to single and multiple components of global change, which includes elevated CO2, warming, nitrate amendment and enhanced precipitation. Each experimental unit at the JRGCE was circular, 2 m in diameter, and equally divided into four quadrants of 0.78 m2. The CO2 and warming treatments are operated at the plot level and N and water treatments were operated at the quadrants level in a factorial design. This creates 16 treatments with 8 replicates each. However, an experimental fire burned four replicate blocks in 2011 to simulate the wildfire effect, thus only the other four replicate blocks (a total of 64 samples) were used in this study.

first: Jizhong

last: Zhou


title: MicroRNA-223 suppresses inflammation by down regulating NF-kB activation in epithelial cells

status: Public on Feb 17 2017

summary: MicroRNA-223, a principal myeloid-specific anti-inflammatory microRNA, is dysregulated in numerous inflammatory conditions and cancers. We found that miR-223 deficient zebrafish displayed augmented neutrophilic inflammation, which was due primarily to elevated activation of the canonical NF-B pathway. To our surprise, the NF-B over-activation was restricted to the basal epithelial cells, a squamous layer permeable to oxygen and chemicals. MiR-223 was expressed in epithelial cells and directly down-regulated multiple components in the NF-B signaling pathway in zebrafish and human. Both phagocytic and epithelial miR-223 suppressed neutrophil wound response and NF-B activation in the basal epithelial cells. Together, our data provide the mechanism of the multifaceted role of miR-223 and highlight the previously overlooked relevance of epithelial cells in miR-223 related diseases.

overall_design:
The zebrafish experiment was conducted in accordance to the internationally accepted standards. The Animal Care and Use Protocol was approved by The Purdue Animal Care and Use Committee (PACUC), adhering to the Guidelines for Use of Zebrafish in the NIH Intramural Research Program (Protocol number: 1401001018).
To generate a miR-223 deficient fish line, two individual single guide RNAs (sgRNAs) targeting pre-miR-223 were designed: sgRNA1 binding site: 5’-TTAGAGTATTTGACAGACTG-3’, and sgRNA2 binding site: 5’-TTTGTCAAATACCCCAAGAG-3’. The T7 promoter and the scaffold were added using overlapping PCR with the following primers listed from 5’ to 3’:
sgRNA1-P1: GCGGCCTCTAATACGACTCACTATAGGGTTAGAGTATTTGACAGACTGGTTTTAGAGCTA, sgRNA1-P2: TGACAGACTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC, sgRNA1-P3: GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACT; sgRNA2-P1: GCGGCCTCTAATACGACTCACTATAGGGTTTGTCAAATACCCCAAGAGGTTTTAGAGCTA, sgRNA2-P2: ACCCCAAGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC, sgRNA2-P3: GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACT. sgRNAs were synthesized by in vitro transcription using MEGAshortscript T7 kit (Invitrogen) using the purified PCR products as template. To make cas9 mRNA, the plasmid pT3TS-nCas9n (Addgene Plasmid #46757)1 was linearized by XbaI and purified. Cas9 mRNA was generated using mMESSAGE mMACHINE T3 kit (Invitrogen). The mix of sgRNA1 (100 ng/ul), sgRNA2 (100 ng/ul) and cas9 mRNA (150 ng/ul) was injected into one-cell stage embryos. The embryos were raised to sexual maturity and the F1s were screened by sequencing the miR-223 locus. The homozygous mutant were obtained after several generations of backcross and incross.
To generate transgenic zebrafish lines, plasmids containing the tol2 backbone was coinjected with transposase mRNA into embryos at one-cell stage as described2. The construct tol2-lyzC-Gal4-Vp16-cyrs-CFP was generated with Gateway Cloning (Thermo Fisher) using the tol2 kit to make Tg(lyzc: Gal4-Vp16; crys: CFP)pu8. Founders were screened with green eyes.
To generate a miR-223 over expression line, Tg(lyzC: RFP-miR-223) pu9, a 317 bp genomic DNA sequence containing miR-223 was PCR amplified using the following primers listed from 5’ to 3’: forward CTGGCAGTACGGGCTCAGGAGGAAGAGGGAGGAGTAAAATTGAAT, reverse TAACAGCAGTTGGCTAATGAATGTTGTCATCCTCCACATTTTGCA, and cloned into the BbsI site in the intron of the vector3. GFP was replaced by RFP and then inserted into a Tol2 backbone with lyzC promoter. A transgenic line expressing the empty backbone, Tg(lyzC: RFP) pu10 was generated as the control.
To generate miR223 sponge line, Tg(krt4: RFP-miR-223 sponge)pu12, a sequence containing six copies of bulged miR-223 binding sites was synthesized as a gBlocks Gene Fragment (Integrated DNA Technologies) and inserted downstream of RFP in a pME construct. The final construct was generated by Gateway Cloning. Founders were selected with red apical epithelial cells. A line with RFP Tg(krt4: RFP) pu13 was made as the control. The line Tg(krt4: GFP) pu11 was also generated for cell sorting.
More than one founders were obtained for each line and their offspring display similar phenotypes. Embryos were collected at 0 hpw, 1 hpw, and 6 hpw. Total RNAs were extracted with Trizol (Invitrogen) and further cleaned up with RNeasy MinElute kit (Qiagen). The quality and integrity of RNAs were confirmed using Agiloent Nano RNA QC chip (Sequencing Center of Purdue University). Transcriptomic microarray analysis was conducted using the one-color hybridization strategy to compare gene expression profiles among the different time points with a zebrafish V3 array (Agilent Technologies).  Following hybridization, arrays were washed and scanned on an Agilent Technologies SureScan Microarray Scanner.  Array image data was extracted using Agilent Feature Extraction Software and data was uploaded onto GeneSpring for statistical analysis.  Microarray analysis was performed following MIAME guidelines.

first: Wenqing

first: Alan

middle: Y

last: Hsu

first: Theodore

last: Gural

first: Xiaoguang

last: Zhu

first: Sara

last: Wirbisky

first: Arpita

last: Pal

first: Andrea

last: Kasinski

first: Qing

last: Deng


title: Proximal methylation features associated with nonrandom changes in gene body methylation

status: Public on Mar 29 2017

city: Cambridge

first: Mary

last: Gehring

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: Gene body methylation at CG dinucleotides is a widely conserved feature of methylated genomes, but remains poorly understood. The A. thaliana strain Cvi has depleted methylation in gene bodies relative to the common reference strains Col and Ler. This naturally occurring hypomethylated strain offers the opportunity to improve understanding of the function and heritablity of genic CG methylation. We bisulfite-sequenced 10 recombinant inbred lines derived from a cross between Col and Cvi, as well as both parent lines, and examined the transmission of the different Col- and Cvi-derived gene body methylation states in these lines. We found that the majority of CG methylation polymorphisms were faithfully transmitted over nine generations according to the parental genotype in the RILs, with only 1-4% of CGs failing to maintain the parental methylation state. We found no evidence to suggest that these differences in gene body methylation led to changes in gene expression. However, we found that sites that fail to maintain the parental methylation state are nonrandom and are associated with intermediate levels of CG methylation and high methylation variability across many A. thaliana strains. These results provide new insights into the features determining the inheritance of gene body methylation and demonstrate that two different methylation equilibria can be maintained within single individuals.

overall_design: Examination of DNA methylation and gene expression in the wild-type A. thaliana strains Col and Cvi as well as in 10 Col/Cvi recombinant inbred lines (RIL) at the F9 generation. High-throughput whole-genome bisulfite-sequencing was used to examine DNA methylation and identify differences between RIL and parent lines, while poly-A-selected mRNA-seq was used to evaluate differences in gene expression. Bisulfite sequencing samples are designated BSseq_{strain}_N, where strain = col, cvi, or a number indicating the RIL line number (e.g. 8, 124 etc.). N = the replicate # (either 1 or 2). Similar naming for RNA-seq samples but with prefix RNAseq_* instead of BS_seq_*. Creation of the RILs is described in Simon et al., Genetics 178:2253-2264 (2008).

first: Colette

last: Picard


title: Detection of differentially expressed genes in broiler Pectoralis major muscle affected by White Striping – Wooden Breast myopathies

status: Public on Nov 30 2016

city: Bologna

first: Roberta

last: Davoli

state: Bologna

address: Viale G. Fanin, 46

department: DISTAL

country: Italy

institute: Bologna University

summary: White Striping and Wooden Breast (WS/WB) are abnormalities increasingly occurring in the fillets of high breast yield and growth rate chicken hybrids. These defects lead to consistent economic losses for poultry meat industry, as affected broilers fillets present an impaired visual appearance that negatively affects consumers’ acceptability. Previous studies have highlighted in affected fillets a deeply damaged muscle, showing profound inflammation, fibrosis and lipidosis. The present study investigated the differentially expressed genes and pathways linked to the compositional changes observed in WS/WB breast muscles, in order to outline a more complete framework of the gene networks related to the occurrence of this complex pathological picture. The biochemical composition was performed on 20 Pectoralis major samples obtained from high breast yield and growth rate broilers (10 affected vs. 10 normal) and 12 out of the 20 samples were used for the microarray gene expression profiling (6 affected vs. 6 normal). The obtained results indicate strong changes in muscle mineral composition, coupled to an increased deposition of fat. In addition, 204 differentially expressed genes (DEG) were found: 102 up-regulated and 102 down-regulated in affected breasts. The gene expression pathways found more altered in WS/WB muscles are those related to muscle development, polysaccharide metabolic processes, proteoglycans synthesis, inflammation and calcium signaling pathway. On the whole, the findings suggest that a multifactorial and complex etiology is associated with the occurrence of WS/WB muscle abnormalities, contributing to further define the transcription patterns associated to these myopathies.

overall_design: Broiler of the same genetic type and reared on the same flock were inspected at slaughterhouse to identfy 6 fillets with normal phenotype (NORM) and 6 fillets with white striping / wooden breast myopathy (ABN). The average transcription profile of the ABN animals wase comapred with that of the NORM broilers in order to detect differentially expressed genes.

first: Paolo

last: Zambonelli

first: Martina

last: Zappaterra

first: Francesca

last: Soglia

first: Massimiliano

last: Petracci

first: Federico

last: Sirri

first: Claudio

last: Cavani


title: DNA methylation in lung cells is a key modulator of asthma endotypes and genetic risk [RNA-seq]

status: Public on Jan 06 2017

city: Chicago

first: Carole

last: Ober

state: IL

address: 920 E 58TH ST

department: Human Genetics

institute: The University of Chicago

summary: We generated genome-wide RNASeq data from freshly isolated airway epithelial cells of asthmatics and non-asthmatics. This data was paired with genome-wide genetic and methylation data from the same individuals allowing for an integrated analysis of genetic, transcriptional, and epigenetic signatures in asthma.

overall_design: examination of genome-wide genome-wide gene expression levels and comparison to phenotypes

first: Jessie

last: Nicodemus-Johnson

first: William

last: Wentworth-Sheilds


title: Embryonic atrazine exposure elicits neuroendocrine dysfunction in adult male zebrafish (gonad)

status: Public on Jul 01 2016

first: Jennifer

last: Freeman

summary: The developmental origins of health and adult disease (DOHaD) hypothesis states that exposure to various environmental stressors early in life can elicit changes to the genome and epigenome thereby resulting in an increased susceptibility of a disease state during adulthood. Atrazine, a common herbicide used throughout the Midwestern United States for control of weeds on various crops frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. Studies suggest atrazine elicits reproductive dysfunction through the hypothalamus-pituitary-gonadal (HPG) axis. In previous studies, zebrafish were exposed to 0.3, 3, or 30 parts per billion (ppb; ug/L) atrazine through embryogenesis, rinsed, and allowed to mature to adulthood. A decrease in spawning was observed in adults with an embryonic exposure. In addition, adult females had an increase in progesterone and ovarian follicular atresia, alterations in levels of a serotonin metabolite and serotonin turnover in brain tissue, and transcriptome alterations in brain and ovarian tissue supporting neuroendocrine alterations from an embryonic atrazine exposure were reported. Furthermore, offspring of the exposed population exhibited morphological alterations. As reproductive dysfunction may also be influenced by males, this study assessed transcriptomic profiles of brain and testes, morphology, histology, and hormone levels in adult males exposed to atrazine during embryogenesis. Transcriptomic profiles of adult male brain tissue revealed alterations in genes associated with cell to cell signaling and interaction of neurotransmission, cell morphology, cellular assembly and organization of neurites, cellular function and maintenance of neuritogenesis and synaptogenesis, and molecular transport of hormones. In addition, the transcriptomic profiles of adult male testes tissue demonstrated alterations in genes associated with lipid metabolism, molecular transport of steroids, small molecule biochemistry of lipids, and cell morphology. The embryonic atrazine exposure resulted in no alterations in body or testes weight, gonadosomatic index, testes histology, or levels of 11-ketotestosterone or testosterone in adult male zebrafish. Thus, although the transcriptomics data indicate later-in-life alterations on the neuroendocrine system of adult males exposed to atrazine during embryogenesis, analysis of additional endpoints is needed to determine functional impairments.

overall_design: We treated zebrafish embryos from approximately 1 hour post fertilization through 72 hours post fertilization (embryogenesis) to 0, 0.3, 3, or 30 parts per billion (ppb) atrazine. Following the 72 hour exposure period, larvae were rinsed and allowed to mature under normal conditions until adulthood (5-6 months post fertilization). At this time, zebrafish were bred weekly for three weeks in order to initiate breeding cycles. Following this peroid, gonadal tissue was dissected and processed for transcriptomic analysis.

first: Maria

middle: S

last: Sepùlveda

first: Gregory

last: Weber

first: Amber

last: Jannasch


title: Embryonic atrazine exposure elicits neuroendocrine dysfunction in adult male zebrafish (brain)

overall_design: We treated zebrafish embryos from approximately 1 hour post fertilization through 72 hours post fertilization (embryogenesis) to 0, 0.3, 3, or 30 parts per billion (ppb) atrazine. Following the 72 hour exposure period, larave were rinsed and allowed to mature under normal conditions until adulthood (5-6 months post fertilization). At this time, zebrafish were bred weekly for three weeks in order to initiate breeding cycles. Following this peroid, gonadal tissue was disseceted and processed for transcriptomic analysis.

first: Sara

last: Wirbisky


title: Embryonic atrazine exposure elicits neuroendocrine dysfunction in adult male zebrafish

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

overall_design: Refer to individual Series


title: An embryonic atrazine exposure results in reproductive dysfunction in adult zebrafish and morphological alterations in their offspring

status: Public on Mar 02 2016

summary: The herbicide atrazine, a suspected endocrine disrupting chemical (EDC), frequently contaminates potable water supplies. Studies suggest alterations in the neuroendocrine system along the hypothalamus-pituitary-gonadal axis; however, most studies address either developmental, pubertal, or adulthood exposures, with few investigations regarding a developmental origins hypothesis. In this study, zebrafish were  exposed to 0, 0.3, 3, or 30 parts per billion (ppb) atrazine through embryogenesis and then allowed to mature with no additional chemical exposure. Reproductive function, histopathology, hormone levels, offspring morphology, and the ovarian transcriptome were assessed. Embryonic atrazine exposure resulted in a significant increase in progesterone levels in the 3 and 30 ppb groups. A significant decrease in spawning and a significant increase in follicular atresia in the 30 ppb group were observed. In offspring, a decrease in the head length to body ratio in the 30 ppb group, along with a significant increase in head width to body ratio in the 0.3 and 3 ppb groups occurred. Transcriptomic alterations involved genes associated with endocrine system development and function, tissue development, and behavior. This study provides evidence to support atrazine as an EDC causing reproductive dysfunction and molecular alterations in adults exposed only during embryogenesis and morphological alterations in their offspring.

overall_design: We treated zebrafish embryos from approximately 1 hour post fertilization through 72 hours post fertilization (embryogenesis) to 0, 0.3, 3, or 30 parts per billion atrazine. Following the 72 hour exposure period, larvae were rinsed and allowed to mature under normal conditions until adulthood (5-6 months post fertilization). At this time, zebrafish were bred weekly for three weeks in order to initiate breeding cycles. Following this peroid, gonadal tissue was dissected and processed for transcriptomic analysis.

first: Tsang-Long

last: Lin


title: Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress

status: Public on Jan 25 2017

city: Rehovot

first: Igor

last: Ulitsky

address: Hertzl St.

country: Israel

institute: Weizmann Institute of Science

summary: Transcription and translation are highly energy consuming processes and both are modulated by the intracellular energy status. Transcription start site (TSS) selection and alternative promoter (AP) usage contribute to the complexity of gene expression but little is known about their impact on translation in response to metabolic deficiencies. Here we performed TSS mapping of the translatome following energy stress. We assessed the contribution of cap-proximal TSS nucleotides and strikingly found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we analyzed its binding to capped-RNAs with different initiating nucleotides and found differential affinity, the lowest being for 5’cytidine, in correlation with the translational response. In addition the number of differentially translated APs was highly elevated following energy stress, suggesting that AP usage is central to the stress response. These include novel glucose-starvation-induced downstream promoters for the translation regulators eIF4A and Pabp which are translationally-induced despite the general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. These findings uncovered several levels of coordination of transcription and translation responses to energy stress.

overall_design: Cap-seq of RNA from three polysomal fractions in control and glucose starvaion (GS) conditions, in two replicates

first: Rivka

last: Dikstein


title: Embryonic exposure to 10 µg/L lead results in female-specific expression changes in genes associated with nervous system development and function and Alzheimer’s disease in aged adult zebrafish brain

status: Public on Feb 08 2016

summary: A developmental lead (Pb) exposure has been proposed as an environmental risk factor for adult neurodegenerative diseases including Alzheimer’s disease (AD). Recent animal studies showed pathological characteristics of AD in adults with a developmental Pb exposure, but additional studies are needed to investigate this phenomenon. To further assess the relationship between an embryonic Pb exposure and latent neurological alterations, the brain of adult female and male zebrafish aged 12 months that were exposed to a control treatment or 10 µg/L Pb only during embryogenesis (1- 72 hours after fertilization) were analyzed on a zebrafish-specific microarray platform. Gene ontology and pathway analysis revealed similarities in the top diseases and functional categories in both sexes, but females had 4.3 times more genes altered than males. In addition, alterations in genes associated with nervous system development and function were more pronounced with a set of 89 genes associated with AD including amyloid precursor protein (APP), apolipoprotein (APOE), and sortlin-related receptor precursor (SORL1) observed to be changed in adult females. Our observations suggest that an  embryonic exposure to Pb at levels as low as 10 μg/L disturb global gene expression patterns in a sex-specific manner that could lead to neurological alterations in later life. With these findings, future studies investigating the adverse neurological outcomes of these changes in gene expression will facilitate our understanding of the impact of an embryonic 10 μg/L Pb exposure on neurological disease pathogenesis and the inclusion of additional concentrations will broaden our knowledge of dose-dependent changes.

overall_design: A developmental exposure to 10ppb Pb induced global gene expression changes were analyzed in 12 months aged male and female zebarfish brains. A developmental exposure was done shortly after fertilization through 72 hours after fertilization.

first: Jinyoung

last: Lee


title: Atrazine Exposure Elicits Copy Number Alterations in the Zebrafish Genome

status: Public on Feb 20 2017

type: Genome variation profiling by genome tiling array

summary: Atrazine is an agricultural herbicide used throughout the Midwestern United States that frequently contaminates potable water supplies resulting in human exposure. Using the zebrafish model system, an embryonic atrazine exposure was previously reported to decrease spawning rates with an increase in progesterone and ovarian follicular atresia in adult females. In addition, alterations in genes associated with distinct molecular pathways of the endocrine system were observed in brain and gonad tissue of the adult females and males. Current hypotheses for mechanistic changes in the developmental origins of health and disease include genetic (e.g., copy number alterations) or epigenetic (e.g., DNA methylation) mechanisms. As such, in the current study we investigated whether an atrazine exposure would generate copy number alterations (CNAs) in the zebrafish genome. A zebrafish fibroblast cell line was used to limit detection to CNAs caused by the chemical exposure. First, cells were exposed to a range of atrazine concentrations and a crystal violet assay was completed, showing confluency decreased by ~60% at 46.3 µM. Cells were then exposed to 0, 0.463, 4.63, or 46.3 µM atrazine and array comparative genomic hybridization completed. Results showed 34, 21, and 44 CNAs in the 0.463, 4.63, and 46.3µM treatments, respectively. Furthermore, CNAs were associated with previously reported gene expression alterations in adult male and female zebrafish. This study demonstrates that atrazine exposure can generate CNAs that are linked to gene expression alterations observed in adult zebrafish exposed to atrazine during embryogenesis providing a mechanism of the developmental origins of atrazine endocrine disruption.

overall_design: The objective of this study was to identify whether an atrazine exposure will generate copy number alterations. First, a zebrafish fibroblast cell line was exposed to a range of atrazine concentrations (0-1.125 mM) to determine concentrations where cell confluency would be altered (n=4 plates with 4 subsamples of each treatment concentration on each plate). From this experiment, treatment concentrations (0, 0.463, 4.63 and 46.3 um atrazine) were chosen to represent a range from no effect on confluency to decreasing cell confuency to ~60% in this cell line. Cells were then exposed to these four treatment concentrations and array CGH was used to detect copy number aberrations (CNAs) in atrazine treatments compared to the control treatment (n=3). qPCR was used to confirm CNAs. CNAs were compared to gene expression microarray data from adult male and adult female zebrafish gonad and brain tissue from individuals that had been exposed to atrazine during embryogenesis (GSE72242, GSE72243, GSE73740).


title: DNA methylation in lung cells is a key modulator of asthma endotypes and genetic risk


title: Embryonic atrazine exposure alters zebrafish and human miRNAs associated with angiogenesis, cancer, and neurodevelopment

status: Public on Apr 21 2016

summary: The study of epigenetic mechanisms of gene regulation and the role of these mechanisms in developmental reprogramming of the genome and disease susceptibility has increased in recent years. Molecular epigenetic mechanisms regulating gene expression include DNA methylation, histone modifications, and small non-coding RNAs (e.g., microRNAs). MicroRNAs (miRNAs) are short, single-stranded RNA that regulate post-transcriptional control of the translation of mRNA. The importance of these epigenetic regulators in controlling developmental processes is becoming clear. However, to date, few studies have investigated the role of miRNAs in toxicological responses limiting knowledge of these critical regulators in mechanisms of toxicity. Atrazine is a herbicide commonly used throughout the Midwestern United States and other areas of the globe for control of weeds in agricultural crops. In certain areas of the United States, atrazine frequently contaminates potable water supplies above the maximum contaminant level of 3 parts per billion (ppb; mg/L) as set by the United States Environmental Protection Agency (U.S. EPA) and is a suspected endocrine disrupting chemical. Studies have begun to investigate the genetic mechanisms of atrazine toxicity; however, studies investigating epigenetic mechanisms are limited. Here we report that expression of both zebrafish and human miRNAs are significantly altered in response to a developmental atrazine exposure of 0.3, 3, or 30 ppb from 1-72 hours post fertilization (hpf). Altered miRNAs are known to play a role in angiogenesis, cancer, or neuronal development, differentiation, and maturation. Targeted analysis of altered human miRNAs with genes previously identified to be altered by atrazine exposure revealed several targets linked to cell cycle and cell signaling. Further analysis of hsa-miRNA-126-3p which had altered expression in all three atrazine treatments at 72 hpf revealed alterations also occurred at 60 hpf in the 30 ppb treatment group. Results from this study indicate miRNA deregulation in zebrafish and human miRNAs following an embryonic atrazine exposure in zebrafish.

platform_organism:
Danio rerio
Homo sapiens

overall_design: We treated zebrafish embryos from approximately 1 hour post fertilization through 72 hours post fertilization (embryogenesis) to 0, 0.3, 3, or 30 parts per billion (ppb) atrazine. Following the 72 hour exposure period, total RNA was extracted and a miRNA microarray was completed.

first: Kelly

last: Schlotman

last: Sepúlveda


title: Embryonic exposure to 10 µg/L lead results in male-specific expression changes in genes associated with nervous system development and function and Alzheimer’s disease in aged adult zebrafish brain


title: Embryonic exposure to 10 µg/L lead results in sex-specific expression changes in aged adult zebrafish brain


title: Muscle transcriptome analysis following Total Knee Arthroplasty with Tourniquet

status: Public on Feb 01 2016

city: Eugene

first: Hans

middle: Christian

last: Dreyer

state: Oregon

address: 1240 University of Oregon

department: Human Physiology

institute: University of Oregon

summary: Transcriptome profiling was performed on muscle biopsies from patients immediately before Total Knee Arthroplasty and two hours after TKA and tourniquet application.

overall_design: RNA was isolated from 10 patients who were give vastus lateralis muscle biopsies immediately before surgery and 2 hours post surgery with tourniquet

first: Douglas

last: Turnbull

first: Jonathan

last: Muyskens


title: Mitochondrial dysfunction, disruption of F-actin polymerization, and transcriptomic alterations in zebrafish larvae exposed to trichloroethylene

summary: Trichloroethylene (TCE) is primarily used as an industrial degreasing agent and has been in use since the 1940s. TCE is released into the soil, surface, and groundwater. From an environmental and regulatory standpoint more than half of Superfund hazardous waste sites on the National Priority List are contaminated with TCE. Occupational exposure to TCE occurs primarily via inhalation, while environmental TCE exposure also occurs through ingestion of contaminated drinking water. Current literature links TCE exposure to various adverse health effects including cardiovascular toxicity. Current studies aiming to address developmental cardiovascular toxicity utilized rodent and avian models with the majority of studies using relatively higher parts per million (ppm; mg/L) doses. In this study to further investigate developmental cardiotoxicity of TCE, zebrafish embryos were treated with 0, 10, 100, or 500 parts per billion (ppb; μg/L) TCE during embryogenesis and/or through early larval stages. After the appropriate exposure period, angiogenesis, F-actin polymerization, and mitochondrial function were assessed. A significant dose response decrease in angiogenesis, F-actin polymerization, and mitochondrial function was observed. To further complement this data, a transcriptomic profile of zebrafish larvae was completed to identify gene alterations associated with the 10 ppb TCE exposure. Results from the transcriptomic data revealed that an embryonic TCE exposure caused significant changes in genes associated with cardiovascular disease, cancer, and organismal injury and abnormalities with a number of targets in the FAK signaling pathway. Overall, results from our study further support TCE as a developmental cardiovascular toxicant and continued priority for environmental regulation.

overall_design: To identify whether an embryonic TCE exposure alters heart development, zebrafish embryos were exposed to 0, 10, 100, or 500 ppb TCE during embryogenesis and/or through early larval stages. Following the exposure periods, angiogenesis, F-actin polymerization, and mitochondrial potential were analyzed. In addition, a transcriptomic profile of zebrafish larvae was completed to identify gene alterations associated with the lowest exposure concentration of 10 ppb of TCE.

first: Nur

last: Damayanti

first: Cecon

middle: T

last: Mahapatra

first: Joseph

last: Irudayaraj


title: Transcriptome and/or marker frequency analysis of S. pneumoniae (control vs. HPUra/Kanamycin/Ciprofloxacin/Hydroxyurea/Rifampicin)

status: Public on Jan 08 2016

city: Groningen

first: Jelle

last: Slager

address: Nijenborgh 7

department: Molecular Genetics

institute: University of Groningen

summary: Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra- and interspecies competition within the human host. What triggers pneumocin expression is poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39 and show that several antibiotics activate the blp-genes. Real-time gene expression measurements showed that while the promoter driving expression of the two-component regulatory system blpS/H was constitutive, the remaining blp-promoters (e.g. controlling pneumocin expression, immunity and the inducer peptide BlpC) was pH-dependent, induced in the late exponential phase and occurred in all cells. Intriguingly, competence for genetic transformation, mediated by the ComD/E two-component quorum system, was induced by the same stimuli. To test for regulatory interplay, we utilized synthetic BlpC and competence-stimulating peptide (CSP). Strikingly, immediately upon addition of CSP, the blp-promoters were activated in a comD/E dependent manner. After a delay, blp-expression was highly induced but strictly dependent on the presence of blpRH and blpC. This raised the question how BlpC is exported since phylogenetic analysis showed that the putative exporter for BlpC, blpAB, is not intact in strain D39 and most other strains. However, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. In fact, we show that high expression of the blp-genes requires comAB. Together, we demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might promote survival under antibiotic stress by killing and liberating DNA from neighboring (antibiotic-resistant) bacteria, which can then be used for transformation or repair.

overall_design: Pairwise comparison of untreated and antibiotic-treated cells (either DNA-seq or RNA-seq). It was performed with deep sequencing, using an Illumina HiSeq 2000 machine with 100 nt paired-end reads. Control and  HPUra-treated samples were analysed from duplicate samples, while other conditions were from single samples.

first: M

last: Kjos

first: E

last: Miller

first: J

first: F

middle: B

last: Lake

first: O

last: Gericke

first: D

last: Rozen

middle: W

last: Veening


title: 5-Day Gavage Study of 4-Methylcyclohexanemethanol (CASRN: 34885-03-5) in Male Harlan Sprague Dawley Rats

status: Public on Dec 31 2015

city: Durham

first: Scott

last: Auerbach

address: 111 TW Alexander Drive

institute: Division of the National Toxicology Program at NIEHS

summary: The goal of this study was to characterize the potential toxicity and genomic benchmark dose  of 4-methylcyclohexylmethanol in liver and kidney of male Harlan Sprague Dawley rats using a 5 day dose-response toxicogenomics study design. The 5 day study is used to quickly identify the dose levels where changes in molecular pathways occur. These dose level where pathway level effects begin to occur have been shown to provide a close approximation of a no effect dose level from more resource intensive guideline toxicological assessments.

overall_design: A total of 58 samples were evaluated in this study. 29 were from liver and 29  were from kidney. Five biological replicate were obtained for the 0 mg/kg/day control group for both liver and kidney. All other non-zero dose groups contain 4 biological replicates. The control groups for liver and kidney are the 0 mg/kg dose groups for those organs.


title: 5-Day Gavage Study of Propylene glycol phenyl ether (PPH; CASRN 770-35-4) in Male Harlan Sprague Dawley Rats

summary: The goal of this study was to characterize the potential toxicity and genomic benchmark dose  of propylene glycol phenyl etherl in liver and kidney of male Harlan Sprague Dawley rats using a 5 day dose-response toxicogenomics study design. The 5 day study is used to quickly identify the dose levels where changes in molecular pathways occur. These dose level where pathway level effects begin to occur have been shown to provide a close approximation of a no effect dose level from more resource intensive guideline toxicological assessments.

overall_design: A total of 49 samples were evaluated in this study. 25 were from liver and 24  were from kidney. Five biological replicate were obtained for the 0 mg/kg/day control group for both liver and kidney. All other non-zero dose groups contain 4 biological replicates. The control groups for liver and kidney are the 0 mg/kg dose groups for those organs.


title: West Virginia Chemical Spill


title: 5-Day Gavage Study of Crude 4-Methylcyclohexanemethanol in Male Harlan Sprague Dawley Rats

summary: The goal of this study was to characterize the potential toxicity and genomic benchmark dose  of crude 4-methylcyclohexylmethanol in liver and kidney of male Harlan Sprague Dawley rats using a 5 day dose-response toxicogenomics study design. The 5 day study is used to quickly identify the dose levels where changes in molecular pathways occur. These dose level where pathway level effects begin to occur have been shown to provide a close approximation of a no effect dose level from more resource intensive guideline toxicological assessments.

overall_design: A total of 44 samples were evaluated in this study. 27 were from liver and 17 were from kidney. Signficant batch effects were detected with a subset of samples and these were removed from the study prior to data analysis and are therefore not included in this GEO series. 3 (kidney) or 4 (liver) biological replicate were obtained for the 0 mg/kg/day control group for both liver and kidney. All other non-zero dose groups contain 2-4 biological replicates. The groups that contain only 2 or 3 replicates do so due to a number of samples not passing QC as byproduct of batch effects. The control groups for liver and kidney are the 0 mg/kg dose groups for those organs.


title: Gene expression analysis in normal and regenerating liver from lean mice and mice with diet induced obesity

status: Public on Jul 14 2015

city: Portland

first: Teresa

last: Zimmers

state: OR

address: 3181 S.W. Sam Jackson Park Road

department: Cell, Developmental & Cancer Biology

institute: Oregon Health & Science University

summary: Analysis of gene expression after sham surgery, 70% hepatectomy or 80% hepatectomy in normal mice or mice made obese through feeding of a high-fat diet.

overall_design: Diet-induced obese (DIO) and lean control male C57BL/6J mice were obtained from Jackson Laboratories and maintained in our colony by feeding of the same dietary forumlation used by Jackson Labs. After several weeks of acclimation, mice were subjected to sham surgery (midline incision and liver manipulation only), 70% hepatectomy or 80% hepatectomy. Mice were euthanized under isoflurane anesthesia 6 hours later. Total RNA was isolated from snap frozen liver and subjected to Illumina gene expression analysis.

first: Xiaoling

last: Jin

first: Zongxiu

last: Zhang

first: Leonidas

last: Koniaris


title: After demyelination in the adult CNS, oligodendrocyte precursor cells revert to an immature mRNA profile and express immune cues favoring their migration

status: Public on Nov 06 2014

city: New York

first: Sarah

last: Moyon

address: 85 St Nicholas Terrace

department: Neuroscience

institute: CUNY ASRC

summary: In multiple sclerosis (MS) and experimental models of demyelination, myelin repair is mostly achieved by oligodendrocyte precursor cells (OPCs). To gain insight into the biological specificity of these adult precursors, in the perspective to better understand why remyelination often fails in MS, we performed a micro-array analysis on purified populations of murine OPCs and oligodendrocytes. We demonstrated that, in a control environment, “quiescent” adult OPCs are more mature than neonatal OPCs, with a mRNA profile closer to oligodendrocytes. However, when demyelination occurs, adult OPCs revert to an immature gene expression profile, acquiring higher migration and faster differentiation capacities. Among the many genes differentially regulated by these “activated” adult OPCs, genes of the innate immune response, IL1β and CCL2 were strongly upregulated after demyelination, with increased protein expression in experimental demyelinating lesions and within MS plaques. We first showed in vitro that both IL1β and CCL2 “account” for increased migration capacities of adult OPCs, an effect that is suppressed by corresponding receptor blockade. Focusing on Ccl2, we then demonstrated that lentiviral over-expression of Ccl2 increases motility of “quiescent” adult OPCs, which become as mobile as “activated” adult OPCs. This in vitro gain of function was further confirmed in vivo, by showing an increased migration of grafted CG4 oligodendroglial cells over-expressing-Ccl2. These results, demonstrating that “activated” adult OPCs upregulate immune cues favouring their migration, open new perspective of demyelination-induced immune-glial interactions, which might influence both oligodendroglial and inflammatory cells, therefore play a key role in lesion fate in multiple sclerosis.

overall_design: In this study, we loaded purified population of progenitors of oligodendrocytes (OPCs) (from neonatal, 2-month-old, 2-month-old and cuprizone-treated brains) and of mature oligodendrocytes (OLs) (2-month-old brains) and compared their gene expression. We compared gene expression of neonatal OPCs, adult OPCs, adult OLs and adult OPCs from control conditions and from demyelinating conditions, using 3 to 4 independant replicates in each group.

first: Marie

last: Aigrot

first: Matthew

last: Trotter

first: Jeffrey

last: Huang

first: Luce

last: Dauphinot

first: Anne

last: Debussy

first: Stéphane

last: Parsadaniantz

first: Robin

last: Franklin

first: Catherine

last: Lubetzki


title: Exposure of rat to a variety of toxicants, thigh muscle assayed by CodeLink microarray

status: Public on Aug 18 2014

summary: DrugMatrix is a comprehensive rat toxicogenomics database and analysis tool developed to facilitate the integration of toxicogenomics into hazard assessment.  Using the whole genome and a diverse set of compounds allows a comprehensive view of most pharmacological and toxicological questions and is applicable to other situations such as disease and development.

overall_design: Complete Drug Matrix dataset for rat thigh muscle. Approximately 600 different compounds were profiled in up to 8 different rat tissues by obtaining tissue samples from test compound-treated and vehicle control-treated rats in biological triplicates for gene expression analysis after 0.25, 1, 3, and 5 days of exposure with daily dosing. In a few studies (1.8%), 7 days of exposure was substituted for 5 days of exposure.  Samples were hybridized to CodeLink RU1 10K rat arrays (Amersham Biosciences).


title: Exposure of rat to a variety of toxicants, spleen assayed by Codelink microarray

overall_design: Complete Drug Matrix dataset for rat spleen. Approximately 600 different compounds were profiled in up to 8 different rat tissues by obtaining tissue samples from test compound-treated and vehicle control-treated rats in biological triplicates for gene expression analysis after 0.25, 1, 3, and 5 days of exposure with daily dosing. In a few studies (1.8%), 7 days of exposure was substituted for 5 days of exposure.  Samples were hybridized to CodeLink RU1 10K rat arrays (Amersham Biosciences).


title: Exposure of rat to a variety of toxicants, intestine assayed by CodeLink microarray

overall_design: Complete Drug Matrix dataset for rat intestine. Approximately 600 different compounds were profiled in up to 8 different rat tissues by obtaining tissue samples from test compound-treated and vehicle control-treated rats in biological triplicates for gene expression analysis after 0.25, 1, 3, and 5 days of exposure with daily dosing. In a few studies (1.8%), 7 days of exposure was substituted for 5 days of exposure.  Samples were hybridized to CodeLink RU1 10K rat arrays (Amersham Biosciences).


title: Human aldosterone producing cell clusters, zona glomerulosa, zona fasciculata, and zona reticularis, from adrenal glands

status: Public on Jun 26 2015

city: Ann Arbor

first: Rork

last: Kuick

state: MI

address: University of Michigan, SPH II, 1415 Washington Heights, Room M2533

institute: University of Michigan

summary: In order to analyze the transcriptome characteristics of aldosterone producing cell clusters (APCC) we compared transcript abundances of APCC, zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors.  The frozen adrenal glands in O.C.T. compound were cut into 7um sections, and every 10-th section immunostained for aldosterone synthase (CYP11B2).  The remaining sections were stained with cresyl violet and used for laser-capture microdissection of tissue to use in the array assays.  APCC and ZG samples were captured from CYP11B2 positive regions based on the CYP11B2-stained sections.  ZF and ZR were captured from lipid-rich cells in the middle layer and compact cells outside of the medulla, respectively.  RNA was isolated using PicoPure RNA isolation kits (Molecular Devices, Sunnyvale, CA). 1-10 ng total RNA was reverse-transcribed and amplified with the Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA).  cDNA was purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and biotin-labeled using Encore Biotin Module (NuGEN Technologies), followed by hybridization to GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). Expression values were calculated using the robust multi-array average method (RMA).  This resulted in base-2 log-transformed data for each of the 4 tissues from each of the 4 people.  In addition to the raw and processed data we also supply a supplementary Excel file holding the data and some statistical analysis, which has features to make simple graphs, and holds probe-set annotation that we used at that time (users may wish to obtain new annotation though). We fit two-way ANOVA models with terms for 4 tissues and 4 people, and compared each probe-set between every pair of tissues using F-tests for pairwise contrasts. We modeled people effects since they were not negligible.  The supplement shows how to calculate the tests.

overall_design: Aldosterone producing cell clusters (APCC), zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors, were individually assayed on 16 Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays.  Expression values were estimated with the Robust Multi-Arry average (RMA) algorithm, which resulted in log-2 transformed values for each of 54675 probe sets.

first: Koshiro

last: Nishimoto

middle: A

last: Tomlins

first: Andi

middle: C

last: Cani

first: Daniel

middle: H

last: Hovelson

first: Chia-Jen

last: Liu

first: Aalok

last: Sanjanwala

first: Michael

last: Edwards

first: Celso

last: Gomez-Sanchez

first: William

last: Rainey