title: Real-time quantitative PCR analysis of human acute myeloid leukemia primary cells

city: TOULOUSE

first: DAVID

last: Laure

address: 2 avenue Hubert CURIEN

institute: Cancer Research Center of Toulouse

summary: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by different recurrent cytogenetic and molecular aberrations that occur in hematopoietic progenitor cells and alter the growth, differentiation, and proliferation capacities of the progenitor cells. The treatment of AML has remained a huge challenge for oncohematologists. The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage is currently used in the treatment of acute myeloid leukemia.     Here, we reasoned that DNA replication represents a less explored source of prognostic markers that could be used to predict AML prognosis and eventually provide potential targets for therapeutic targeting. Accurate execution of the DNA replication program limits cancer risk by preserving genome integrity. Multiple studies of solid cancers have provided evidence that defective or dysregulated DNA replication program triggers replicative stress, leading to the accumulation of genetic alterations. However, defective DNA replication as a source of markers in hematological malignancies has not been explored. Thus we explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy.

overall_design: Patients samples  Between Jan 1, 2000, and Dec 31, 2010, the use of fresh and thawed samples from 198 AML patients samples have been performed after informed consent and stored at the HIMIP collection (BB-0033-00060) of the Cancer Research Center of Toulouse. According to the French law, HIMIP collections have been declared to the Ministry of Higher Education and Research (DC 2008-307 collection 1) and we obtained a transfer agreement (AC 2008-129) after approbation by the “Comité de Protection des Personnes Sud-Ouest et Outremer II” (ethical committee). Clinical and biological annotations of the samples have been declared to the CNIL (Comité National Informatique et Libertés ie Data processing and Liberties National Committee), and these are supported by CAPTOR (Cancer Pharmacology of Toulouse-Oncopole and Region). This study was approved by the institutional review board (Ethical Committee of Research).  Total RNA extraction and Reverse Transcription-PCR  Total RNA were extracted from frozen cells (7 to 15 million of cells) stored in 1 ml of Tri Reagent RNA/DNA/protein isolation reagent (Molecular Research Center). The extraction was done by adding 200 µl of cold Ready Red-Chloroform isoamyl alcohol (MP Biomedicals, France) and vigorously shaking for 15 s using a vortex, then the sample was incubated on ice for at least 5 min. After centrifugation at 13 000 rpm for 15 min at 4°C, the upper aqueous phase was transferred into a new vial. One volume of isopropanol was added, the sample was vortexed and incubated for one hour at -20°C. After centrifugation at 13 000 rpm for 15 min at 4°C, the pellet was dried and 1 ml of cold 75% ethanol added. After centrifugation at 8000 rpm for 10 min at 4°C, the pellet was dried and incubated for 4 min at 65°C. The pellet was resuspended in 30 µl of RNAse-free water with RiboLock RNase Inhibitor (40U, Fermentas, France). RNA concentration was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc, Thermo Scientific, Brebières, France). RNA quality and purity was assessed on the Agilent 2100 BioAnalyser by using the Agilent RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). Only RNA presenting a RIN (RNA Integrity Number) > 6.5 were selected for expression analysis (~ 90% of the samples had a RIN > 8). cDNA was generated from 1 µg of RNA with the SuperScript VILO cDNA Synthesis Kit (Invitrogen) for reverse transcription-PCR following the manufacturer’s suggestions. In order to ensure a good quality reverse transcription step, one part of each cDNA was used to check ABL1 (“TaqMan Gene Expression Assay”, Applied Biosystems, Hs01104728_m1) expression using ABI Prism 7300 HT (Applied Biosystems, Carlsbad, CA, USA).  Specific target amplification and Quantitative PCR   The other part of each cDNA was diluted in water to 5 ng/µl and used for target amplification by Biomark Dynamic Arrays (Fluidigm, BioMark, Pueblo, CO, USA). Inventoried TaqMan assays (Applied Biosystems) were pooled using 84 probes and primers pairs (72 DNA replication genes, 9 housekeeping genes - ABL, ACTB, B2MC, G6PD, GAPDH, GUSB, HPBS, TBP, UBC -, and BAALC, ERG, and MN1 genes) to a final concentration of 0.2X for each of the 84 assays. To increase sensitivity, a multiplexed pre-amplification process was performed for the pool on every 1.25 µl cDNA using 14 cycles cDNA pre-amplification step (95°C for 15s and 60°C 4 min) and Taqman PreAmp Master Mix (Applied Biosystems) in a standard PCR Thermocycler. Pre-amplified cDNA was diluted 1:5 in 10 mmol/L Tris, 1 mmol/L EDTA. Diluted cDNA (2.25 µl) was added to 2.5 µl Taqman Universal PCR Master Mix (Applied Biosystems) and 0.25 µl GE Sample Loading Reagent (Fluidigm). In a separate tube, 3.5 µl of Taqman Assay was added to 3.5 µl Sample Loading Reagent. 5 µl cDNA samples were loaded into the sample inlet wells, and 5 µl assay samples were loaded into assay detector inlets. Because 198 samples were to be analyzed in duplicates, five 96.96 Dynamic Arrays (Fluidigm) were used. For each plate, 1 well was loaded with H2O as a control for contamination. Genomic DNA (gDNA) from 3 different patients was loaded in order to check if TaqMan assays can also amplify gDNA. To verify STA (Specific Target Amplification) efficiency, a sample control gDNA and assay control RNAse P Taqman probe was treated (Lifetech PN 4316844), pre-amplified and quantified using the same master mix from the kit. The expected value cq (quantification cycle) was between 12 and 13.  To perform inter-plate calibration, a sample calibrator made of cDNA from the patient #1 was included in duplicate in each plate. The chip was primed and placed into the NanoFlex Integrated fluidic circuit controller where 8 nl of cDNA and 1 nl of Assay were mixed. Real time PCR analysis was completed on the BioMark System (Fluidigm). The experiment was done on 2 series with 3 plates in the first serie and 2 plates in the second one. So to perform inter-plates  calibration, a patient samples made of cDNA from patient #1 was used for the first serie and cDNA from samples #1, #30, #60, #90, #95 and #111 for the second one. The cDN1 froma patient sample #1 was also used as a between series control.    Data processing  Raw data obtained from the system’s software, using the auto detector function to establish the threshold setting (BioMark Realtime PCR Analysis V2.1.1, Fluidigm), were checked using the graphical representation of the plate layout. Among all reactions investigated, 0 were rejected due to bubbles or instable ROX (Carboxy-X-Rhodamin) signal. All amplification curves were displayed for each well of the calibrator sample. When the threshold for cycle did not meet quality criteria (that is the threshold occurred in the linear phase of the amplification curve instead of the exponential phase), the threshold value was set manually. The threshold established for the first Dynamic Array were applied to the 4 other Dynamic Arrays. Wells with very high (> 26), absent (999), or very low (< 2) endogenous Ct were excluded.    Normalization method  These raw data wre then normalized with the data obtained with the housekeeping genes. Then we performed the inter-plate calibration using the qbase+ algorithm as described (Hellemans, 2007). Among the 9 housekeeping genes tested [GUSB (Hs99999908_m1), ACTB (Hs99999903_m1), ABL1 (Hs01104728_m1), G6PD (Hs00166169_m1), TBP (Hs00427621_m1), GAPDH (Hs03929097_g1), HMBS (Hs00609293_g1), B2M (Hs00984230_m1), UBC (Hs00824723_m1)], GeNorm algorithm determined the four most stable, which were GAPDH, GUSB, TBP and ABL1, and were used to calculate the gene expression normalization factor. Expression values are given in CNRQ  which corresponds to a value normalised and calibrated (2^ - deltaCT) (the reference value is 1, and every value can varie between 0 and the infinity).  References:   Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007;8(2):R19.

first: Laure

last: David

last: Fernandez-Vidal

first: Sarah

last: Bertoli

first: Srdana

last: Grgurevic

first: Benoît

last: Lepage

first: Dominique

last: Deshaies

first: Naïs

last: Prade

first: Maëlle

last: Cartel

first: Clément

last: Larrue

first: Jean-Emmanuel

last: Sarry

last: Delabesse

first: Christophe

last: Cazaux

first: Christine

last: Didier

first: Christian

last: Récher

first: Stéphane

last: Manenti

first: Jean-Sébastien

last: Hoffmann


title: Proteomic and genomic characterization of a yeast model for Ogden syndrome [microarray]

status: Public on Sep 03 2016

city: Seattle

first: Maitreya

middle: J.

last: Dunham

state: WA

address: Foege Building, S403B, Box 355065

department: Genome Sciences

country: USA

institute: University of Washington

summary: Gene expression microarrays accompanying "Proteomic and genomic characterization of a yeast model for Ogden syndrome" by Doerfel et al 2016 in press at Yeast.  Naa10 is a Na-terminal acetyltransferase that, in a complex with its auxiliary subunit Naa15, co-translationally acetylates the a-amino group of newly synthetized proteins as they emerge from the ribosome. Roughly 40-50% of the human proteome is acetylated by Naa10, rendering this an enzyme with one of the most broad substrate ranges known. Recently, we reported an X-linked disorder of infancy, Ogden syndrome, in two families harboring a c.109T>C (p.Ser37Pro) variant in NAA10. In the present study we performed in-depth characterization of a yeast model of Ogden syndrome. Stress tests and proteomic analyses suggest that the S37P mutation disrupts Naa10 function and reduces cellular fitness during heat shock, possibly due to dysregulation of chaperone expression and accumulation. Microarray and RNA-seq revealed a pseudo-diploid gene expression profile in DNaa10 cells, likely responsible for a mating defect. In conclusion, the data presented here further support the disruptive nature of the S37P/Ogden mutation and identify affected cellular processes potentially contributing to the severe phenotype seen in Ogden syndrome.

overall_design: Each microarray compares gene expression between two strains.

first: Jamie

last: Pogachar

first: Max

last: Doerfel

first: Gholson

last: Lyon


title: Differential expression of miRNAs in 3-Deazaneplanocin-A (DZNep) treated normal pancreatic and pancreatic cancer cells

city: Houston

first: Kyle

middle: Ryan

last: Navel

state: Texas

address: 2575 W. Bellfort Ave. Ste 270

department: Sales

institute: LC Sciences

summary:
MiRNAs differentially expressed in normal pancreatic cell line (Human Pancreatic Ductal Epithelial cell line) vs MIA PaCa-2 (Pancreatic cancer cell line) were first identified by profiling >1900 miRNAs using an array approach.
Subsequently, miRNAs differentially expressed in DZNep treated cells (as compared to untreated) were also identified. We also checked for miRNA changes with gemcitabine treatment as well as combination treatment.

overall_design: MiRNAs differnetially expressed between Normal (HPDE) vs Cancer Control (MIA PaCa-2), Normal - Control vs Treated (DZNep) or Cancer - control vs either Treated 1 (DZNep; 8 h) or Treated 2 (DZNep; 72 h) or Treated 3 (Gemcitabine; 72 h) or Treated 4 (Gem:DZNep: 1:10) was carried out

first: Hardik

last: Mody


title: Expression data from Northeastern Indigenous and Large White longissimus dorsi

status: Public on Sep 02 2016

city: changchun

first: yan

last: gao

address: 5333 xian road

country: China

institute: Jilin University

summary:
To understand the molecular basis of distinct pork quality in Chinese indigenous and Western breed, longissimus dorsi samples were collected from three adult Northeastern Indigenous and from three adult Large White. Total RNA was extracted and subjected to porcine Affymetrix Genechip.
The study helps to elucidate the genetic mechnism of divergent pork quality and provide the theory basis for selection and genetic improvement of meat quality traits in porcine.

overall_design: Six longissimus dorsi samples were collected from three Northeastern Indigenous and from three Large White. Three Large White were control samples. Total RNA was extracted from each sample.Gene-expression profiling was performed for each RNA sample separately on the GeneChip® Porcine Genome Array at CapitalBio Corporation (Beijing, China).

first: Yan

last: Gao

first: Yonghong

last: Zhang

first: Jiabao


title: Meiosis timecourse Rap1-depletion Rap1 ChIP-chip

status: Public on Sep 01 2016

city: Chicago

first: Sean

middle: Erik

last: Hanlon

state: IL

address: 920 E. 58th Street – CLSC 515E

department: Department of Human Genetics

institute: University of Chicago

type: Genome binding/occupancy profiling by array

summary:
To confirm that Rap1-depletion resulted in decreased binding of Rap1 to its target sites, we performed compared Rap1 ChIP-chip experiments in respiratory cells (pre-meiotic/YPA 20 hours) depleted (plus doxycycline for 12 hours) or not depleted (minus doxycycline) of Rap1.
There is growing recognition that the binding of a transcription factor near a gene does not always indicate regulatory function, and further that a single factor may function to either activate or repress its targets depending on the cellular context. We examined these issues through a series of experiments involving the S. cerevisiae transcription factor Rap1, and its function throughout critical metabolic and developmental transitions between vegetative growth, respiratory growth, meiosis and sporulation. We simultaneously monitored the expression of all genes and the genomic binding locations of Rap1 throughout the timecourse. Genes downstream of Rap1 binding were activated and repressed dynamically, but a change - or lack of change - in Rap1 binding status was not predictive of activation, repression, or no change in regulation. Despite this, we show that Rap1 is required, at a given point in time, for both activation and repression of different gene targets, within the same cell. Specification of the transcriptional consequences of Rap1 binding is thus highly promoter-specific. The presence of other transcription factor binding motifs, the subtype of Rap1 motif, and the underlying chromatin structure of the promoter cannot fully account for the observed transcriptional outcomes. There is a better accounting for the dynamic binding behavior of Rap1 including specification of an expanded meiotic target set through a Tup1- dependent nucleosome-loss mechanism. The variable and dynamic association between binding and transcription in this simple unicellular system portends a similarly volatile relationship in more complex eukaryotes. Biological interpretations of transcription factor occupancy should be made cautiously and in conjunction with supporting data obtained under the precise condition of interest.

overall_design: SK1 yeast strain SHy20 (MATa/MATalpha lys2/lys2 ura3/ura3 ho::LYS2/ho::LYS2 leu2::hisG/leu2::hisG his4x/his4b URA3::CMV-tetTA/URA3::CMV-tetTA RAP1promoter::kanR-tetO7-TATA/RAP1promoter::kanR-tetO7-TATA).  An overnight culture of YPD was used to innoculate a YPA culture (minus doxycycline; Rap1 on). The YPA culture was grown for 8 hours and then split into minus doxycyline (Rap1 on) and plus doxycylcine (Rap1 depleted) samples. Minus and plus doxycyline samples were collected after an additional 12 hours in YPA (respiratory growth/pre-meiotic; YPA 20 hours).  6 separate biological replicates were used.  Labeled ChIP DNA from Rap1 depeleted and non-depleted samples were competitively hybridized to yeast whole genome PCR based spotted arrays (resolution ~1kb).  The Rap1-depletion ChIP and RNA abundance/expression analysis were carried out on the same biological samples. The three replicates of Rap1-depletion RNA abundance/expression analysis correspond to replicates 2, 3, and 5 of the Rap1-depletion Rap1 ChIP experiment.

first: Hanlon

last: Sean Erik

first: Lieb

last: Jason D.


title: Meiosis timecourse Rap1-depletion expression profile

summary:
To investigate the impact of Rap1 binding on nearby gene expression during the transition from vegetative growth to meiosis, we measured RNA abundance in cells depleted (plus doxycycline) or not depleted (minus doxycycline) of Rap1. RNA abundance was measured in depleted and non-depleted condition in respiratory cells (pre-meiotic/YPA 20 hours) and in cells undergoing synchronous meiosis/sporulation in the budding yeast Saccharomyces cerevisiae.
There is growing recognition that the binding of a transcription factor near a gene does not always indicate regulatory function, and further that a single factor may function to either activate or repress its targets depending on the cellular context. We examined these issues through a series of experiments involving the S. cerevisiae transcription factor Rap1, and its function throughout critical metabolic and developmental transitions between vegetative growth, respiratory growth, meiosis and sporulation. We simultaneously monitored the expression of all genes and the genomic binding locations of Rap1 throughout the timecourse. Genes downstream of Rap1 binding were activated and repressed dynamically, but a change - or lack of change - in Rap1 binding status was not predictive of activation, repression, or no change in regulation. Despite this, we show that Rap1 is required, at a given point in time, for both activation and repression of different gene targets, within the same cell. Specification of the transcriptional consequences of Rap1 binding is thus highly promoter-specific. The presence of other transcription factor binding motifs, the subtype of Rap1 motif, and the underlying chromatin structure of the promoter cannot fully account for the observed transcriptional outcomes. There is a better accounting for the dynamic binding behavior of Rap1 including specification of an expanded meiotic target set through a Tup1- dependent nucleosome-loss mechanism. The variable and dynamic association between binding and transcription in this simple unicellular system portends a similarly volatile relationship in more complex eukaryotes. Biological interpretations of transcription factor occupancy should be made cautiously and in conjunction with supporting data obtained under the precise condition of interest.

overall_design: SK1 yeast strain SHy20 (MATa/MATalpha lys2/lys2 ura3/ura3 ho::LYS2/ho::LYS2 leu2::hisG/leu2::hisG his4x/his4b URA3::CMV-tetTA/URA3::CMV-tetTA RAP1promoter::kanR-tetO7-TATA/RAP1promoter::kanR-tetO7-TATA).  An overnight culture of YPD was used to innoculate a YPA culture (minus doxycycline; Rap1 on). The YPA culture was grown for 8 hours and then split into minus doxycyline (Rap1 on) and plus doxycylcine (Rap1 depleted) samples. Minus and plus doxycyline samples were collected after an additional 12 hours in YPA (respiratory growth/pre-meiotic; YPA 20 hours) and 0 hr, 3hr, 6hr, 9hr and 12hr after transfer to sporulation media (SM) with or without doxycycline.  3 separate biological replicates were used for each condition/time-point.  Labeled total RNA from Rap1 depeleted and non-depleted samples were competitively hybridized to yeast whole genome PCR based spotted arrays (resolution ~1kb).  The Rap1-depletion ChIP and RNA abundance/expression analysis were carried out on the same biological samples. The three replicates of Rap1-depletion RNA abundance/expression analysis correspond to replicates 2, 3, and 5 of the Rap1-depletion Rap1 ChIP experiment.

first: S

middle: E

first: J

middle: D

last: Lieb


title: Gene expression profiles in CD44- and CD44+ hepatocellular carcinoma HuH7 cells

city: Yonago

first: Tomohiko

last: Sakabe

state: Tottori

address: Nishi-cho 86

country: Japan

institute: Tottori University

summary: To investigate the significance of CD44+ hepatocellular carcinoma (HCC) HuH7 cells, gene expression profiles of CD44-positive HuH7, CD44-negative HuH7, and human nomal hepatocyes were analyzed. Results provide the insight into the significance of CD44-positive HCC cells as the liver CSCs.

overall_design: CD44-positive and CD44-negative HuH7 cells were sorted by flow cytometry. Total RNA were extracted from CD44-positive HuH7, CD44-negative HuH7 cells, and human normal hepatocytes. Total RNA isolated from each cell labeled using Cy5 and hybridized onto the 3D-Gene human mRNA oligo chip (human 25k, Ver 2.10).

first: Goshi

last: Shiota


title: Human Corneal Epithelial Cells (SV40-HCEC) 6h Treatment with 100nM Vitamin D (1,25D3) vs. Vehicle Control

city: Stockholm

first: Cecilila

last: Williams

address: Tomtebodavägen 23A, Solna

department: Molecular & Cellular Proteomics

country: Sweden

institute: KTH

summary:
Inflammatory signals must be regulated and kept in check in order to prevent tissue damage.  This is especially true in the cornea, where damage can induce loss of transparency, essential for vision.  Toll-like receptors (TLRs) are present at the ocular surface and, in addition to being protective against infection, have also been implicated in the pathogenesis of dry eye syndrome, an inflammatory condition that affects millions of individuals in the United States each year.   Therefore, an important area of research is the development of new anti-inflammatory therapeutics that limit aberrant ocular surface inflammation.  Vitamin D has been studied for its role in suppressing inflammation in other tissues.  In previous studies, we have demonstrated that vitamin D is able to decrease proinflammatory mediators induced by TLRs in human corneal epithelial cells (HCEC).  Therefore, the goal of the current study was to examine this mechanism further through an evaluation of vitamin D’s influence on gene expression in two different HCEC cell lines.
Microarray comparing SV40-HCEC, human corneal epithelial cells, treated for 6 hours with Vitamin D (1,25D3) and control (0.1% ethanol)

overall_design: Two-condition arrays: SV40-HCEC cells were treated with either 100nM 1,25D3 or 0.1% Ethanol for 6h. Two biological replicates, one per array, with a dye swap (technical replicate).

first: Fahmi

last: Mesmar

first: Rose

last: Reins


title: Human Corneal Epithelial Cells (hTCEpi) 6h Treatment with 100nM Vitamin D (1,25D3) vs. Vehicle Control

summary:
Inflammatory signals must be regulated and kept in check in order to prevent tissue damage.  This is especially true in the cornea, where damage can induce loss of transparency, essential for vision.  Toll-like receptors (TLRs) are present at the ocular surface and, in addition to being protective against infection, have also been implicated in the pathogenesis of dry eye syndrome, an inflammatory condition that affects millions of individuals in the United States each year.   Therefore, an important area of research is the development of new anti-inflammatory therapeutics that limit aberrant ocular surface inflammation.  Vitamin D has been studied for its role in suppressing inflammation in other tissues.  In previous studies, we have demonstrated that vitamin D is able to decrease proinflammatory mediators induced by TLRs in human corneal epithelial cells (HCEC).  Therefore, the goal of the current study was to examine this mechanism further through an evaluation of vitamin D’s influence on gene expression in two different HCEC cell lines.
Microarray comparing hTCEpi, human corneal epithelial cells, treated for 6 hours with Vitamin D (1,25D3) and control (0.1% ethanol)

overall_design: Two-condition arrays: hTCEpi cells were treated with either 100nM 1,25D3 or 0.1% Ethanol for 6h. Two biological replicates, one per array, with a dye swap (technical replicate).


title: RNA Microarray of 36H liver stages of Pb WT and Pb SSPELD Knock out parasites

city: Hyderabad

first: Segireddy

middle: Rameswara

last: Reddy

state: Telangana

address: Gachibowli

department: Animal Biology

country: India

institute: University of Hyderabad

summary: Global gene expression profiling of P. berghei liver stages at 36h from WT and Pb SSPELD KO parasites to investigate the changes in RNA expression levels in Pb SSPELD parasites.

overall_design: HepG2 cells were infected with Plasmodium berghei Wild type and SSPELD knock out sporozoites

first: Faisal

middle: M

last: Al-Nihmi

first: Kota

middle: A

last: Kumar


title: The expression of lncRNA in cow mammary glands infected with different pathogens

city: Yangzhou

first: Yongjiang

last: Mao

address: No.48 East Wenhui Road

institute: Yangzhou University

summary: To identify the expression of lncRNA in cow mammary glands infected with different pathogens, we used our custom lncRNA gene chip to explore expression of lncRNA from three Chinese cows. Compared with the control individual, a total of 629 and 139 lncRNAs showed significant different expression in mammary gland infected with S. aureus, and S. uberis, respectively. In addition, we found 3610, and 720 pairs of lincRNA/proteinencoding genes showing a highly significant correlated expression in mammary gland infected with S. aureus and S. uberis, respectively. Else, 309 and 98 pairs of lincRNA/proteinencoding genes showing a highly significant cis-regulation in mammary gland infected with S. aureus and S. uberis, respectively. Some of the lincRNAs expressed in mammary gland are located within quantitative trait loci for mastitis resistance. Our study first provides a glimpse into the lncRNA content of bovine mammary gland and will facilitate future experimental studies to unravel the function of these molecules. It may prove useful to elucidate their effects on mechanisms underlying the genetic variability of mastitis resistance.

overall_design: Three primiparous Holstein cows in mid-lactation were used for this study .At the beginning of the study, these cows were clinically healthy for > 2 consecutive weeks based on composite milk SCC < 200,000 cells/mL and having no clinical signs of mastitis or any other disease during early lactation. A1 as control, B3 was infected with a 5 mL suspension of 1000000 CFU/ml S. aureus (ATCC29213),C3 was infected with S. uberis.  Systemic and local inflammatory indicators were used to monitor the clinical response to intramammary challenge. At 24 h post-inoculation, and before peak clinical signs, the mammary gland tissues both the infected and un-infected quarters were sampled using surgical operation by experienced veterinarian.

first: Xiaorui

last: Zhu

first: Shiyu

last: Xin

first: Rui

last: Li

first: Dan

last: Chen

first: Chenlong

first: Dejun

last: Ji

first: Huiming

first: Zhangping

last: Yang


title: Meiosis timecourse Rap1 ChIP-chip

first: Sean

last: Hanlon

summary:
To investigate the genome-wide binding dynamics of Rap1 during  the transition from vegetative growth to meiosis, we performed Rap1 ChIP-chip experiments in vegetative cells (YPD), respiratory cells (pre-meiotic/YPA 8 hours), and in cells undergoing synchronous meiosis/sporulation in the budding yeast Saccharomyces cerevisiae.
There is growing recognition that the binding of a transcription factor near a gene does not always indicate regulatory function, and further that a single factor may function to either activate or repress its targets depending on the cellular context. We examined these issues through a series of experiments involving the S. cerevisiae transcription factor Rap1, and its function throughout critical metabolic and developmental transitions between vegetative growth, respiratory growth, meiosis and sporulation. We simultaneously monitored the expression of all genes and the genomic binding locations of Rap1 throughout the timecourse. Genes downstream of Rap1 binding were activated and repressed dynamically, but a change - or lack of change - in Rap1 binding status was not predictive of activation, repression, or no change in regulation. Despite this, we show that Rap1 is required, at a given point in time, for both activation and repression of different gene targets, within the same cell. Specification of the transcriptional consequences of Rap1 binding is thus highly promoter-specific. The presence of other transcription factor binding motifs, the subtype of Rap1 motif, and the underlying chromatin structure of the promoter cannot fully account for the observed transcriptional outcomes. There is a better accounting for the dynamic binding behavior of Rap1 including specification of an expanded meiotic target set through a Tup1- dependent nucleosome-loss mechanism. The variable and dynamic association between binding and transcription in this simple unicellular system portends a similarly volatile relationship in more complex eukaryotes. Biological interpretations of transcription factor occupancy should be made cautiously and in conjunction with supporting data obtained under the precise condition of interest.

overall_design: SK1 yeast strain SHy002 (MATa/MATα ho::LYS2/ho::LYS2 leu2::hisG/leu2::hisG lys2/lys2 ura3/ura3).  Meiosis timecourse with samples collected during vegetative growth (YPD), respiratory growth (pre-meiotic/YPA 8 hours) and 0 hr, 1.5hr, 3hr, 4.5hr, 6hr, 9hr and 12hr after transfer to sporulation media (SM).  7 separate biological replicates were used for each condition/time-point.  Labeled ChIP and input samples were competitively hybridized to yeast whole genome PCR based spotted arrays (resolution ~1kb).  The Rap1 ChIP, RNA abundance/expression analysis, and FAIRE analysis were all carried out on the same biological samples. The two Mock Rap1 ChIP timecourse replicates correspond to timecourse replicates 3 and 4.

first: Hanlon

last: Sean Erik

first: Lieb

last: Jason D.


title: Transcriptional profiling of Escherichia coli after addition of CO gas to aerobically and anaerobically growing cells

city: Sheffield

first: Robert

last: Poole

state: South Yorkshire

address: Firth Court, Western Bank

department: Molecular Biology & Biotechnology

country: United Kingdom

institute: University of Sheffield

summary:
Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in Evans media containing 2 mM glucose as the sole and limiting source of energy and carbon. The working volume was 200 ml, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 0.1 l/min, and the dissolved oxygen tension was maintained at ~7% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbe® electrode. For anaerobic growth, cells are grown on 100 % nitrogen gas bubbled at 0.1 l/min Cells were grown as above to steady-state, At steady-state, CO gas was bubbled through the culture at 0.1L/min.
Samples were taken immediately prior to the addition of CO gas and over a time course of 2.5, 5, 10, 20, 40 and 80 min exposure to CO gas for subsequent analysis using microarrays. Cells were harvested directly into phenol:ethanol to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers.

overall_design: Time course experiment with samples taken immediately prior to or 2.5, 5, 10, 20, 40 or 80 minutes after addition of CO gas under either 0 or 100 % perceived aerobiosis. 2 biological repeats were performed for the aerobic condition with 2 technical (dye swap) repeats per biological repeat, and three biological repeats were perfomed for the anaerobic condition with 2 technical (dye swap) repeats per biological repeat.

first: Lauren

last: Wareham

first: Samantha

last: McLean

middle: K


title: Effect of Itolizumab on gene expression profiles of patients enrolled in phase 2 psoriasis clinical trial study (Study number: T1hAb‐CT1‐001‐07).

city: Bangalore

first: Genotypic

last: technology

state: Karnataka

address: 259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE

institute: Genotypic Technology

summary: Punch biopsies of patients enrolled in phase II clinical trial study was obtained at various time points. Skin biopsies from 16 responders and 8 non responders and partial responders, chosen after the completion of clinical trial, was evaluated before Itolizumab treatment (Day 1) and at Day 57. Responders had a PASI improvement >75% while non responders had a PASI improvement <60%. The gene expression values at Day 57 were normalised with values obtained at Day 1 of the respective patient sample. The genes with a minimum fold change of 1.8 spontaneously clustered into predominantly responders and non responders.

overall_design: Agilent Custom Human Gene Expression 8X15k (AMADID: 16332) designed by Genotypic Technology Private Limited .

first: Pradip

last: Nair

first: Disha

last: Rao


title: MiRNA expression profiling of skeletal muscles in aged rodents

status: Public on Aug 31 2016

city: Toyohashi

first: Toshio

last: Kojima

address: 1-1 Hibarigaoka Tenpaku-cho

department: Health Care Center

institute: Toyohashi University of Technology

summary: Skeletal muscle atrophy is one of the critical issues which elderly people face. The precise mechanism underlying muscle atrophy during aging is not fully understood. In order to identify miRNA whose expression is changed in age-associated muscle atrophy, we performed miRNA expression profiling of skeletal muscles in young and aged rats. Microarray analysis revealed differential miRNA expression in EDL and soleus muscles of aged rats compared with those of young rats. We next investigated whether the age-associated changes of miRNA expression observed in rats were recapitulated in mice and found that the expression level of miR-206 in EDL muscle and that of miR-196a in EDL and soleus muscles were respectively higher and lower in aged rodents than in young rodents. In mouse C2C12 myoblasts and myotubes, introduction of miR-196a decreased the protein level of Forkhead-box transcription factor Foxo1, a known target of miR-196a, indicating that miR-196a may regulate Foxo1 expression also in skeletal muscles. Furthermore, miR-196a overexpression exacerbated cell death caused by an exposure to hydrogen peroxide. Lastly, we demonstrated that expression of Foxo1 was elevated in EDL and soleus muscles of aged mice compared with those of young mice. These results suggest that miRNAs are involved in skeletal muscle atrophy during aging and that decreased miR-196a expression may protect skeletal muscle cells from oxidative stress in part through induction of Foxo1.

overall_design: Male Fischer 344 rats were obtained at one month of age from Charles River Laboratories (Yokohama, Japan). Rats were maintained under the standard animal laboratory condition with 12-h light-dark cycle at 23 ºC and were provided water and food ad libitum. Rats were sacrificed at 6-7 and 25-28 months of age and hindlimb muscles were dissected. The weight of the dissected extensor digitorum longus (EDL) muscle, and soleus muscle were measured. Skeletal muscles dissected from rodents were immersed in the RNAlater RNAstabilization reagent (Qiagen, Hilden, Germany) to prevent RNA degradation. The experimental procedures were approved by the Animal Ethics Committee of the Tokyo Metropolitan Institute of Gerontology.

first: Yasunori

last: Fujita

first: Kyojiro

last: Kawakami

first: Masafumi

last: Ito


title: Meiosis timecourse expression profile

summary:
To investigate transcriptome dynamics during the transition from vegetative growth to meiosis, we measured RNA abundance in vegetative cells (YPD), respiratory cells (pre-meiotic/YPA 8 hours), and in cells undergoing synchronous meiosis/sporulation in the budding yeast Saccharomyces cerevisiae.
There is growing recognition that the binding of a transcription factor near a gene does not always indicate regulatory function, and further that a single factor may function to either activate or repress its targets depending on the cellular context. We examined these issues through a series of experiments involving the S. cerevisiae transcription factor Rap1, and its function throughout critical metabolic and developmental transitions between vegetative growth, respiratory growth, meiosis and sporulation. We simultaneously monitored the expression of all genes and the genomic binding locations of Rap1 throughout the timecourse. Genes downstream of Rap1 binding were activated and repressed dynamically, but a change - or lack of change - in Rap1 binding status was not predictive of activation, repression, or no change in regulation. Despite this, we show that Rap1 is required, at a given point in time, for both activation and repression of different gene targets, within the same cell. Specification of the transcriptional consequences of Rap1 binding is thus highly promoter-specific. The presence of other transcription factor binding motifs, the subtype of Rap1 motif, and the underlying chromatin structure of the promoter cannot fully account for the observed transcriptional outcomes. There is a better accounting for the dynamic binding behavior of Rap1 including specification of an expanded meiotic target set through a Tup1- dependent nucleosome-loss mechanism. The variable and dynamic association between binding and transcription in this simple unicellular system portends a similarly volatile relationship in more complex eukaryotes. Biological interpretations of transcription factor occupancy should be made cautiously and in conjunction with supporting data obtained under the precise condition of interest.

overall_design: SK1 yeast strain SHy002 (MATa/MATα ho::LYS2/ho::LYS2 leu2::hisG/leu2::hisG lys2/lys2 ura3/ura3).  Meiosis timecourse with samples collected during vegetative growth (YPD), respiratory growth (pre-meiotic/YPA 8 hours) and 0 hr, 1.5hr, 3hr, 4.5hr, 6hr, 9hr and 12hr after transfer to sporulation media (SM).  7 separate biological replicates were used for each condition/time-point.  Labeled total RNA from each sample and a labeled genomic DNA common reference were competitively hybridized to yeast whole genome PCR based spotted arrays (resolution ~1kb).  The Rap1 ChIP, RNA abundance/expression analysis, and FAIRE analysis were all carried out on the same biological samples.

first: S

first: J

last: Lieb


title: Gene expression in transgenic line overexpressing Arabidopsis AT5G40830 (ICA)

city: Pohang

first: Ildoo

last: Hwang

address: Chungam-ro

department: Department of life science

country: South Korea

institute: POSTECH

summary: To investigate the genome-wide effects of ica transgenic line overexpressing AT5G40830 (ICA) involved in the cambial activity and shoot growth, we used cDNA microarrays to establish global changes in the gene expression. We selected the uppermost 1 cm of vegetative nodes in the inflorescence stems at the sixth silique stage. These showed the most distinctive changes in phenotypes and endogenous hormone levels between ica and Col-0. Expression of 9 genes (ARR5, ARR6, ARR7, EXP1, GASA4, GASA6, PME1, PME44, AT3G49220) based on microarray analysis was quantified by real-time PCR.

overall_design: Total RNA was isolated from the inflorescence stem at the stage of the sixth silique development in Col-0 and ica

first: Hyunsook

last: Kim

first: Daeseok

last: Choi


title: Zonal Gene Expression and Gene Regulatory Network Responses in Centrilobular and Periportal Hepatocytes Following Repeated Exposure to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)

city: Research Triangle Park

first: Michael

middle: B.

last: Black

state: North Carolina

address: 100 Capitola Dr., Suite 106

institute: Scitovation, LLC.

summary:
Wild-type and AHR-KO rats harboring a 29-bp deletion mutation in exon 2 of the AHR gene were used in this study.  Wild-type and AHR-KO rats were generated from heterozygous breeding stocks generated on a Sprague-Dawley outbred background and maintained at the Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina
Persistent activation of the aryl hydrocarbon receptor (AHR) is believed to play a key role in the mode-of-action for TCDD induced rat liver tumorigenesis. It has been hypothesized that the cellular responses of hepatocytes to AHR activation may differ across regions of the liver lobule and that zone-specific effects of AHR agonists may play a role in the pathogenesis of rat liver tumorigenesis. Dose-dependent changes in gene expression were observed in both populations of hepatocytes collected from WT rats which were consistent with activation of AHR signaling.  No significant or dose-dependent changes in gene expression were observed in samples from AHR-KO rats.  In addition, evidence of inflammatory signaling pathway activation was observed only in centrilobular hepatocytes.  Evidence of cell adhesion pathway enrichment was observed only in periportial hepatocytes.  Benchmark dose analysis demonstrated that dose-dependent changes in gene expression occurred at lower doses in centrilobular as compared to periportal hepatocytes.  These results indicate zone-specific differences in the sensitivity and response of hepatocytes to persistent AHR activation.

overall_design: AHR-KO and WT rats (n = 10 rats/dose/genotype) were dosed by oral gavage (4-5 doses / week, 19 total doses) with varying concentrations of TCDD in corn oil (0, 3, 22, 100, 300, 1000 ng/kg/day). Cryopreserved liver slices collected at necropsy were thawed, cryosectioned (12 µm thickness), fixed with ethanol and “quick-stained” with hematoxylin. Centrilobular and periportal hepatocytes were collected using laser capture microdissection. Total RNA was extracted, amplified and labeled using low-yield techniques and global transcriptomic profiles were obtained using Affymetrix Gene-Titan peg arrays.

middle: B

first: Joshua

last: Harrill


title: Aging-associated inflammatory and oxidative changes in the rat urinary bladder and dorsal root ganglia - preventive effect of caloric restriction [bladder]

summary: Purpose: To disclose the molecular mechanisms of aging-related functional impairments of the rat bladder, and to determine whether long-term caloric restriction (CR) may have preventive effects on these mechanisms.    Materials and Methods: Male Fischer 344 rats were divided into three groups: young (6 months-old) fed ad libitum (Y, N = 16), old (25-28 months-old) fed ad libitum (O+AL, N = 15), and old (25-28 months-old) fed restrictedly three days a week since 6 weeks-old (O+CR, N = 16). cDNA microarray analysis and real-time polymerase chain reaction (RT-PCR) of the bladder and L6 dorsal root ganglia (L6DRG) were performed. The bladder was subjected to oxidative stress measurement and immunohistochemistry.  Results: In the O+AL group, 83 genes in the bladder and 48 genes in the L6DRG were up-regulated than compared with those of the Y and O+CR groups (fold change > 2). These genes were mostly related to immune- and inflammatory-responses. Immunohistochemistry showed that Granzyme B, a cytotoxic T-lymphocyte-associated serine protease, and matrix metalloproteinase 13 (Mmp13), an interstitial collagenase, were more strongly expressed in the bladder of the O+AL than the Y and O+CR groups. The level of Malondialdehyde (MDA), an oxidative stress marker, was higher in the O+AL than the Y group, whereas there were no significant differences between the O+CR and Y groups.   Conclusions: In the rat, aging is associated with up-regulation of immune and inflammatory genes in the bladder and DRG as well as with oxidative and fibrotic changes of the bladder. Long-time CR reduced these aging-related changes.

overall_design: Male Fischer 344 rats (Charles River Laboratories International, Inc. Yokohama, Japan) were divided into three groups: young (6 months-old) ad libitum fed with normal food (Y, N = 4), old (25-28 months-old) ad libitum fed with normal food (O+AL, N = 4) and old (25-28 months-old) that had been fed three days (on Monday, Wednesday, and Friday) a week since 6 weeks-old (O+CR, N = 4). The O+CR groups showed approximately 40% caloric restriction compared with the O+AL groups. Rats were maintained under standard laboratory conditions with a 12:12 h light: dark cycle, and free access to food pellets and tap water. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Tokyo and Tokyo Metropolitan Hospital and Institute of Gerontology, and conformed to NIH guidelines for the care and use of experimental animals.

first: Hiroki

first: Naoki

last: Aizawa

first: Motofumi

last: Suzuki

first: Hiroshi

last: Fukuhara

first: Yukio

last: Homma

first: Yoshinobu

last: Kubota

first: Karl-Erik

last: Andersson

first: Yasuhiko

last: Igawa


title: Gene expression profiles of esophageal squamous cell carcinoma cell lines

city: Kyoto

first: T

last: Fujiwara

address: 46-29 Yoshida Shimoadachi-cho Sakyo-ku

institute: Kyoto University

summary: Profiles of esophageal squamous cell carcinoma and normal esophageal normal epithelium normal cell line. Analysis provides validation of novel microRNA targets prediction algorithms.

overall_design: esophageal squamous cell carcinoma:14, normal epithelium cell:2

first: Takeshi

first: Soken

last: Tsuchiya

first: Fumiaki

last: Sato

first: Yutaka

last: Shimada

first: Gozoh

last: Tsujimoto

first: Kazuharu

last: Shimizu


title: Aging-associated inflammatory and oxidative changes in the rat urinary bladder and dorsal root ganglia - preventive effect of caloric restriction

first: Toshio

last: Kojima

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

overall_design: Refer to individual Series


title: Aging-associated inflammatory and oxidative changes in the rat urinary bladder and dorsal root ganglia - preventive effect of caloric restriction [L6 dorsal root ganglia]

last: Ito

first: Yasunori

last: Fujita

first: Masafumi


title: Loss of beta-catenin triggers oxidative stress and impairs hematopoietic regeneration

city: Durham

first: William

last: Lento

state: NC

address: 213 Research Drive

department: Medicine/Cellular Therapy

institute: Duke University

summary: Accidental or deliberate ionizing radiation exposure is fatal due to widespread hematopoietic destruction. However, little is known about either the course of injury or the definitive molecular pathways that regulate the subsequent regenerative response. We have investigated the spatial and temporal consequences of radiation injury on  the hematopoietic system and show the Wnt signaling pathway is critially important for regeneration.

overall_design: Sorted KLS34- hematopoietic stem cells from 3 control samples and 3 beta catenin floxed vav-Cre positive  were analyzed.

first: Takahiro

first: Chen

last: Zhao

first: Jeffrey

middle: R

last: Harris

last: Jiang

first: Kouros

last: Owzar

first: Nelson

last: Chao

first: Tannishtha

last: Reya


title: miRNA expression profiles of HPV-infected cervical cancer patients in the Uyghur population in China

status: Public on Aug 27 2016

city: xinjiang

first: he

last: ying

state: State...

address: urlumuqi

institute: xinjiang medical university

summary: The study examined the infection state of HPV in the Uyghur population with cervical cancer, followed by genotyping to determine the variation in the types of HPV. Using microRNA microarray technology, differential gene expression between HPV-infected cervical cancer and uninfected normal cervical tissues was determined. The microarray results were verified by qRT-PCR using 20 sets of HPV-infected cervical cancer and uninfected cervical tissues.

overall_design: Cervical cancer tissues and uninfected normal mucosa tissues were obtained from the Obstetrics and Gynecology Department of Xinjiang Medical University Cancer Hospital. All tissues were obtained between June 2012 and June 2013 as fresh specimens from surgical resection, and stored at -80°C until use. There were 30 cervical cancer tissues and 26 normal mucosa tissues. All patients were from the Uyghur population of Xinjiang, China. All cervical specimens were confirmed as squamous cell carcinoma through pathological examination. Prior to tissue sampling, patients had not received any chemotherapy treatment. Samples from the control group were determined to be normal by ThinPrep cytologic tests. This study was approved by the Ethics Committee of Xinjiang Medical University Cancer Hospital (K-201343), and informed consent was obtained from all patients.

first: Dongmei

last: Gao


title: Hydrogen cyanamide breaks grapevine bud dormancy in the summer

status: Public on Aug 30 2016

city: Chiayi City

first: Yung-fu

last: Yen

address: 300, University Road

department: Bioagricultural Science

country: Taiwan

institute: National Chiayi University

summary: Microarray analysis revealed specific alterations in gene expression in dormancy breaking buds induced by pruning (P), hydrogen cyanamide (HC), pruning plus hydrogen cyanamide (PHC) after 24 h of treatment. PHC treatment altered the expression of the largest number of genes and rapid accumulation of a sublethal level of reactive oxygen species and reactive nitrogen species subsequently induces cell wall loosening and expansion for bud sprouting

overall_design: The dormancy buds treated by P, HC, PHC and water as control. Biological replicates: 3 replicates.

first: Boonyawat

last: Sudawa

first: Maurice

middle: S

last: Ku

first: Hsiu-fung

first: Chih-Sheng

last: Chang


title: C. elegans gene expression in response to acute Pseudomonas aeruginosa infection (Part 1) and recovery (Part 2) by Neomycin/Streptomycin treatment

status: Public on Aug 11 2016

first: Brian

middle: P

last: Head

address: Box 3580 DUMC

department: Molecular Genetics and Microbiology

institute: Duke University Medical Center

summary:
(Part 1) Gene expression profiles of C. elegans in response to P. aeruginosa. Synchronized larval stage 1 (L1) worms were raised on E. coli OP50 for 72 hours. These synchronized young adult (YA) animals were subsequently exposed to P. aeruginosa PA14 for 4 hours. 25dC.
(Part 2) Gene expression profiles of C. elegans in response to Neomycin/Streptomycin-mediated recovery after a 4 hour exposure to P. aeruginosa. Synchronized larval stage 1 (L1) worms were raised on E. coli OP50 for 72 hours. These synchronized young adult (YA) animals were subsequently exposed to P. aeruginosa PA14 for 4 hours and then treated with Neomycin and shifted to E. coli OP50 plus Streptomycin plates for 6, 12, or 24 hours to resolve the infection. As a control for Neomycin/Streptomycin exposure, synchronized larval stage 1 (L1) worms were raised on E. coli OP50 for 72 hours and either shifted to fresh E. coli OP50 plates for 6 hours or treated with Neomycin and shifted to E. coli OP50 plus Streptomycin plates for 6 hours. 25dC.

overall_design: Samples include 72 hours on OP50 (72hOP), 72 hours on OP50 plus 4 hours on PA14 (76hPA), 72 hours on OP50 plus 4 hours on PA14 plus 6 hours of Neomycin/Streptomycin-mediated recovery (82hR), 72 hours on OP50 plus 4 hours on PA14 plus 12 hours of Neomycin/Streptomycin-mediated recovery (88hR), 72 hours on OP50 plus 4 hours on PA14 plus 24 hours of Neomycin/Streptomycin-mediated recovery (100hR), 82 hours on OP50 (82hOP), and 72 hours on OP50 plus 6 hours of Neomycin/Streptomycin (82hOPC). Triplicate biological samples were included for each timepoint except for 82hOP (which utilized duplicates). 20 total samples. Agilent C. elegans expression microarrays (GPL11346).

first: Alejandro

last: Aballay


title: Functional genomics study of Aspergillus niger biofilms for the identification and characterization of genes of lignocellulolytic enzymes of industrial interest

status: Public on Aug 23 2016

city: La Moilina

first: Marcel

last: Gutiérrez-Correa

address: Av. La Molina

country: Peru

institute: UNALM

summary: Aspergillus niger produces a variety of lignocellulolytic enzymes (cellulases, hemicellulases, among others) and is regarded as cell factory for the production of heterologous proteins. Therefore, there is a growing interest in the study of its genes and the understanding of the cellular mechanisms in order to expand its applications. On the other hand, we have shown that enzyme production by A. niger is higher when grown forming biofilms than when grown conventionally in submerged systems. The objective of this study was to perform a global transcriptomic analysis and an expression analysis of both lignocellulases and biofilm regulatory genes as compared to A. niger in submerged culture. DNA microarray assays were performed to investigate the global gene expression which yielded information on the expression of more than 90% of A. niger genes. To further this comparison, the two culture systems were supplemented with different carbon sources (glucose, lactose, xylose and maltose) to establish a differential gene expression under different culture conditions. Also, to validate the differential expression qPCR was performed for quantitative comparison of the transcriptional level of genes in both culture systems.

overall_design:
Organism : Aspergillus niger,  Agilent Aspergillus niger Gene expression 4x44k Array AMADID: 032510
Grant Information: Grant Nº 072-FINCyT-PIN2008 from the National Program of Science and Technology of Peru
Contributor: Institut Pasteur de Montevideo, Uruguay

first: Yevtte

last: Ludeña

first: Sujay

last: Paul

first: Gretty

last: Villena


title: Investigation of miRNA expression signatures in mTORC1 -stimulated osteoarthritis

status: Public on Jul 30 2016

city: guanghzou

first: hua

last: wang

state: guangdong

address: gongzhou dadao bei

department: cell biology

institute: southern medical university

summary: To further explain pathology of mTORC1 -stimulated osteoarthritis, we have employed whole genome microarray expression profiling as a discovery platform to identify miRNAs which involved in development of mTORC1-stimulated osteoarthritis

overall_design: We generated Col2a1-specific deletion of Tsc1 mice. mTORC1 induced miRNAs expression in development of osteoarthritis was measured at eight weeks after birth. Independent experiments were performed using knee joint cartilage from Col2a1Tsc1KO and control mice.

first: Xiaochun

last: Bai

first: Hua

last: Wang


title: Disease Severity and Immune Activity Relate to Inter-Kingdom Gut Microbiome States in Ethnically Distinct Ulcerative Colitis Patients

status: Public on Jul 25 2016

city: San Francisco

first: Jordan

middle: Samuel

last: Mar

state: Ca

address: 513 Parnassus Ave., S-357

department: Medicine, Division of Gastroenterology

institute: University Of California, San Francisco

summary: Significant gut microbiota heterogeneity exists amongst UC patients though the clinical implications of this variance are unknown. European and South Asian UC patients exhibit distinct disease risk alleles, many of which regulate immune function and relate to variation in gut microbiota β-diversity. We hypothesized ethnically distinct UC patients exhibit discrete gut microbiotas with unique luminal metabolic programming that influence adaptive immune responses and relate to clinical status. Using parallel bacterial 16S rRNA and fungal ITS2 sequencing of fecal samples (UC n=30; healthy n=13), we corroborated previous observations of UC-associated depletion of bacterial diversity and demonstrated significant gastrointestinal expansion of Saccharomycetales as a novel UC characteristic. We identified four distinct microbial community states (MCS 1-4), confirmed their existence using microbiota data from an independent UC cohort, and show they co-associate with patient ethnicity and degree of disease severity. Each MCS was predicted to be uniquely enriched for specific amino acid, carbohydrate, and lipid metabolism pathways and exhibited significant luminal enrichment of metabolic products from these pathways. Using a novel in vitro human DC/T-cell assay we show that DC exposure to patient fecal water led to MCS -specific changes in T-cell populations, particularly the Th1:Th2 ratio, and that patients with the most severe disease exhibited the greatest Th2 skewing. Thus, based on ethnicity, microbiome composition, and associated metabolic dysfunction, UC patients may be stratified in a clinically and immunologically meaningful manner, providing a platform for the development of FMC-focused therapy.

platform_organism: Bacteria Archaea

overall_design: Fecal microbiome was assessed with Affymetrix PhyloChip arrays from patients with ulcerative colitis and healthy controls.

first: Brandon

middle: J

last: LaMere

first: Din

middle: L

last: Lin

first: Sophia

last: Levan

first: Michelle

last: Nazareth

first: Uma

last: Mahadevan

first: Susan

middle: V

last: Lynch


title: The effect of PRRSV on modulation of porcine monocyte-derived dendritic cell (MoDC)

city: Bangkok

first: Teerawut

last: Nedumpun

department: Veterinary Microbiology

country: Thailand

institute: Chulalongkorn University

summary: To investigate the gene expression of MoDC after PRRSV stimulation and nucleocapsid protein transfection

overall_design: MoDC were cultured with 0.1 m.o.i. of PRRSV or mock-infected MARC 145 cell lysate for 24 hr. In another experiment, MoDC were transfected with plasmid encoding nucleocapsid protein or null plasmid for 24 hr. Total mRNA were subjected to Microarray analysis.

first: Sanipa

last: Suradhat


title: Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis [dtatC-vs-ip_37C-stat]

status: Public on Jul 01 2016

city: Braunschweig

first: Ann

middle: Kathrin

last: Heroven

state: Niedersachsen

address: Inhoffenstr. 7

department: Molecular Infection Biology

country: Germany

institute: Helmholtz Centre for Infection Research

summary: The Twin-arginine translocation (Tat) system promotes secretion of folded proteins that in bacteria are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. However, in silico predictions did not reveal any obvious virulence factors among the potential Tat substrates encoded by Y. pseudotuberculosis. Aiming to identify Tat dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26°C during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper concentrations as well as sensitivity to acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypes of the Tat deficient strain were generally more pronounced than for the individual mutants of the genes encoding the specific Tat substrates. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.

overall_design: Y. pseudotuberculosis IP32953 was grown in LB medium supplemented with 2.5 mM CaCl2 under aeration for one hour at 26°C and then shifted to 37°C. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.

first: Ummehan

last: Avican

first: Michael

last: Beckstette


title: Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis [dtatC-vs-ip_37C-log]


title: Two Populations of Glucocorticoid Receptor-Binding Sites in the Male Rat Hippocampal Genome

status: Public on Jul 09 2016

city: Boston

first: Richard

middle: G.

last: Hunter

state: Massachusetts

address: 100 William Morrissey Blvd.

department: Psychology

institute: University of Massachusetts Bo

type: Genome binding/occupancy profiling by high throughput sequencing

summary: In the present study, genomic binding sites of glucocorticoid receptors (GR) were identified in vivo in the rat hippocampus applying chromatin immunoprecipitation followed by next-generation sequencing. We identified 2470 significant GR-binding sites (GBS) and were able to confirm GR binding to a random selection of these GBS covering a wide range of P values. Analysis of the genomic distribution of the significant GBS revealed a high prevalence of intragenic GBS. Gene ontology clusters involved in neuronal plasticity and other essential neuronal processes were overrepresented among the genes harboring a GBS or located in the vicinity of a GBS. Male adrenalectomized rats were challenged with increasing doses of the GR agonist corticosterone (CORT) ranging from 3 to 3000 μg/kg, resulting in clear differences in the GR-binding profile to individual GBS. Two groups of GBS could be distinguished: a low-CORT group that displayed GR binding across the full range of CORT concentrations, and a second high-CORT group that displayed significant GR binding only after administering the highest concentration of CORT. All validated GBS, in both the low-CORT and high-CORT groups, displayed mineralocorticoid receptor binding, which remained relatively constant from 30 μg/kg CORT upward. Motif analysis revealed that almost all GBS contained a glucocorticoid response element resembling the consensus motif in literature. In addition, motifs corresponding with new potential GR-interacting proteins were identified, such as zinc finger and BTB domain containing 3 (Zbtb3) and CUP (CG11181 gene product from transcript CG11181-RB), which may be involved in GR-dependent transactivation and transrepression, respectively. In conclusion, our results highlight the existence of 2 populations of GBS in the rat hippocampal genome. - See more at: http://press.endocrine.org/doi/10.1210/en.2012-2187?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#sthash.LqK088DP.dpuf

overall_design:
Hippocampal Glucocorticoid receptor ChIP-seq using Santa Cruz H-300 Ab, for 300ug.kg and 3mg/kg corticosterone treatment and associated IgG controls

Raw data are unavailable for this series because the raw data files have been lost. Available data include read alignments in bed format and genome coverage data in wiggle format.

middle: G

first: Nicole

last: Datson


title: Effects of exendin-4 in Brunner's glands of male GLP-1R-/-  and wild-type control mice

city: Måløv

first: Klaus Stensgaard

last: Frederiksen

address: Novo Nordisk Park F9.1.07

department: Molecular Genetics

country: Denmark

institute: Novo Nordisk A/S

summary: To gain insight into the biological functions of the highly expressed GLP-1R in Brunner’s glands, transcriptome analyses were conducted in male GLP-1R-/-  and wild-type control mice. Analyses were performed 6 hours after a single s.c. dose of exendin-4 (1.0mg/kg s.c.),  following 18 hours of two doses of exendin-4 (1.0 mg/kg s.c., administered at 0 and 9 hours), and in untreated controls.  Brunner’s glands were isolated by laser capture micro dissection and extracted total RNA was used for microarray profiling.

overall_design: A total of 18 samples consisting of laser captured microdissected Brunner's glands from individual male GLP-1R-/- and wild-type (CD-1) mice. Before the isolation of Brunner's glands, mice were dosed with exendin-4 for 6 and 18 hours. The extracted total RNA from Brunner's glands were compared by full transcriptome profiling.

first: Klaus

first: Rolf

last: Søkilde

first: Claus

middle: H

last: Bang-Berthelsen

first: Lasse

last: Folkersen


title: Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis [dtatC-vs-ip_26C-stat]

overall_design: Y. pseudotuberculosis IP32953 was grown at 26°C in LB medium under aeration. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.


title: Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis


title: Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis [dtatC-vs-ip_26C-log]

overall_design: Y. pseudotuberculosis IP32953 was grown at 26°C in LB medium under aeration on a rotary shaker. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.


title: Microarray analysis of liver and mammary gland biopsies of dairy cows supplemented with vitamins B9 and B12 alone or in combination

status: Public on Jun 30 2016

city: Sherbrooke

first: Bazoumana

last: Ouattara

state: Quebec

address: 2000 College

department: Agriculture and Agri-Food Canada

country: Canada

institute: Sherbrooke Research and Development Centre

summary:
Data from one animal from B9_B12 group  (Cow 31) were not taken into account for the liver samples
Treated animals were compared to control (non supplemented cows)

overall_design: Twenty four multiparous Holstein cows were assigned to 6 blocks of 4 animals according to their 305-d milk production during the previous lactation to one of the following treatments: injections saline 0.9% NaCl (Ctl); folic acid (B9); vitamin B12 (B12) or folic acid and vitamin B12 (B9_B12). Biopsies of hepatic (foie for liver) and mammary (mam for mammary gland) tissues were taken from these 24 cows. Microarray analyses were performed on samples from both tissues for 3 animals per treatment in CTL, B9 and B9_B12 and for 4 animals in B12 treatment. Data for hepatic tissue of animal 31 (group B9_B12) were not included. For both tissues, treated animals were compared to control.

first: Nathalie

last: Bissonnette

first: Melissa

last: Duplessis

first: Christiane

last: Girard


title: Cell type dependent comparison and strain specific comparison

city: Birmingham

last: Turan

address: University of Birmingham, Edgbaston

institute: University of Birmingham

summary:
Enterohemorrhagic Escherichia coli (EHEC) is a gram negative enteric bacterial pathogen that can cause hemorrhagic colitis and heamolytic uremic syndrome (HUS) in humans and is the cause of bloody diarrhoea and acute renal failure in children. We have studied the transcriptional response of a colon cell line (CaCo2) to infection by EHEC and another closely related enteric pathogen Enteropathogenic Escherichia coli (EPEC) and compared its response to a cervical cell line (Hela).
We carried out microarray analysis on CaCo2 infected with EHEC O157H:7 EDL933 and EPEC E2348/69 at 4 hours of infection and analysis on Hela infected with EHEC also at 4 hours of infection

overall_design: CaCo2 cells and Hela cells were grown to 80% confluency and infected with the bacteria for 4 hours before samples were collected for microarray analysis.

first: Francesco

last: Falciani

first: Nazish

last: Daudi


title: Genome wide gene expression during lung development in three inbred mouse strains

city: Bar Harbor

first: Carol

last: Bult

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

summary:
To better understand the temporal dynamics of gene expression during normal murine lung development we characterized global gene expression at 26 time points in three common inbred strains of mice (A/J, C57BL/6J, and C3H/HeJ). The data set provides a unique resource for identifying patterns of gene expression changes during normal lung development and for investigating the developmental origins of respiratory disease.
The transcriptional profiles generated for lung development in three inbred strains of the laboratory mouse revealed concordance with pre-natal stages of lung development defined by anatomy and morphology.  The genomic data support the view that the postnatal alveolar development is composed of 4 distinct molecular stages. The data revealed strain specific differences in the expression of genes related to respiratory cell differentiation, pulmonary innervation, metabolic pathway interactions, and immune system function.

overall_design: Mouse lungs or whole embryos (e09.5 only) were surgically dissected at 26 time points across all six canonical stages of lung development (embryonic-EMB, pseudoglandular-PSG, canalicular-CAN, saccular-SAC, alveolar-ALV, homeostatic-HOM) to identify transcriptional profiles associated with 9 molecular divisions of lung development (embryonic-EMB, pseudoglandular-PSG, canalicular-CAN, saccular-SAC, alveolar1-ALV1, alveolar2-ALV2, alveolar3-ALV3, alveolar4-ALV4, homeostatic-HOM). Lung tissue from three or four embryos collected at e11.5 or e12.5, respectively, was pooled for each sample to obtain sufficient RNA for array analysis. Tissue from at least three animals was collected for each time point (biological replicates). Only male mice were used in the study.

first: Kyle

last: Beauchemin

first: Julie

last: Wells


title: Host pathogen interaction at a transcriptional level

overall_design: Refer to individual Series


title: Comparison of acid treated and untreated

summary:
Enterohemorrhagic Escherichia coli (EHEC) is a gram negative enteric bacterial pathogen that can cause hemorrhagic colitis and heamolytic uremic syndrome (HUS) in humans and is the cause of bloody diarrhoea and acute renal failure in children. We have studied the transcriptional response of a colon cell line (CaCo2) to infection by EHEC and compared its profile to infection by EHEC shocked in acid at pH 2.5.
We carried out microarray analysis on CaCo2 infected with EHEC O157H:7 EDL933 and EHEC shocked at pH 2.5 at 4 hours post infection.

overall_design: CaCo2 cells were grown to 80% confluency and infected with acid shocked and unschocked bacteria for 4 hours before samples were collected for microarray analysis.


title: Identification of Predictive Gene Markers for Multipotent Stromal Cell Proliferation [RT-PCR]

city: Silver Spring

first: Raj

last: Puri

state: Maryland

address: 10903 New Hampshire Ave

institute: FDA

summary: Multipotent stromal cells (MSCs) are known for their distinctive ability to differentiate into different cell lineages such as adipocytes, chondrocytes and osteocytes.  They can be isolated from numerous tissue sources including bone marrow, adipose tissue, skeletal muscle and others.  Because of their differentiation potential and their secretion of growth factors, MSCs are believed to have an inherent quality of regeneration and immune suppression, which are considered advantageous in treating multiple disorders such as graft-versus-host disease.  Since the number of MSCs derived from a tissue source is low, cellular expansion is necessary to obtain sufficient numbers for a desired cell therapy.  However, after several rounds of passaging, our previous results have shown that MSCs exhibit reduced capacity for proliferation and differentiation.  In this study, gene markers of MSC proliferation were identified and evaluated for their ability to predict the cell population proliferative quality.  Microarray data of human bone marrow-derived MSCs were correlated with two proliferation assays.  A collection of 24 genes were observed to significantly correlate with both proliferation assays (|r| > 0.70) for 8 MSC donors at multiple passages.  These 24 identified genes were then confirmed using an additional set of MSCs from 8 new donors using RT-qPCR.  The proliferative potential of the second set of MSCs was measured for each donor/passage by three proliferation assays for confluency fraction, fraction of EdU+ cells and population doubling time.  The second set of MSCs exhibited a greater proliferative potential at passage 4 in comparison to passage 8, which was distinguishable by 15 genes; however, only 7 of the genes (BIRC5, CCNA2, CDC20, CDK1, PBK, PLK1, SPC25) demonstrated significant correlation (FDR: q < 0.05 and |r| > 0.62) with MSC proliferation regardless of passage.  These 7 genes and the proliferative capacity of different non-MSC cell lines were assessed for comparison.  Our analyses revealed that correlation between gene expression and proliferation was consistently reduced with the introduction of non-MSC cell lines; therefore this set of 7 genes may be more strongly associated with MSC proliferative quality.  These correlative methods may be further used to identify additional markers that exhibit strong correlation with a particular MSC quality such as differentiation or immune suppression potential.  Our results pave the way toward the identification of specific gene markers that could rapidly determine the quality of an MSC population for a particular cellular therapy in lieu of an extended in vitro or in vivo assay.

overall_design: Microarray gene expression analysis with confirmation of genes by RT-qPCR

first: Ian

last: Bellayr

first: Ross

last: Marklein

first: Jess

last: Lo Surdo

first: Steven

last: Bauer


title: Transcriptional Response to Sulfur Limitation in Pelagibacter ubique

status: Public on Jun 26 2016

city: Corvallis

first: Daniel

middle: Patrick

last: Smith

state: OR

address: 220 Nash Hall

department: Microbiology Department

institute: Oregon State University

summary: Candidatus Pelagibacter ubique is the most abundant marine microorganism, but is unable to utilize inorganic sulfur compounds that are plentiful in the ocean. To investigate how these cells adapt to organic sulfur limitation, batch cultures were grown in defined media containing either limiting or non-limiting amounts of dimethylsulfoniopropionate (DMSP) as the sole sulfur source. Protein and mRNA expression were measured during exponential growth, immediately prior to stationary phase, and in late stationary phase. Two distinct responses were observed: one as DMSP approached exhaustion, and another after the DMSP supply was depleted. The first response was characterized by increased transcription and translation of all Ca. P. ubique genes downstream of previously confirmed S-adenosyl methionine (SAM) riboswitches: bhmT, mmuM, and metY. These genes were up to 33 times more abundant during low DMSP conditions and shunt all available sulfur to methionine. The osmotically inducible organic hydroperoxidase OsmC was the most up-regulated protein as DMSP (an osmolyte) became scarce. The second response, during sulfur-depleted stationary phase, saw increased transcription of the heme c shuttle ccmC and two small genes of unknown function (SAR11_1163 and SAR11_1164) which were 6-10 times higher in sulfur-starved cultures. No known membrane transporters were up-regulated in response to sulfur limitation, suggesting that this bacterium's strategy for coping with sulfur stress focuses on intracellularly redistributing, rather than importing, organic sulfur compounds. This supports the conclusion that the few organosulfur molecules that Ca. P. ubique is able to metabolize are rarely limiting in the marine environment.

platform_organism:
Candidatus Pelagibacter ubique HTCC1002
Candidatus Pelagibacter ubique HTCC1062
Candidatus Pelagibacter sp. HTCC7211

overall_design: Batch cultures of P. ubique were grown in a defined arificial seawater media. Five cultures were amended with a limiting concentration of DMSP as the sole sulfur source and another four control cultures were amended with a non-limiting DMSP concentration. Cultures were harvested for microarray analyses at multiple timepoints for the purpose of observing differences in gene expression related to sulfur limitation. Proteomic analyses were conducted in parallel and are available at https://www.ebi.ac.uk/pride/archive/projects/PXD003672 .

first: Stephen

last: Giovannoni


title: CD103+ Conventional Dendritic Cells are Critical for TLR-dependent Host Defense against an Endemic Fungal Pathogen

status: Public on Jun 28 2016

first: Mark

last: Voorhies

state: CA

address: 513 Parnassus, S472

department: Microbiology and Immunology

institute: University of California

summary:
Innate immune cells shape the host response to microbial
pathogens. Here we studied the role of TLR7/9 in dendritic cell (DC)
responses to Histoplasma capsulatum, an intracellular fungal pathogen
of humans. Whereas macrophages succumbed to Histoplasma infection,
TLR7/9 were critical for DCs to produce Type I interferons (IFN-I),
restrict fungal growth, and survive fungal infection. In the mouse
model of infection, optimal IFN-I and IFN gamma production, host survival,
and restriction of cerebral fungal burden were also dependent on
TLR7/9.  Interestingly, CD103+ conventional DCs were the major
producer of IFN-I in the lungs of infected mice, revealing a
previously unknown role for these cells and uncovering their pivotal
function in modulating the host immune response to endemic fungi.

overall_design:
In 6-well tissue culture treated dishes, 1 x 10 6 BMDCs or BMMs were
subjected to either mock infection, infection with UV-treated
Histoplasma, or infection with live Histoplasma yeasts at an MOI of
4. UV-treated (UVT) yeasts were prepared by subjecting G217B to UV
light (UV Stratalinker 1800) for 1 hour; these yeasts failed to
generate colonies when plated on HMM agarose.

first: Nancy

last: Van Prooyen

first: Allen

last: Henderson

first: Davina

last: Hocking Murray

first: Anita

last: Sil


title: Mycobacterium tuberculosis Phosphate Uptake System Component PstA2 is Not Required for Gene Regulation or Virulence

status: Public on Jun 29 2016

city: Aurora

first: Martin

middle: I

last: Voskuil

state: CO

department: Microbiology

institute: University of Colorado Denver

summary: The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability. To determine if the alternative Pst system is similarly required for virulence or gene regulation, we constructed a deletion of pstA2. Transcriptome analysis revealed that PstA2 is not required for regulation of gene expression in phosphate-replete growth conditions. PstA2 was also dispensable for replication and virulence of M. tuberculosis in a mouse aerosol infection model. However, a ∆pstA1∆pstA2 double mutant was attenuated in mice lacking the cytokine interferon-gamma, suggesting that M. tuberculosis requires high-affinity phosphate transport to survive phosphate limitation encountered in the host. Surprisingly, ∆pstA2 bacteria were more resistant to acid stress in vitro. This phenotype is intrinsic to the alternative Pst transporter since a ∆pstS1 mutant exhibited similar acid resistance. Our data indicate that the two M. tuberculosis Pst transporters have distinct physiological functions, with the PstA1 transporter being specifically involved in phosphate sensing and gene regulation while the PstA2 transporter influences survival in acidic conditions.

sample_organism: Mycobacterium tuberculosis str. Erdman = ATCC 35801

platform_organism:
Mycobacterium tuberculosis CDC1551
Mycobacterium tuberculosis H37Rv

overall_design: Aerobically growing logarithmic phase Wt or pstA2 mutant or pstA1A2 double mutant strains were grown in phosphate replete media and analyzed after several hours. Experiments were repeated in triplicate.

first: Anna

middle: D

last: Tischler

first: Rachel

last: Leistikow

first: Pavithra

last: Ramakrishnan

first: John

last: McKinney


title: Filtering of RNA-seq datasets and differences between cell types in global coordination of splicing and proportion of highly expressed genes

city: Farmington

first: Ephraim

middle: F

last: Trakhtenberg

state: CT

address: 263 Farmington Ave. RM L4005

department: Neuroscience

institute: University of Connecticut School of Medicine

type: Expression profiling by high throughput sequencing

summary: The goal of this study was to investigate whether mammalian cell types intrinsically differ in global coordination of gene splicing and expression levels. We analyzed RNA-seq transcriptome profiles of 8 different purified mouse cell types. We found that different cell types vary in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, and that the cell types that express more variants of alternatively spliced transcripts per gene are those that have higher proportion of highly expressed genes. Cell types segregated into two clusters based on high or low proportion of highly expressed genes. Biological functions involved in negative regulation of gene expression were enriched in the group of cell types with low proportion of highly expressed genes, and biological functions involved in regulation of transcription and RNA splicing were enriched in the group of cell types with high proportion of highly expressed genes. These data reveal specific candidate genes, which may be involved in global coordination of balance in the transcriptome.

overall_design:
The following samples were reprocessed and reanalyzed:

Astrocytes, GSE52564/GSM1269903/GSM1269904
Endothelial cells, GSE52564/GSM1269915/GSM1269916
Cortical neurons, GSE52564/GSM1269905/GSM1269906
Oligodendrocytes, GSE52564/GSM1269911/GSM1269912
Microglia, GSE52564/GSM1269913/GSM1269914
Megakaryocyte-erythroid progenitors, GSE40522/GSM995525
Erythroid-committed precursors Gata1 KO, GSE40522/GSM995536

Libraries for all samples included two biological replicates, were prepared using polyA-selected RNA, and paired reads sequenced 100 bp from each end on HiSeq 2000 Sequencer (Illumina). The raw reads were reprocessed as follows. Reads were mapped to mouse reference genome mm10 (UCSC Genome Browser) and a comprehensive transcriptome annotation database GTF file, which was assembled by using the UCSC Table Browser Intersection utility to merge the GENCODE M4 transcripts in a non-redundant manner with the UCSC Gene Track transcripts that did not overlap more than 90% with the GENCODE transcripts. The raw reads were mapped using the TopHat/Bowtie2/Cufflinks pipeline, with -g option, to construct merged GTF file that included the annotated and novel transcript structures from all samples. Then, the IntersectBed tool (Bedtools) was used to retain only the reads that mapped to the merged GTF, which was converted to BED with Gtf2bed tool (Bedops). This filtering step allowed selecting the reads which contributed to the identified gene structures, and exclude noise and artifacts even if they mapped to the genome but did not contribute to gene structure. Next, only uniquely mapped and properly paired reads were selected using View -bq 4 -bh -f2 -F12 command (Samtools). After this step, DownsampleSam tool (Picard) was used to randomly subsample equal number of paired reads, which provided representative samples of the same size for all samples (34.6M per sample/replicate; reads count with Flagstat, Samtools). The reprocessed samples were reanalyzed as follows: The TopHat/Bowtie2/Cufflinks/Cuffdiff pipeline with -g option was used for determining normalized expression in FPKMs in each replicate of each sample with Cuffdiff’s across-sample normalization. After the cell types were segregated into two clusters based on higher or lower proportion of the highly expressed genes, the differential expression analysis was preformed between the two groups, which were treated as two conditions. For this analysis, each replicate of each cell type was assigned to one of only two cluster groups. For differential expression analysis, the Cuffdiff q-value cut off was set to 0.05. Software versions used: Tophat 2.0.12, Bowtie 2.2.4, Cufflinks 2.2.1, Samtools 0.1.19, Picard 1.79, Bedops 2.4.2, Bedtools 2.19.0. Analyses were performed on the Orchestra High Performance Compute Cluster at Harvard Medical School NIH supported shared facility.

first: Nam

last: Pho

first: Kristina

middle: M

last: Holton

first: Thomas

middle: W

last: Chittenden

first: Jeffrey

last: Goldberg

first: Lingsheng

last: Dong


title: Real-time quantitative PCR analysis of goat mammary glands in peak-lactation and late-lactation periods

status: Public on Jun 25 2016

city: Xi'an

first: Zhi

last: Chen

state: Select a State or Province

address: 22 Xinong Road, Yangling

department: Shaanxi Key Laboratory of Molecular Biology for Agriculture

institute: College of Animal Science and Technology, Northwest A&F University,  China

summary:
In comparison with cow milk, goat (Capra hircus) milk contains much higher levels of unsaturated fatty acids, as well as higher levels of total fat, proteins, carbohydrates, calcium, and vitamins.The main objective of the present study was to better define the relationship of known miRNAs regulating milk fat metabolism.
Our main purpose is to search for some known miRNAs regulating milk fat metabolism, to this end, we screened potential miRNAs with differential expression between peak-lactation and non-lactation.

overall_design: qPCR gene expression profiling.  Monocytes from three healthy goats (3 year old) of similar body weight. We screened a series of potential miRNAs involved in regulation of milk metabolism.

first: Jun

last: Luo


title: Parasite in vivo blood transcriptomes from Malawian children with cerebral malaria

status: Public on Jun 24 2016

city: Bronx

first: Johanna

middle: Patricia

last: Daily

state: NY

address: 1301 Morris Park Avenue, Price Center

department: Medicine (Infectious Disease)

institute: Albert Einstein College of Medicine

summary: Paper: Transcriptional Profiling of Plasmodium falciparum Parasites from Patients with Severe Malaria Identifies Distinct Low vs. High Parasitemic Clusters Milner et al Plos One July 18, 2012

platform_organism:
Plasmodium berghei
Plasmodium falciparum


title: Global transcriptional profiling of SG200 and the SA sensing mutant SG200∆rss1 during biotrophic growth

status: Public on Jun 23 2016

city: Vienna

first: Armin

last: Djamei

address: Dr. Bohr-Gasse 3

country: Austria

institute: Gregor Mendel Institute of Molecular Plant Biology GmbH

summary: The phenolic compound salicylic acid (SA) is a key signaling molecule regulating local and systemic plant defense responses against biotrophs. Many microbial organisms, including pathogens, share the ability to degrade SA. However, the mechanism by which they perceive SA is unknown. We showed that Ustilago maydis, the causal agent of corn smut disease, employs a so far uncharacterized SA sensing mechanism. We identified and characterized the novel SA sensing regulator, Rss1, a binuclear zinc cluster protein with dual functions as putative SA receptor and transcriptional activator. To determine global transcriptional changes between the rss1 deletion mutant SG200∆rss1 and its progenitor strain SG200 during biotrophic growth and to identify potential Rss1 target genes, we conducted a microarray analysis at four days post infection. We identified only nine genes that were differentially regulated (adjusted p-value < 0.05), six of those were significantly repressed while three were significantly induced in SG200∆rss1. This set includes genes important for SA and tryptophan degradation indicating that Rss1 could play a role in the regulation of respective metabolic processes.

sample_organism:
Zea mays
Mycosarcoma maydis

platform_organism:
Zea mays
Mycosarcoma maydis

overall_design: Maize plants (Early Golden Bantam) were infected with SG200 or SG200Δrss1. Four days post infection, samples were collected in three independently conducted experiments by sampling 12 plants per experiment. After RNA extraction Agilent microarrays were performed.

first: Franziska

last: Rabe

first: Denise

last: Seitner

first: Lisa

last: Bauer

first: Fernando

last: Navarrete

first: Angelika

last: Czedik-Eysenberg

middle: A

last: Rabanal


title: Identification of specific xWNT5A and xWNT11specific target genes in dorsal marginal zone explants of Xenopus laevis

status: Public on Jun 21 2016

city: Karlsruhe

first: Dietmar

last: Gradl

address: Fritz Haber Weg 2

institute: Karlsruhe Institute of Technology

summary: The non-canonical signaling branches of the WNT signaling network realize different movements during Xenopus laevis gastrulation movements. The WNT5A branch is responsible for the constriction whereas the WNT11 branch triggers the elongation of the dorsal mesoderm. Therefore we used dorsal marginal zone explants which undergo convergent extension movements as readout system to identify differences in gene expression pattern between the WNT5A and WNT11 signaling branch via microarray analysis.We identified pbk as specific WNT5A target gene and rab11fip5 as specific WNT11 target gene. Therefore the non-canonical branches of the WNT signaling network do not only regulate cell behavior but also the expression of different target genes.

overall_design: Dorsal marginal zone explants of COMO, WNT5AMO and WNT11MO injected embryos were analysed in three biological replicates.

first: Veronika

last: Wallkamm


title: Corynebacterium glutamicum as a powerful host for the production of 5-aminovalerate from glucose fermentation

status: Public on Jun 17 2016

city: Bielefeld

first: João

middle: M. P.

last: Jorge

address: Universitätsstraße 25

institute: University of Bielefeld

summary: 5-aminovalerate (5AVA), L-lysine derived compound, represents a potential building block for the production of the bio-plastic nylon-5. Escherichia coli has been engineered for the production of 5AVA, but Corynebacterium glutamicum has never been engineered for the production of 5AVA, but, a lot of work was done in the last decades to optimize the production of the precursor L-lysine and more recently cadaverine. 5AVA added to the growth medium hardly affected growth rate of C. glutamicum, since, a half-inhibitory concentration of 1.1 M 5AVA was determined. While in E. coli, 5AVA production was engineered by using the DavBA pathway from Pseudomonas putida, here a pathway based on the route described in P. aeruginosa was established. C. glutamicum wild type was converted into a 5AVA producing strain by heterologous expression of L-lysine decarboxylase (LdcC), putrescine transaminase (PatA) and γ-aminobutyraldehyde dehydrogenase (PatD) genes from E. coli. 5AVA production was improved by using a strain previously engineered for high L-lysine production, by de-repressing phosphoenolpyruvate phosphotransferase system (PTS) and glycolysis and by avoiding formation of the by-product L-lactate. 5AVA accumulation by this strain was increased to 44.9 mM, representing a yield of 202 mmol mol-1 glucose, which is about three times higher than the highest yield achieved in E. coli for the production of 5AVA from glucose fermentation.

overall_design: Two conditions tested: 200 mM of NaCl vs 200 mM 5AVA supplemented in the culture medium. Control experiment done with the addition of 200 mM NaCl. 3 technical replicates

last: Pérez-García

first: Volker

last: Wendisch


title: Pol II and CTD Phosphorylation ChIP-chip of ATP-analog sensitive Kin28 budding yeast

status: Public on Jun 16 2016

city: Madison

first: Aseem

last: Ansari

state: WI

address: 440 Henry Mall

department: Biochemistry and The Genome Center

institute: University of Wisconsin-Madison

type: Genome binding/occupancy profiling by genome tiling array

summary: During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a “checkpoint” during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves long-standing controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo.

sample_organism:
Saccharomyces cerevisiae
Saccharomyces cerevisiae BY4741

overall_design: Rpb3-TAP, Ser2P, Ser5P, and Ser7P ChIP of irreversibly sensitive kin28is strain treated with inhibitors 1-NAPP-1 and CMK or solvent control (DMSO)

middle: Z

first: Juan

middle: B

last: Rodríguez-Molina


title: Comparison of Transient + articular cartilage (TC+AC) over pure transient cartilage (TC) from both E12 and E14 chicken tibia

status: Public on Jun 09 2016

city: Kanpur

first: Amitabha

last: Bandyopadhyay

state: Uttar Pradesh

address: IIT Kanpur

department: BSBE

institute: Indian Institute of Technology Kanpur

summary:
During the past two decades although many genes e.g.,Gdf5, Wnt9a, Noggin etc. have been identified and characterized in joint development, still a comprehensive understanding of molecular network operational in articular cartilage morphogenesis is far from being drawn. This might be due to incompleteness in the number of molecules identified.
We used microarray profiling to identify chicken articular cartilage specific genes. We decided to conduct microarray-based transcriptome comparison of HH38 (E12) and HH40 (E14). As it is difficult to clearly dissect out articular cartilage even at these late stages we decided to identify the transcripts that are enriched in the transient + articular cartilage (TC+AC) over pure transient cartilage (TC) thus using TC as control or reference samples

overall_design: Transient + articular cartilage (TC+AC) and pure transient cartilage (TC) were dissected out of both E12 and E14 chicken tibia. Many such tissues were pooled together and RNA was isolated using Trizol method. The RNA solution was cleaned-up using Qiagen micro KIT. 5 microgram of total RNA for each sample was used. Pure transient cartilage (TC) was used as reference samples

first: Pratik

middle: N

last: Singh