title: The Complex Architecture of Transgene Insertions

status: Public on Mar 16 2018

city: La Jolla

first: Joseph

middle: R

last: Ecker

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

summary: Over the last 30 years the soil bacterium Agrobacterium tumefaciens has been the workhorse tool for plant genome engineering. Replacement of native tumor-inducing (Ti) plasmid elements with customizable cassettes enabled insertion of a sequence of interest as “Transfer DNA” (T-DNA) into the plant genome of interest. Although these T-DNA transfer mechanisms are well understood, detailed understanding of the structure and epigenomic status of insertion events was limited by current technologies. To fill this gap, we analyzed transgenic Arabidopsis thaliana lines from three widely used collections (SALK, SAIL and WISC) with two single molecule technologies, optical genome mapping and nanopore sequencing. Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements with unexpectedly large sizes ranging from 27 to 236 kilobases. De novo nanopore-based genome assemblies for two heterozygous lines resolved T-DNA structures up to 36 kb and revealed large-scale T-DNA associated translocations and exchange of chromosome arm ends. The multiple internally rearranged nature of T-DNA arrays, consisting of identical T-DNA/backbone concatemers made full assembly even for long nanopore reads impossible. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. This unprecedented nucleotide-level definition of T-DNA insertions enabled the mapping of epigenome data. The SALK_059379 T-DNA insertions were enriched for 24nt siRNAs and contained dense cytosine DNA methylation. Transgene silencing via the RNA directed DNA methylation pathway was confirmed by in planta assays. In contrast, SAIL_232 T-DNA sequence was predominantly targeted by 21/22nt siRNAs, and DNA methylation and silencing was limited to the GUS gene, but not the resistance gene. With the emergence of genome editing technologies that rely on Agrobacterium for gene delivery, this study provides new insights into the structural impact of engineering plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.

overall_design: 2 biological samples, each RNAseq, smallRNAseq and bisulfite sequencing

first: Florian

last: Jupe

first: Todd

last: Michael

first: Angeline

last: Rivkin

first: Huaming

last: Chen


title: Regenerative adaptation to electrochemical perturbation in planaria:  a molecular analysis of physiological plasticity

status: Public on Aug 31 2017

city: Gainesville

first: Christopher

last: Martyniuk

state: FL

address: 8891 SW 79th Avenue

country: USA

institute: University of Florida

type: Expression profiling by high throughput sequencing

summary: The development and maintenance of organismal form results from dynamic interactions between genome, protein interaction, physiology and the external environment.  Bioelectric signaling represents an important epigenetic layer of control over processes of large-scale patterning during development and regeneration. However it is still largely unknown how physiological plasticity and adaptation operate during regenerative processes. Planarian flatworms are an exceptional system in which to study the interplay between genetics and environment, as they are able to regenerate any tissue or organ after traumatic injury. Here, we study acquired tolerance to a bioelectric homeostasis-altering pharmacological treatment. After exposure to the non-selective potassium channel blocker BaCl2, D. japonica flatworms display extreme depolarization of the head and then violently degenerate their anterior tissues via an apoptotic mechanism. Remarkably, when kept in fresh barium solution, they then regenerate a head that displays normal bioelectric state and is non-responsive to the channel blocker. Transcriptomics using RNAseq was used to characterize transcriptional changes that occur during this adaptation process, identifying a number of ion translocators that are differentially expressed with BaCl2, including the BK channel which is resistant to barium blockade. Many of these channels were identified as cation transporters (cation-transporting ATPases, small conductance calcium activated potassium (SK) channels, and sodium/hydrogen exchangers), suggesting that the physiological buffering of a depolarizing treatment is dependent on employing alternative means of transporting cations into and out of anterior cells and tissues.  Moreover, pathway enrichment analysis revealed that transcriptional networks related to synaptic plasticity, nervous system development, neurotransmission, and nerve development were increased ~2 to 3-fold. Transcriptome responses were also activated for membrane steady potential, excitatory junction potential, and membrane hyperpolarization, all associated with the modulation of Vmem. The ability to adjust to physiological conditions that are strongly injurious to normal anatomy, by altering the transcriptome is a powerful adaptive capability of physiological circuits. Further studies on this plasticity will shed light on numerous regenerative and patterning mechanisms in the context of evolution, and will contribute to biomedical therapies that exploit this innate robustness.

overall_design: Two Control (WT) and two BaCl2 treated.  Total RNA was extracted from wild type D. japonica, D. japonica starved for 35 days, and D. japonica with BaCl2-immune heads. Three biological replicates were obtained from each treatment, with 25 worms per replicate. For the RNAseq experiment, worms were kept in BaCl2 for 35 days, when insensitive heads had fully regenerated. Heads were then amputated, and RNA extracted. Total RNA from whole worms, and worm head fragments was extracted using Trizol (Ambion/ThermoFisher) purification (0.5mL/25 worms). Samples were suspended in two washes of chloroform, and a final isopropanol wash was carried out after pelleting and removal of phenol-chloroform phase. Total RNA was pelleted and then suspended in 80% EtOH. Samples were then shipped to the MIT sequencing core. Quality control for RNA was conducted on an 2100 Agilent Bioanalyzer and RNA was deemed high quality.

first: Maya

last: Emmons-Bell

first: Fallon

last: Durant

middle: J

first: Junji

last: Morokuma

first: Michael

last: Levin


title: Thyroid cancer: tumor vs. precancerous lesions

status: Public on Aug 30 2017

city: chengdu

first: qiheng

last: gou

address: southern people street 3rd 17

country: China

institute: sichuan university

summary: Multiple lines of evidence have highlighted that long noncoding RNAs (lncRNAs) serve as a novel class of regulators of cancer biological processes. Currently, our knowledge of lncRNAs in papillary thyroid carcinoma (PTC) is still limited. Using microarray analysis, we identified a lncRNA (lncAB), which is downregulated in PTC. The decreased expression of lncAB is induced by CpG hypermethylation in the flanking region of lncAB. Overexpression of lncAB inhibited tumor growth and arrested G2/M phase in vitro and in vivo, whereas knockdown of lncAB promoted cell proliferation, colony formation, DNA replication ability, and increased G0/G1 phase. Mechanistic analyses revealed that lncAB binds to KHSRP, resulting in the mRNA degradation, high expression of CDKN1a and low expression of CDK2, and thereby repressed cell proliferation. Taken together, we provide evidence that lncAB is a tumor suppressor in PTC tumorigenesis and delineate a novel mechanism of lncRNA in PTC progression.

overall_design:
The present study include three adults diagnosed as thyroid cancers. Total RNA was isolated for gene expression analysis that compares the two groups - tumor (n=3) and precancerous lesions (n=3) - in a pairwise manner.

"lncAB" is a novel isoform of lncRNA AB074169, which was identified by RACE in thyroid cell line Nthy-ori 3. We have uploaded both isoform sequences to Genbank: BankIt2041199 Seq1(MF681781) and Seq2 (MF681782).

first: Qiheng

last: Gou

first: Linbo

last: Gao

first: Xinwen

last: Nie


title: Dieldrin augments mTOR signaling and inhibits lysosomal acidification in the adult zebrafish heart (Danio rerio)

status: Public on Sep 24 2016

summary:
Rationale: Dieldrin is a legacy organochlorine pesticide that is persistent in the environment, despite being discontinued from use in North America since the 1970s. Some epidemiological studies suggest that exposure to dieldrin is associated with increased risks of neurodegenerative disease and breast cancer by inducing inflammatory responses in tissues as well as oxidative stress. However, the direct effects of organochlorine pesticides on the heart have not been addressed adequately to date. This is a significant knowledge gap because these chemicals are detectable in human serum and are environmentally persistent, thus individuals may show latent adverse effects due to chronic, low dose exposure over time.

Objective: To determine whether low level exposure to dieldrin at an environmentally relevant dose results in aberrant molecular signaling in the vertebrate heart.

Methods and Results: Using transcriptomic profiling and immunoblotting, we determined the global gene and targeted protein expression response to dieldrin treatment, and show that dieldrin effects gene regulator networks in the heart that are associated with protein degradation. Our results show that genes related to the development of cardiovascular disease, specifically cardiac arrest and ventricular fibrillation, are affected by environmentally relevant levels of dieldrin. We find that genes regulating inflammatory responses, a significant risk factor for cardiovascular disease, are upregulated by dieldrin treatments. Transcripts related to lysosomal function, also important to heart function, are significantly downregulated. To verify these findings, proteins in these pathways were examined with immunoblotting, and our data suggest that dieldrin constitutively activates Akt/mTOR signalling and downregulates lysosomal genes, which is hypothesized to be mediated through Transcription Factor EB (TFEB), the master regulator of lysosome function.  Conclusions: This study is one of few to report that dieldrin alters molecular signaling cascades in the cardiovascular system and proposes a novel mechanism for pesticide-induced cardiotoxicity.

overall_design: Female zebrafish hearts were pooled into 15 tubes. Final sample sizes for microarray analysis were as follows: Control group (n=5), fish fed the 0.15 μg/g dose diet (n=6), and (n=4) fish fed the 1.8 μg/g dose diet. The fish were fed these diets for 21d.

first: L

last: Slade

first: A

last: Cowie

first: C

first: P

middle: C

last: Kienesberger

first: T

last: Pulinilkunnil


title: The effects of fresh oil sands process water, ages oil sands process water, and pure naphthenic acids on fathead minnow larval transcriptome.

status: Public on Oct 31 2016

summary: In this study, fathead minnow (FHM) embryos were exposed to two doses of fresh, aged and pure naphthenic acids mixtures until hatch. Larvae were scored for developmental deformities prior to preservation for gene expression analyses. 5 larvae were pooled per gene expression replicate.

overall_design:
There were 27 samples analyzed:  3) fresh oil sands, salt control;  3)  fresh oil sands, 10ppm; 3) fresh oil sands, 5ppm; 3) aged oil sands, salt control; 3) aged oil sands 10ppm; 3) aged oil sands, 20ppm; 3) pure naphthenic acids, salt control; 3) pure naphthenic acids 1.25ppm; 3) pure naphthenic acids 2.5ppm
In this study, a dose response of three sources of naphthenic acids were investigated in larval fathead minnow after an embryo to hatch exposure period.

first: Julie

last: Marrentette

first: Jennifer

last: Loughery

first: Chris


title: The effects of waterbourne phenanthrene exposure on teleost hypothalamic transcriptome

status: Public on Aug 25 2016

first: Christopher

summary: In this study, female fathead minnows (FHM) were exposed to waterbourne phenanthrene (201.8 µg/L) or a solvent control for 7 weeks. Fish were tested for behavioral differences in a modified behavioral test prior to euthansia. Hypothalami were excised and stored for microarray analyses.

overall_design:
Fish were exposed to one dose of phenanthrene. Female and male hypothalami were analyzed (n=8 per group, control vs. treatment); liver was also analyzed. N=7 control and 8 phenanthrene.
In this study, a subchronic exposure to phenanthrene was investigated


title: Gene expression analyses of the differentiating hMSC into Osteoblasts and Adipocytes.

status: Public on Mar 15 2017

city: Rotterdam

first: Jeroen

last: van de Peppel

address: 's Gravendijkwal 230

country: Netherlands

institute: ErasmusMC

summary: Age-related skeletal degeneration in patients with osteoporosis is characterized by decreased bone mass and occurs concomitant with an increase in bone marrow adipocytes. Using microarray expression profiling with high temporal resolution, we identified gene regulatory events in early stages of osteogenic and adipogenic lineage commitment of human mesenchymal stromal cells (hMSCs). Data analysis reveal three distinct phases when cells adopt a committed expression phenotype: initiation of differentiation (0-3h, Phase I), lineage-acquisition (6-24h, Phase II) and early lineage-progression (48-96h, Phase III). Upstream regulator analysis identifies 34 transcription factors (TFs) in Phase I with a role in hMSC differentiation. Interestingly, expression levels of identified TFs did not always change and indicate additional post-transcriptional regulatory mechanisms. Functional analysis reveals that forced expression of IRF2 enhances osteogenic differentiation. Thus, IRF2 and other ‘early-responder‘ TFs may control osteogenic cell fate of MSCs and should be considered in mechanistic models that clarify bone-anabolic changes during clinical progression of osteoporosis.

overall_design: Total RNA obtained from hMSC cultured in Osteogenic or Adipogenic differentiation medium . Each samples consist of 3 pooled wells and for each timepoint we have generated 3 biologcial replicates. (for the non-differentiated cells 6 replicates)

first: Tanja

last: Strini

first: Julia

last: Tilburg

first: Hans

last: Westerhoff

first: Andre

last: van Wijnen

first: Johannes

middle: P

last: van Leeuwen


title: Determining the effects of the loss of sympathetic drive in bone, hypothalamus, and gut in adrenergic B1 and B2 receptor knockout mice [hypothalamus]

status: Public on Jul 01 2016

last: Martyniuk

summary: Eight weeks old male Adrb1tm1BkkAdrb2tm1Bkk/J , stock number 003810, were purchased from The Jackson Laboratory. These mice are homozygous null for the Adrb1 and Adrb2 genes, and are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. Mice were euthanized, and the whole bone marrow was extracted using established methods.1 Whole bone marrow cells from Adrb1tm1BkkAdrb2tm1Bkk/J mice were reconstituted into lethally- irradiated (950 Rad) C57BL/6J mice using a single retro-orbital injection at a ratio of 1:4 (Adrb1tm1BkkAdrb2tm1Bkk/J to C57BL/6J). All reconstituted mice were recovered for 2-3 months prior to tissue collection for mRNA arrays (1) The FASEB Journal 2015:29(1),652.13.

overall_design: Eight control animals and eight animals that received new bone marrow from double adrenergic receptor knockout (treated KO chimera). Following recovery, tissues that were harvested included bone marrow, brain, and distal colon.

first: Niousha

last: Ahmari

first: Jordan

last: Schmidt

last: Martnyiuk

first: Jasenka

last: Zubcevic


title: The non-coding variant rs1800734 enhances DCLK3 expression through long-range interaction and promotes colorectal cancer progression

status: Public on Feb 15 2017

first: Ning Qing

last: Liu

address: Wytemaweg 80

department: Hematology

institute: Erasmus University Medical Center Rotterdam

type: Genome binding/occupancy profiling by high throughput sequencing

type: Non-coding RNA profiling by high throughput sequencing

summary: Genome-wide association studies (GWAS) have identified a great number of non-coding risk variants for colorectal cancer (CRC). To date, the majority of these variants have not been functionally studied. Identification of allele-specific transcription factor (TF) binding is of great importance to understand regulatory consequences of such variants. A recently developed proteome-wide analysis of disease-associated SNPs (PWAS) enables identification of DNA-TF interactions in an unbiased fashion. Here, we performed a large-scale PWAS studies to comprehensively characterize TF binding related to CRC, which identified 731 allele-specific TF binding at 116 CRC risk loci. This screen identified rs1800734 within the promoter region of MLH1 as perturbing the binding of TFAP4 and consequently increaseing DCLK3 expression through a long-range interaction, which  promotes cancer progression through enhancing expression of the genes related to epithelial-to-mesenchymal transition (EMT).

overall_design:
Determine functional SNPs related to colorectal cancer risk, and investigate functional interaction between rs1800734 and DCLK3 in colorectal cancer progression as well as the molecular basis behind this interaction
Histone ChIP-seq (HCT116 and LoVo) and DNase I-seq (CaCO2, COLO205, GP5d, HT-29, HUTU80, RKO, SK-CO-1, SW480, SW1116, T-84 and LoVo) were used to define regulatory SNPs. Transcription factor ChIP-seq (SNU175 and COLO320), targeted RNA-/DNA-seq (SNU175 and COLO320), and ATAC-seq (SNU175 and COLO320) were used to inspect allele-specific binding, expression and hypersensitivity of rs1800734. 4C-seq and ATAC-seq on isogenic COLO320 cells (G/G, G/A, and A/A genotypes at rs1800734) were used to study the molecular basis of rs1800734 in regulating DCLK3 expression. Sequencing were performed using HiSeq2000 and NextSeq 500. Targeted RNA-seq on DCLK3 mRNA confirmed the transcription of this gene in isogenic COLO320 cells.

first: Ning

middle: Q

first: Hendrik

middle: G

last: Stunnenberg

first: Jian

last: Yan

first: Jussi

last: Taipale


title: Secretoneurin A regulates neurogenic and inflammatory transcriptional networks in radial glia of the goldfish brain

status: Public on Feb 22 2018

summary: Radial glial cells (RGCs) are the most abundant macroglia in the teleost brain and have established roles in neurogenesis and neurosteroidogenesis; however, their transcriptome remains unstudied, limiting functional understanding of this crucial cell type. Using cultured goldfish RGCs, RNA sequencing and de novo transcriptome assembly were performed, generating the first reference transcriptome for fish RGCs with 17,620 unique genes identified. These data revealed that RGCs express a diverse repertoire of receptors and signaling molecules, suggesting that RGCs may respond to and synthesize an array of hormones, peptides, cytokines, and growth factors. Expanding on recent neuroanatomical data and possible direct neuronal regulation of RGC physiology, differential gene expression analysis identified transcriptional networks that are responsive to the conserved secretogranin II-derived neuropeptide secretoneurin A (SNa). Pathway analysis indicated that cellular processes related to the central nervous system (e.g., neurogenesis, synaptic plasticity, glial cell development) and immune functions (e.g., immune system activation, leukocyte function, macrophage response) were preferentially modulated by SNa.  These data reveal an array of new functions that are proposed to be critical to neuronal-glial interactions through the mediator SNa.

overall_design: Four control, three SNa (1000nM) treated and three SKF (10uM). SNa is sectretoneurin A peptide and SKF is a selective dopamine D1 receptor agonist SKF 38393. Passage three cells were exposed for 24hrs. Total RNA was extracted using the RNeasy Micro Kit (Qiagen) including an on-column DNase treatment to remove genomic DNA. The concentration of total RNA was determined using the Qubit RNA Assay Kit (Life Technologies). In order to evaluate the quality of the total RNA, RNA integrity number (RIN) was assessed using Agilent RNA 600 Nano Reagents and RNA Nano Chips in Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing of 10 RGC cultures was performed by MR DNA (www.mrdnalab.com) following the manufacturer’s guidelines (Illumina HiSeq) for paired end sequencing (151 bp). RNA with poly(A) tails were purified and fragmented into shorter sequences that were used as templates for cDNA synthesis. A total of 10 cDNA libraries were constructed using random-hexamer primers from 1 μg of total RNA from each sample using TruSeq RNA LT Sample Preparation Kits (Illumina).

first: Dillon

middle: F

last: Da Fonte

first: Lei

last: Xing

first: Adrian

last: Pelin

first: Nicolas

last: Corradi

first: Wei

last: Hu

first: Vance

middle: L

last: Trudeau


title: Interlab microarray comparison Lab E

status: Public on May 31 2016

first: Christopher

summary: Microarrays are used to assess the impacts of aquatic contaminants in both laboratory and field studies, which necessitates the validation of the comparability of microarray data generated by different laboratories before this tool is adopted for regulatory toxicology or environmental monitoring. Fundamental issues which need answering before microarray technology can be used in environmental monitoring programs include defining whether these molecular changes translate into adverse effect at the individual or population level, as well as how large a magnitude (threshold) and how to quantify (fold change versus p value) a modification or modifications in gene expression which constitute an adverse effect. Here, male FHM were exposed to 25 ng/L EE2 for 96 hr, and six laboratories received flash frozen liver to conduct a microarray analysis on using a 60K Agilent microarray. This study aimed to assess the congruency of microarray data across six independent laboratories given both control and EE2 exposed samples, and to identify gold standard estrogen-responsive genes triggered by treatment with an environmentally relevant EE2 dose.

overall_design: 8 biological replicates per group. 2 groups, control and EE2 treated.

first: Natalia

last: Garcia-Reyero


title: Interlab microarray comparison Lab B

summary: Microarrays are used to assess the impacts of aquatic contaminants in both laboratory and field studies, which necessitates the validation of the comparability of microarray data generated by different laboratories before this tool is adopted for regulatory toxicology or environmental monitoring.  Fundamental issues which need answering before microarray technology can be used in environmental monitoring programs include defining whether these molecular changes translate into adverse effect at the individual or population level, as well as how large a magnitude (threshold) and how to quantify (fold change versus p value) a modification or modifications in gene expression which constitute an adverse effect. Here, male FHM were exposed to 25 ng/L EE2 for 96 hr, and six laboratories received flash frozen liver to conduct a microarray analysis on using a 60K Agilent microarray. This study aimed to assess the congruency of microarray data across six independent laboratories given both control and EE2 exposed samples, and to identify gold standard estrogen-responsive genes triggered by treatment with an environmentally relevant EE2 dose.

overall_design: 8 biological replicates per group.  2 groups, control and EE2 treated.

first: April

last: Feswick

first: Meghan

last: Issacs

first: Chris

first: Kelly

last: Munkittrick


title: Determining the effects of the loss of sympathetic drive in bone, hypothalamus, and gut in adrenergic B1 and B2 receptor knockout mice [distal colon]

last: Martyniuk

overall_design: Seven control animals and seven animals that received new bone marrow from double adrenergic receptor knockout (treated KO chimera). Following recovery, tissues that were harvested included bone marrow, brain, and distal colon.

first: Niousha

last: Ahmari

first: Jordan

last: Schmidt

first: Jasenka


title: Interlab microarray comparison Lab C

first: Marianna

last: Brown-Augustine

last: Vulpe


title: Interlab microarray comparison Lab D

first: Lorraine

last: Brown

first: Graham

last: van Aggelen


title: Expression data from dendritic cells treated with IFNβ for 2.5 hours and control

status: Public on Feb 02 2016

city: Rochester

first: Juilee

last: Thakar

state: New York

address: 601 Elmwood Ave

department: Microbiology and Immunology

institute: University of Rochester

summary: We used microarray to characterize interferon stimulated genes in dendritic cells

overall_design: Monocyte-derived DCs were obtained from healthy human blood donors and were treated with IFNβ for 2.5 hours. Naive DCs served as negative control.

first: Steven

middle: H

last: Kleinstein


title: Low-dose exposure to 17alpha-ethinylestradiol rapidly regulates expression networks of gonad differentiation in the adult fathead minnow testes

status: Public on May 11 2016

summary: Environmental estrogens are ubiquitous in aquatic systems worldwide.  In wild fish inhabiting wastewater effluent with estrogenic components, the presence of both oocytes and spermatogenic tissue in the testes has been documented. However, the molecular networks underlying the manifestation of the intersex condition are not completely characterized. In this study, we used an environmentally relevant concentration of 17alpha-ethinylestradiol (EE2), the active component of the birth control pill, to determine the response of the fathead minnow testis transcriptome after 96 hrs. The goal was to capture early molecular initiating events that may lead to the intersex condition following exposure to an estrogen that is present in wastewater effluent. There were no effects of EE2 on morphometric endpoints such as gonadosomatic index nor proportion of gametes within the testes.  However, in vitro production of 11-ketotestosterone and testosterone in the testis was decreased relative to controls following EE2 exposure.  Gene expression profiling revealed that there were 10 transcripts that were differentially expressed in the testis using a 8x60K fathead minnow microarray, the most dramatic change being that of coagulation factor XIII A chain (20-fold increase).  Transcripts that included guanine nucleotide binding protein (Beta Polypeptide 2), peroxisome proliferator-activated receptor delta, and WNK lysine deficient protein kinase 1a, were down-regulated by EE2. Subnetwork enrichment analysis revealed that EE2 suppressed transcriptional networks associated with reproduction such as steroid metabolism, hormone biosynthesis, and sperm mobility. Most interesting was that gene networks associated with doublesex and mab-3 related transcription factor 1 (dmrt1) were suppressed in the adult testis, despite the fact that dnmt1 itself was not differentially expressed from controls. Moreover cell processes involving forkhead box L2 (foxL2) such as ovarian follicle development and granulosa cell development were increased in expression in the testis. Cell processes that included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway were decreased with EE2 while lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased, shedding light on the transcriptional responses that may be perturbed prior to gonadal remodelling following exposure to estrogenic contaminants.  These data demonstrate that low concentrations of EE2 rapidly suppresses male hormone secretion, suppresses gene networks related to male sex differentiation, and induces transcriptional networks related to female sexual differentiation in the adult testis. These responses are hypothesized to be the molecular initiating events that occur prior to the development of the intersex phenotype after prolonged exposures to estrogens.

first: Jennifer

last: Loughery


title: Interlab microarray comparison

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

overall_design: Refer to individual Series


title: Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation [human data]

status: Public on Jan 01 2016

summary: Endogenous bioelectric signaling via changes in cellular resting potential (Vmem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of Vmem has been functionally implicated in de-differentiation, tumorigenesis, anatomical re-specification, and appendage regeneration.  However, no unbiased analyses have been performed to understand genome-wide transcriptional responses to Vmem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to specific Vmem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (Axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both  sub-network enrichment and PANTHER analyses identified a number of key genetic modules as targets of Vmem change, and also revealed important (well-conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that Vmem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies.

overall_design: Whole bone marrow aspirate from a 25 year-old healthy man was purchased from Lonza through their Research Bone Marrow Donor Program, following approved guidelines of informed consent as previously documented (Sundelacruz et al. 2008, 2013b). Aspirate was plated at a density of 10 mL of aspirate per cm2 in control medium (DMEM with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 mg/mL), and 0.1 mM nonessential amino acids) supplemented with basic fibroblast growth factor (1 ng/mL) (Invitrogen). Cells were maintained in a humidified incubator at 37°C with 5% CO2. The hMSCs were isolated on the basis of their adherence to tissue culture plastic and were used for experiments between passages two and four.  Undifferentiated hMSCs were cultured in control medium. Osteogenic (OS) differentiation medium consisted of αMEM medium supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), 10mM β-glycerophosphate, 0.05mM L-ascorbic acid-2-phosphate, and 100nM dexamethasone (Sigma-Aldrich).  Vmem was depolarized by (1) addition of Na+/K+ - ATPase-inhibitor ouabain (OB, 10 nM; Sigma-Aldrich) to differentiation medium or (2) elevation of extracellular K+ by adding potassium gluconate (High K+, HK, 40 mM; Sigma-Aldrich) to differentiation medium. Depolarization induced by these concentrations of OB and K+ has been confirmed using voltage-sensitive dyes and/or sharp intracellular recordings [(Sundelacruz et al. 2008) and unpublished data]. OS cells were pre-differentiated for three weeks before the addition of OB or HK for an additional three weeks of culture. The depolarizers were added to the differentiation medium and replenished in subsequent media changes.

first: Vaibhav

last: Pai

first: Karen

last: Echeverri

first: Sarah

last: Sundelacruz

first: David

last: Kaplan

first: Michael

last: Levin


title: Determining the effects of the loss of sympathetic drive in bone, hypothalamus, and gut in adrenergic B1 and B2 receptor knockout mice [bone]

first: Christopher

last: Martnyiuk

last: Zubcevic


title: Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation [frog data]

last: Martyniuk

overall_design: Xenopus laevis embryos were fertilized in vitro according to standard protocols (Sive 2000) in 0.1X Marc’s Modified Ringer’s (MMR; 10mM Na+, 0.2mM K+,10.5mM Cl, 0.2 mM Ca2+, pH 7.8). Intracellular ion concentrations in Xenopus embryo are: 21mM Na+, 90mM K+, 60mM Cl-, 0.5mM Ca2+ (Gillespie 1983). Xenopus embryos were housed at 14-18oC and staged according to Nieuwkoop and Faber (Nieuwkoop 1967). All experiments were approved by the Tufts University Animal Research Committee (M2014-79) in accordance with the guide for care and use of laboratory animals. Capped synthetic mRNAs generated using mMessage mMachine kit (Ambion) were dissolved in nuclease free water and injected into the embryos in 3% Ficoll using standard methods (Sive 2000). Each injection delivered between 1-2 nL or 1– 2 ng of mRNA (per blastomere) into the embryos, usually at 4-cell stage into the middle of the cell in the animal pole. Constructs used were: GlyR (Davies et al. 2003) and 666 chimera (Hough et al. 2000).


title: Comprehensive assessment of shockwave intensity:  transcriptomic biomarker discovery for primary blast-induced mild traumatic brain injury (mTBI) using the mammalian hair follicle

status: Public on May 07 2016

summary: Primary blast-induced mild traumatic brain injury (mTBI) represents a common injury experienced during modern warfare. With virtually no apparent physical damage or symptoms presented, an effective source with minimum-invasion for mTBI biomarker discovery is required. In this study, we measure the transcriptomic sensitivity of the hair follicles in relation to the severity of primary blast-derived TBI.

overall_design: An Advanced Blast Simulator system was used to expose rats to single pulse shockwaves with a static overpressure ranging from 15 psi to 30 psi in a head-only fashion. Blood and hair were collected for microarray analysis.

first: Jing

last: Zhang

first: Rosalinda

last: Knight

first: Yushan

last: Wang

first: Thomas

middle: W

last: Sawyer

first: Valerie

middle: S

last: Langlois


title: Estrogenic compounds reduce influenza A virus replication in primary human nasal epithelial cells derived from female, but not male, donors

status: Public on Dec 31 2015

city: Baltimore

first: Anne

middle: E.

last: Jedlicka

state: MD

address: 615 N. Wolfe street

department: Molecular Microbiology and Immunology

institute: Johns Hopkins University, School of Public Health

summary:
E2 exposure significantly decreased peak viral titer in hNECs from female donors. We used microarray analyses to identify global gene expression patterns between E2 and vehicle exposed hNECs from female donors
Influenza causes an acute infection characterized by virus replication in respiratory epithelial cells. The severity of influenza and other respiratory diseases changes over the life course and during pregnancy in women, suggesting that sex steroid hormones, such as estrogens, may be involved. Using primary, differentiated human nasal epithelial cell (hNEC) cultures from adult male and female donors, we exposed cultures to the endogenous 17Î²-estradiol (E2) or select estrogen receptor modulators (SERMs), then infected cultures with a seasonal influenza A virus (IAV) to determine whether estrogenic signaling could affect the outcome of IAV infection and whether these effects where sex-dependent. Estradiol, raloxifene, and bisphenol A decreased IAV titers in hNECs from female, but not male, donors. The estrogenic decrease in viral titer was dependent on the genomic estrogen receptor- 2 (ESR2) as neither genomic ESR1 nor non- genomic GPR30 were expressed in hNEC cultures and addition of the genomic ER antagonist ICI 182,780 reversed the antiviral effects of E2. Treatment of hNECs with E2 had no effect on interferon or chemokine secretion, but significantly downregulated cell metabolic processes, including genes that encode for zinc finger proteins, many of which contain estrogen response elements in their promoters. These data provide novel insights into the cellular and molecular mechanisms of how natural and synthetic estrogens impact IAV infection in respiratory epithelial cells derived from humans.

overall_design: Primary human nasal epithelial cells from females were exposed to E2 for 24h prior to infection, then infected with an H3N2 strain of influenza a virus for 2 hours. At 24 and 48h post infection, hNECs were collected in Trizol for RNA extraction and hybridization on Affymetrix Human Gene ST 2.0 microarrays.

first: Jackye

last: Peretz

first: Andrew

last: Pekosz

last: Lane

first: Sabra

last: Klein

first: Amanda

last: Dziedzic

middle: E


title: Interlab microarray comparison Lab A

first: Nancy

middle: D

last: Denslow


title: Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation [axolotl data]

overall_design:
All axolotls used in these experiments were bred in the axolotl facility at the University of Minnesota under the IACUC protocol #1201A08381. Axolotls of 2–3 cm were used for all in vivo experiments, and animals were kept in separate containers and fed daily with artemia; water was changed daily. Animals were anesthetized in 0.01% p-amino benzocaine (Sigma) before microinjection was performed.  There were 9 microarrays conducted, and these were ivermectin-treated  (n=3).

Experimental Design: Ivermectin injection
Ivermectin or vehicle only (water) was pressure injected into the central canal of the spinal cord, and this was visualized by the addition of Fast Green into the solution. Directly after injection, a portion of the spinal cord was surgically removed and the animals were placed back into water in individual containers. One day post injury (1dpi) animals were anesthetized again and the area of the injury was removed. Tissue from 10 animals were pooled for each microarray replicate.

Mature - Uninjured spinal cord tissue
Sc Crush- spinal cord tissue 1 day after injury
Ivermectin - Ivermectin injected spinal cord tissue 1 day after injury.

first: Vaibhav

last: Pai

last: Echeverri

last: Sundelacruz

last: Kaplan


title: Deep Sequencing Identifies Multiple Fusion Transcripts, Differentially Expressed Genes, and Generalized Immune Suppression in BRAF (V600E) mutant vs. BRAF wild-type Papillary Thyroid Carcinoma

status: Public on Jul 01 2015

city: Jacksonville

middle: Patrick

last: Walsh

state: Florida

address: 4500 San Pablo Road

department: Cancer Basic Science

institute: Mayo Clinic Jacksonville

summary: CONTEXT:  BRAF V600E mutation (BRAF-mut.) confers aggressiveness in papillary thyroid carcinoma, but unidentified genomic abnormalities may be required for full phenotypic expression.  OBJECTIVE:  To perform deep sequencing to identify genes differentially expressed between BRAF-mut. and BRAF-wild-type (BRAF-WT) tumors, and to compare to patient clinical status.  DESIGN:  BRAF-mut. and BRAF-WT tumors were identified in patients with T1N0 and with T23N1 tumors.  Expression levels of genes were determined from RNA sequencing (RNA-Seq) data and fusion transcripts were detected.  NanoString was used to validate the RNA-Seq data for immune genes.  SETTING:  Patients were seen at two sites of a major referral medical center.  PATIENTS:  Twenty patients were studied.  BRAF-mut. patients included 9 women, 3 men; 9 were TNM stage I and 3 were stage III; 3 (25%) had lymphocytic thyroiditis.  BRAF-WT included 5 women; 3 men; all were stage I; 5 (62.5%) had lymphocytic thyroiditis.  RESULTS:  560 of 13,085 genes were differentially expressed by RNA-Seq, and MetaCore analysis identified  55 immune function genes that were differentially expressed as a function of  BRAF mutational status.  Immune function genes were broadly underexpressed in BRAF-mut. tumors, with only 4 genes (HLA-G, CXCL14, TIMP1, IL1RAP) more highly expressed.  NanoString validated the RNA Seq data for immune genes.  Eleven high confidence fusion transcripts were detected, four being inter-chromosomal and seven intra-chromosomal.  CONCLUSION:  BRAF-mut. papillary thyroid cancers have less expression of immune and inflammatory response genes than BRAF-WT tumors.  Thirteen of 20 (65%) tumors had between one and three fusion transcripts.  Functional studies will be required to determine the potential role of the newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-mut. and wild-type tumors.

overall_design: RNA-seq was performed on 20 thyroid carcinoma tumors

first: Robert

last: Smallridge

first: Ana

middle: M

last: Chindris

first: Yan

last: Asmann

first: E

middle: A

last: Thompson


title: Effect of chronic MDPV on striatal gene expression

status: Public on Jul 31 2017

first: Christopher

last: Martyniuk

summary: The bath salt constituent 3,4-methylenedioxypyrovalerone (MDPV) is often detected in blood of patient’s presenting at emergency rooms with a psychosis-like syndrome involving agitation, panic, confusion/cognitive impairment, violent behavior, depression, and other adverse behavioral symptoms. The mechanisms underlying MDPV’s behavioral disturbances and its effect on brain gene expression patterns, particularly effects of chronic use on genetic changes in striatum and cortex are unknown. Such changes in gene expression are expected to reflect altered changes in brain function and structure as a consequence of MDPV abuse. A major objective of these experiments is to determine the relationship between repeated MDPV exposure and genes encoding dopaminergic, vascular, central inflammatory markers (e.g., IL6, IL10, IL1β, IFNγ, TNFα), particularly in prefrontal cortex, striatum, hypothalamus, and amygdala.

overall_design: 3 treatment groups: saline, single MDPV/acute, repeated MDPV/chronic (n = 6 per group)

first: Chris

first: Marcelo

last: Febo

first: Marjory

last: Pompilus


title: Stress-induced gene expression alterations in the amygdala of C57BL/6J and SWR/J mice.

city: Krakow

first: Marcin

last: Piechota

address: Smetna 12

department: Molecular Neuropharmacology

country: Poland

institute: Institute of Pharmacology PAS

summary: Comparison of changes in gene expression between the mouse strains (C57BL/6J and SWR/J) with diverse phenotype provides the possibility to find associations between transcriptional and behavioral response to the stress. We used whole-genome Illumina microarrays to analyze the effects on transcription of different aversive stimuli : restraint and/or footshocks, or after the exposure to the fear conditioning context.

overall_design: The microarray experiment was performed to compare transcriptional response in the amygdala to stress between two inbred mouse strains  C57BL/6J and SWR/J. The effects on gene expression 2 h after the restraint (0.5 h) stress (R) and/or exposure to 5 x 1 mA footshocks (FC) and after the exposure to the fear conditioning context (CTX)(14 days post footshocks). Individual amygdala samples were used per microarray, 5-8 biological replicates were used per experimental group. To provide appropriate balance in the whole dataset groups were equally divided between the array hybridization batches.

first: K

last: Szklarczyk

first: M

last: Korostynski


title: Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system

status: Public on Sep 01 2015

city: Athens

first: Brad

last: Nelms

state: GA

address: 120 E Green St

institute: University of Georgia

summary: To identify the gene expression profile of enteric glia and assess the transcriptional similarity between enteric and extraenteric glia, we performed RNA sequencing analysis on PLP1-expressing cells in the mouse intestine. This analysis shows that enteric glia are transcriptionally unique and distinct from other cell types in the nervous system. Enteric glia express many genes characteristic of the myelinating glia, Schwann cells and oli- godendrocytes, although there is no evidence of myelination in the murine ENS.

overall_design: Total RNA expression profiles of PLP1 expressing enteric glial cells (GFP+) and non-glial cells (GFP-negative) were obtained from the ileum and colon of juvenile PLP1-eGFP transgenic mice.

first: Bradlee

first: Meenakshi

last: Rao


title: The effects of a dietary organic selenium exposure of juvenile rainbow trout livers.

status: Public on Jun 30 2015

summary: In this study, juvenile rainbow trout were exposed four dietary doses of organic selenium (7.1, 10.7 19.5 and 31.8 mg/kg Selenium) over 60 days. The RNA was exctracted from liver tissue and used for further gene expression analysis.

overall_design: There were 39 samples analyzed  (8) control liver tissues   (8) 7.1 mg/kg Se dosed fish liver tissues  (7) 10.7 mg/kg Se dosed fish liver tissues (8) 19.5 mg/kg Se dosed fish liver tissues (9) 31.8 mg/kg Se dosed fish liver tissues.  There was a total of 39 microarrays processed.

first: Rosalinda

middle: O

last: Knight

first: Vicki

last: Marlatt

first: James

last: Elphick

first: Josh

last: Baker

first: Adrian

last: de Bruyn

first: Emily-Jane

last: Costa


title: Differential hepatic gene expression in zebrafish (Danio rerio) exposed to the fungicide chlorothalonil reveals estrogenicity

status: Public on Jun 17 2015

summary: Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is a broad spectrum fungicide used extensively in agricultural crops . The aim of this study is to analyse the effects of Chorothalonil on the gene expression profiles in zebrafish (Danio rerio), exposed to two concentrations of the fungicide in the water. Nominal concentrations were 1) Low 0.007mg/l  (environmentally relevent) and 2) High 0.035mg/ml . A commercial third generation microarray for Danio rerio (Agielnt V3, 4x44k) was used to identify patterns of gene expression in male  livers during a 96h toxicological assay.

overall_design: Replicates: Six control, five low and four high concentrations ; 15 samples examined. Expression profiles of male livers compared. Two concentrations of the fungicide chlorothalonil were compared.

first: Anny

middle: B

last: Sánchez

first: Paulina

last: Bahamonde

middle: J

first: Kelly

middle: R

last: Munkittrick

first: Miguel

last: Betancourt


title: TNFα Signaling Exposes Latent Estrogen Receptor Binding Sites in Breast Cancer Cells

status: Public on Mar 05 2015

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center


title: Spheroid growth in ovarian cancer promotes gene expression involved in epithelial-mesenchymal transition, stem cell promotion, and multiple stress responses

status: Public on Apr 18 2016

first: Christopher

last: Martyniuk

summary: In this study, we examine gene expression changes through microarray analysis in spheroid versus monolayer ovarian cancer cells treated with TGFβ to induce EMT.  Interestingly, EMT, stress response, and stem cell differentiation pathways were all significantly affected by 3D growth.  These findings support the hypothesis that three dimensional ovarian cell culturing is physiologically different from its monolayer counterpart.

overall_design: HEY Ovarian Cancer cells were treated with TGFβ and either cultured as a monolayer or using the hanging drop method to create spheroids. Microarray analysis was conducted on spheroid (3D) versus monolayer cells (2D).

first: Trillitye

last: Paullin

first: Chase

last: Powell

last: Menzie

first: Robert

last: Hill

first: Feng

last: Cheng

first: Sandy

last: Westerheide


title: Gene expression alteration by different cell lines derived from lung SCC of IKKα mutant mice

status: Public on Jan 27 2015

city: Frederick

first: zuoxiang

last: xiao

address: Bldg 567, Rm. 274, 1050 Boyles Street

institute: NCI

summary: In this study, we found that kinase-dead IKKα knockin (KAL) mice.  develop spontaneous lung squamous cell carcinomas (SCCs) associated with IKKα downregulation and marked pulmonary inflammation.KKα downregulation dysregulates the expression of multiple oncogenes and tumor suppressors in K5+ lung epithelial cells. The mutant macrophages increase inflammatory responses and oxidative stress to promote DNA damage in IKKα-mutant K5+ lung epithelial cells, which further dysregulate the levels of multiple oncogenes, tumor suppressors, and stem cell genes, thereby promoting the IKKαlowK5+p63hi cell transition to tumor cells in L-IkkαKA/KA lungs. To further investigate the underlying mechanisms by which the lung SCC development, we generated two cell lines, named as S1 and S2 individually, which were derived from KAL lung SCC. The S1 cells express high level of Sca1 and exhibit tumorigenic phenotype, while the S2 cells express low level of Sca1 and exhibit less tumorigenic phenotype. The aim of this microarray assay is to identify differentially expressed genes between S1 and S2 cells, which may highlight the important genes or pathways involved in inflammation-associated lung SCC carcinogenesis.

overall_design: There were two cell lines were used to perform Gene chip assay with Affymetrix. The two cell lines, named as S1 and S2 individually, were derived from KAL lung SCC. The S1 cells express high level of Sca1 and exhibit tumorigenic phenotype, while the S2 cells express low level of Sca1 and exhibit less tumorigenic phenotype.

first: yinling

last: hu


title: Gene expression classifier in papillary thyroid carcinoma: validation and application of a classifier for prognostication

status: Public on Jan 20 2015

city: Aarhus C

first: Jan

last: Alsner

address: Nørrebrogade 44, Bldg. 5

department: Experimental clinical Oncology

country: Denmark

institute: Aarhus University Hospital

summary:
Background: With the incidence of papillary thyroid carcinoma rising worldwide, the decision about lobectomy versus total thyroidectomy will become relevant for an increasing number of patients. There exists no reliable biomarker for metastatic potential or tendency of recurrence that could assist in the risk stratification of patients.

Objective: To develop a gene expression classifier for metastatic potential by measuring RNA expression in the primary tumor at the time of cancer surgery. Further, to investigate the ability of the gene expression classifier to identify metastatic and recurrent cases.

Method: Genome-wide expression analyses. The development cohort consisted of freshly frozen tissues from 38 patients collected between the years 1986 and 2009. Validation was performed on a consecutive cohort of formalin fixated paraffin embedded surgical tissue specimens from 183 patients treated at Odense and Aarhus University Hospitals.

Results: A 17 gene classifier (ADAMTS1, ANTXR1, C7, CXCL12, EBF1, FBLN2, FOSL2, GGT5, GPR124, JAM3, LRIG1, NDRG1, PRRX1, ROBO1, SORL1, TCF4, and ZEB1) was identified based on the expression values of these genes in the groups with and without metastasis in the development cohort. The 17 gene classifier for regional and/or distant metastasis identified was tested against the clinical status in the validation cohort. Sensitivity was 51.6% (95% CI 41.2%-61.8) and specificity 61.6 % (95% CI 50.5%-71.9%). Further, the Kaplan-Meyer method was used to estimate whether the classifier was useful as a prognostic marker for recurrences. Log-rank testing failed to identify any significance (p=0.32).

Conclusion: A 17 gene classifier for metastatic potential was developed, and the results showed a clear biological difference between groups. However, through validation, the prognostic significance of this classifier could not be shown in identifying metastatic cases or in the ability of dichotomizing patients according to risk of recurrence after primary treatment.

overall_design: 38 patients with papillary thyroid carcinoma from a consecutive cohort of patients, no replicates

first: Stefano

middle: C

last: Londero

first: Marie

last: Jespersen

first: Annelise

last: Krogdahl

first: Lars

last: Bastholt

first: Jens

last: Overgaard

first: Sten

last: Schytte

first: Christian

last: Godballe


title: Genome-wide expression analysis of E18.5 and P30 olfactory bulbs of control and Tshz1 mutant mice

status: Public on Apr 01 2014

city: Heidelberg

first: Hagen

last: Wende

address: Im Neuenheimer Feld 366

department: Institute of Pharmacology

country: Germany

institute: Heidelberg University

summary: Analysis of genes regulated by Tshz1 in olfactory bulb neurons.

overall_design:
Total RNA from olfactory bulbs of embryonic day E18.5 and postnatal day 30 control mice was compared to Tshz1 mutant mice.

NOTE: On Nov 18, 2022, the titles of GSM1252068-GSM1252075 were changed from "Tshz1 mutant" to "control", and the titles of GSM1252076-GSM1252083 were changed from "control" to "Tshz1 mutant", in accordance with corrections provided by the submitter. The genotype tshz1 characteristic of these Samples was also changed accordingly.

first: Ragancokova

last: Daniela

first: Wende

last: Hagen

first: Garratt

last: Alistair


title: Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation

first: Christopher

last: Martyniuk

sample_organism:
Ambystoma mexicanum
Xenopus laevis
Homo sapiens


title: Effects of sample size on differential gene expression, rank order and prediction accuracy of a gene signature

status: Public on May 15 2014

city: Edmonton

first: Kathryn

last: Graham

state: Alberta

address: 11560 University Ave

department: Oncology

country: Canada

institute: University of Alberta

summary:
Gene expression profiles were generated from muscle biopsies from 134 individuals, and differences in expression based on sex were explored.

Top differentially expressed gene lists are often inconsistent between studies and it has been suggested that small sample sizes contribute to lack of reproducibility and poor prediction accuracy in discriminative models. We considered sex differences (69♂, 65♀) in 134 human skeletal muscle biopsies using DNA microarray. The full dataset and subsamples (n= 10 (5♂, 5♀) to n=120 (60♂, 60♀)) thereof were used to assess the effect of sample size on the differential expression of single genes, gene rank order and prediction accuracy. Using our full dataset (n=134), we identified 717 differentially expressed transcripts (p-value < 0.0001; false discovery rate < 0.006) and we were able to predict sex with 92% accuracy, both within our dataset and on external datasets. Both p-values and rank order of top differentially expressed genes became more variable using smaller subsamples. For example, at n=10 (5♂, 5♀), no gene was considered differentially expressed at p<0.0001 and prediction accuracy was ~50% (no better than chance). We found that sample size clearly affects microarray analysis results; small sample sizes result in unstable gene lists and poor prediction accuracy. We anticipate this will apply to other phenotypes, in addition to sex.

overall_design: RNA was isolated from 134 muscle samples. Gene expression is compared between males and females.

first: Cynthia

last: Stretch

first: Vickie

last: Baracos


title: Serotonergic regulation of melanocyte conversion: a bioelectric network explains stochastic all-or-none hyperpigmentation

status: Public on Nov 03 2015

summary: Depolarization of resting membrane potential in select cells in Xenopus larvae induces striking hyperpigmentation due to dysregulation of melanocytes. Here, we show that this non-cell-autonomous process is mediated by cAMP, CREB, and the transcription factors Sox10 and Slug. Our microarray analysis reveals specific transcripts responsive to Vmem levels within a few hours of depolarization, and a set of 517 transcripts whose expression remains altered during the full hyperpigmented phenotype over a week later, linking instructor cell-depolarization to a range of developmental processes and disease states. We also show that voltage-dependent conversion of melanocytes involves the MSH-secreting melanotrope cells of the pituitary, and formulate a model for the molecular pathway linking the bioelectric properties of melanocyte cells’ microenvironment in vivo to the genetic and cellular changes induced in this melanoma-like phenotype. Remarkably, the phenotype is all-or-none: each individual animal either undergoes melanocyte conversion or not, as a whole. This group decision is stochastic, resulting in varying percentages of hyperpigmented individuals for a given experimental treatment. To understand the stochasticity and dynamic properties of this complex signaling system, we developed a novel computational method that automates the reverse-engineering of stochastic dynamic signaling models. We used this method to discover a network model that quantitatively explained our complex dataset, and even made correct predictions for new experiments that we validated in vivo. Taken together, these data (1) reveal new molecular details about a novel trigger of metastatic-like developmental cell behavior in vivo, (2) suggest new targets for biomedical intervention, and (3) demonstrate proof-of-principle of a computational method for understanding stochastic decision-making by cells during embryonic development and metastasis.

overall_design: Gene expression analysis was performed using samples treated with ivermectin from NF stage 10 onwards, collected at two developmental stages; stage 15 (early neurula) and stage 45 (free-swimming tadpole). Embryos were collected in eppendorf tubes (N=50 for stage 15, N=5 for stage 45) and frozen at -80°C. RNA extraction and microarray analysis were performed by the Beth Israel Deaconess Medical Center Genomics and Proteomics Center (Harvard) according to standard Affymetrix protocol, using a high throughput hybridization and scanning system. Microarray hybridization was performed using the Affy 3’ IVT Express Kit. Fragmented and biotin labeled and amplified RNA was hybridized to the GeneChip® Xenopus laevis Genome 2.0 array as per manufacturer’s protocol. The Affymetrix GeneChip® X. laevis Genome 2.0 Array, has 32,400 probe sets representing more than 29,900 X. laevis transcripts.

first: Maria

last: Lobikin

first: Daniel

last: Lobo

first: Douglas

last: Blackiston

first: Elizabeth

last: Tkachenko

first: Michael

last: Levin


title: Transcriptome sequencing of Brassica rapa tissues

status: Public on Oct 09 2013

city: Shenzhen

first: Zhangyan

last: Wu

address: Yantian district, Shenzhen

institute: Beijing Genomics Institute

summary: Deep sequencing of mRNA from six different tissues

overall_design: Analysis of poly(A)+ RNA of multiple different tissues of Brassica rapa containing Callus, Root, Stem, Leaf, Flower and Silique.

first: Chaobo

last: Tong

first: Jian

first: Xiaowu

last: Wang

first: Xinhua

last: Yang

first: Shengyi

last: Liu


title: Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells

status: Public on Dec 23 2011

city: Boston

first: Kevin

last: Dong

state: MASSACHUSETTS

address: 360 Longwood Ave

institute: Dana-Farber Cancer Institute

summary:
Hundreds of Chromatin Regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs co-localize in characteristic patterns at distinct chromatin environments, genes of coherent functions and distal regulatory elements. When comparing between cell types, CRs redistribute to different loci, but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: Study of genom-wide binding of 29 CRs in two cell types.

first: Oren

last: Ram

first: Alon

last: Goren

first: Bradley

last: Bernstein


title: Activation of the Interferon Pathway is Dependent upon Autoantibodies in African-American SLE Patients, but not in European-American SLE Patients

status: Public on Nov 15 2013

first: Timothy

middle: B.

last: Niewold

state: MN

address: 200 1st St. SW

department: Division of Rheumatology and Department of Immunology

institute: Mayo Clinic

summary: Systemic lupus erythematosus (SLE) is a heterogeneous disease which leads to different levels of serum autoantibodies to RNA-binding proteins (anti-RBP) and interferon-α (IFN-α), which plays an important pathogenic role in SLE, between European-American (EA) and African-American (AA) patients. We aimed to explore how IFN-related gene expression pathways differ in patients according to their ancestry and anti-RBP profile

overall_design: Whole blood from 33 female SLE patients and 16 matched controls from EA and AA ancestral backgrounds was analyzed through Affymetrix Gene 1.0 ST gene expression arrays and the data were further studied through Ingenuity Pathways Analysis for canonical pathway comparison. An independent replication cohort of more than 100 SLE patient samples and 30 controls was used to test the hypotheses generated by the microarray data, using qPCR to quantify gene expression.

first: Kichul

last: Ko

first: Yelena

last: Koldobskaya

last: Rosenzweig


title: Validation cohort for genomic predictor of response and survival following neoadjuvant taxane-anthracycline chemotherapy in breast cancer

status: Public on May 11 2011

city: Woburn

first: Christos

last: Hatzis

state: MA

address: 400 West Cummings Park, Suite 5350

institute: Nuvera Biosciences

summary:
PURPOSE: To develop a predictive test for response and survival following neoadjuvant taxane-anthracycline chemotherapy for HER2-negative invasive breast cancer.
METHODS: We developed a microarray-based gene expression test from pre-treatment tumor biopsies (310 patients) to predict favorable outcome based on estrogen receptor (ER) status,pathologic response to chemotherapy, 3-year disease outcomes, and sensitivity to endocrine therapy. Tumors were classified as treatment-sensitive if predicted to have pathologic response (and not resistance) to chemotherapy, or sensitive to endocrine therapy. We tested predictive accuracy, with 95% confidence interval (CI), for pathologic response (PPV, positive predictive value), distant relapse-free survival (DRFS), and absolute risk reduction at median follow-up in 198 other patients. Independence from clinical-pathologic factors was assessed in a multivariate Cox regression analysis based on the likelihood ratio test. Other evaluable, published response predictors (genomic grade index (GGI), intrinsic subtype (PAM50), pCR predictor (DLDA30)) were compared.

overall_design: Neoadjuvant validation cohort of 198 HER2-negative breast cancer cases treated with taxane-anthracycline chemotherapy pre-operatively and endocrine therapy if ER-positive.  Response was assessed at the end of neoadjuvant treatment and distant-relapse-free survival was followed for at least 3 years post-surgery.

first: W

middle: F

last: Symmans

first: Frankie

last: Holmes

first: Tatiana

last: Vidaurre

first: Miguel

last: Martin

first: Eduardo

last: Souchon


title: Genomic predictor of response and survival following neoadjuvant taxane-anthracycline chemotherapy in breast cancer

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

overall_design: Refer to individual Series


title: Inner Ear Tissues

status: Public on Oct 01 2006

city: Bethesda

first: Robert

last: Morell

address: 35A Convent Drive, Room 1F-103

institute: National Institute on Deafness and Other Communication Disorders

summary:
MPSS analysis of signatures from microdissections of mouse inner ear tissues.
Keywords: other

overall_design: Microdissections of day 7 C57Bl/6 animals, male and female, both ears, pooled together. Approximately 14 - 20 ears for each of three libraries:  organ of Corti, sensory epithelia of vestibular system, and stria vascularis.

first: Inna

last: Belyantseva

first: Thomas

last: Friedman

first: James

last: Battey


title: Discovery cohort for genomic predictor of response and survival following neoadjuvant taxane-anthracycline chemotherapy in breast cancer

overall_design: Neoadjuvant study of 310 HER2-negative breast cancer cases treated with taxane-anthracycline chemotherapy pre-operatively and endocrine therapy if ER-positive.  Response was assessed at the end of neoadjuvant treatment and distant-relapse-free survival was followed for at least 3 years post-surgery.

first: Laura

last: Esserman


title: Basal gene expression data from Human Variation Panel

status: Public on Jul 24 2010

first: Liewei

last: Wang

address: 200 First street SW

department: Molecular Pharmacology and Experimental Therapeutics

summary:
We used microarrays to identify the variation of basal gene expression level among 287 lymphoblastoid cell lines.
Radiation therapy is used to treat half of all cancer patients.  Response to radiation therapy varies widely among patients.  Therefore, we performed a genome-wide association study (GWAS) to identify biomarkers to help predict radiation response using 277 ethnically defined human lymphoblastoid cell lines (LCLs).  Basal gene expression levels and 1.3 million genome-wide SNP markers from both Affymetrix and Illumina platforms were assayed for all 277 human LCLs.  MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays for radiation cytotoxicity were also performed to obtain area under the curve (AUC) as a radiation response phenotype for use in the association studies.  Functional validation of candidate genes, selected from an integrated analysis that used SNP, expression and AUC data, was performed with multiple cancer cell lines using specific siRNA knockdown, followed by MTS and colony-forming assays.  27 loci, each containing at least 2 SNPs within 50kb with p-values <10-4, were associated with radiation AUC.  270 expression probe sets were associated with radiation AUC with p<10-3.  The integrated analysis identified 50 SNPs in 14 of the 27 loci that were associated with both AUC and the expression of 39 genes that were also associated with radiation AUC (p<10-3).  Functional validation using siRNA knockdown in multiple tumor cell lines showed that C13orf34, MAD2L1, PLK4, TPD52 and DEPDC1B each significantly altered radiation sensitivity in at least 2 cancer cell lines.  Studies performed with LCLs can help to identify novel biomarkers that might contribute to variation in response to radiation therapy and enhance our understanding of mechanisms underlying that variation.

overall_design: EBV-transformed LCLs from 95 African-American (AA), 96 Caucasian-American (CA), and 96 Han Chinese-American (HCA) unrelated healthy subjects (sample sets HD100AA, HD100CAU, HD100CHI) were purchased from the Coriell Cell Repository (Camden, NJ).

first: Richard

last: Weinshilboum


title: Human gene expression compendium containing 9395 samples from the HG_U133PlusV2 platform

status: Public on Jan 15 2015

first: Brad

last: Nelms

type:
Expression profiling by array
Third-party reanalysis

summary: Re-analysis of human gene expression data generated on the Affymetrix HG_U133PlusV2 platform. The original data were downloaded from GEO on May 28, 2008 and normalized to obtain an integrated gene expression atlas across diverse biological sample types and conditions.

overall_design: All available GEO series on the Affymetrix Human Genome U133 Plus 2.0 array (GEO accession GPL570) were downloaded on May 28,2008. For each series, we aggregated and normalized probe level information using robust multi-array average (RMA), transformed each expression value using log base 2, and removed technical bias resulting from variation in hybridization conditions and starting material using the R package bias 0.0.3. This with-in-series normalization identified probe or arrays outliers within each dataset. We mapped probes to genes using the Bioconductor annotation package hgu133plus2 1.16.0, and calculated expression values for each gene by averaging the values of probe- sets measuring the same gene. Finally, we performed quantile normalization on the entire compendium using the limma R package (version 2.18.2) in order to reconcile broader differences between datatsets and ensure that all arrays were on the same scale. The entire compendium comprises 20,099 genes and 9395 arrays.

first: Bradlee

first: Jesse

last: Engreitz


title: H2A.Z landscapes and dual modifications in pluripotent and multipotent stem cells underlie complex genome regulatory functions

status: Public on Jul 20 2012

first: Kevin

last: Dong

summary: The histone variant H2A.Z has been implicated in nucleosome exchange, transcriptional activation and Polycomb repression. However, the relationships among these seemingly disparate functions remain obscure. We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers, and correlates strongly with H3K4 methylation. Accordingly, H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation, but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z, including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin. Our findings associate H2A.Z with functionally distinct genomic elements, and suggest that post-translational modifications may reconcile its contrasting locations and roles.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Examination of histone variant, histone modifications and transcription machinery in 3 cell types

first: Manching

last: Ku

middle: P

last: Koche

first: Timothy

last: Durham

first: Bradley

last: Bernstein


title: Regulation of gene expression with thyorid hormone in rats with myocardial infarction

status: Public on May 01 2012

city: Brookings

first: Xijin

last: Ge

state: SD

address: SDSU box 2220C

department: Mathematics and Statistics

institute: South Dakota State University

summary: Thyroid hormone improves left ventricular remodeling and cardiac performance after myocardial infarction (MI), but the molecular basis is unknown. This study was designed to detect gene expression changes in left ventricular non-infarcted areas at 4 weeks following myocardial infarction with and without thyroid hormone treatment. The results suggest that altered expression of genes for molecular function and biological process may be involved in the beneficial effects of thyroid hormone treatment following myocardial infarction in rats.

overall_design: MI was produced by ligation of the left anterior descending coronary artery in female SD rats. Rats were divided into the following groups: (1) Sham MI, (2) MI, and (3) MI+T4 treatment (T4 pellet 3.3mg, 60 days release, implanted subcutaneously immediately following MI). Four weeks after surgery, total RNA was isolated from left ventricular non-infarcted areas for microarray analysis using the Illumina RatRef-12 Expression BeadChip Platform.

first: Chen

last: Yuefeng

middle: V

last: Pottala

first: Nathan

middle: Y

last: Weltman

first: Olga

last: Savinova

first: A

last: Gerdes


title: GC-rich Sequence Elements Recruit Polycomb Repressive Complex 2 (PRC2) in ES cells

status: Public on Nov 23 2010

summary: Polycomb proteins are epigenetic regulators that localize to developmental loci in the early embryo where they mediate lineage-specific gene repression. In Drosophila, these repressors are recruited to sequence elements by DNA binding proteins associated with Polycomb repressive complex 2 (PRC2). However, the sequences that recruit PRC2 in mammalian cells have remained obscure. To address this, we integrated a series of engineered bacterial artificial chromosomes into embryonic stem (ES) cells and examined their chromatin. We found that a 44 kb region corresponding to the Zfpm2 locus initiates de novo recruitment of PRC2. We then pinpointed a CpG island within this locus as both necessary and sufficient for PRC2 recruitment. Based on this causal demonstration and prior genomic analyses, we hypothesized that large GC-rich elements depleted of activating transcription factor motifs mediate PRC2 recruitment in mammals. We validated this model in two ways. First, we showed that a constitutively active CpG island is able to recruit PRC2 after excision of a cluster of activating motifs. Second, we showed that two 1 kb sequence intervals from the E. coli genome with GC-contents comparable to a mammalian CpG island are both capable of recruiting PRC2 when integrated into the ES cell genome. Our findings demonstrate a causal role for GC-rich sequences in PRC2 recruitment and implicate a specific subset of CpG islands depleted of activating motifs as instrumental for the initial localization of this key regulator in mammalian genomes.

overall_design: Analysis of YY1 binding in two cell types

first: Eric

last: Mendenhall