ABSTRACT: Analysis of noncoding RNA processing phenotypes in mutant yeast strains, with isogenic wildtype strains as controls two-color microarrays, total RNA labeled directly with Alexa Ulysis 546 and 648 dyes, dye-swap, eight replicates of each oligo per slide, Lowess smoother, Hedge et al. 2002 protocols, see Peng, Robinson et al., 2003.
Project description:Analysis of noncoding RNA processing phenotypes in mutant yeast strains, with isogenic wildtype strains as controls two-color microarrays, total RNA labeled directly with Alexa Ulysis 546 and 648 dyes, dye-swap, eight replicates of each oligo per slide, Lowess smoother, Hedge et al. 2002 protocols, see Peng, Robinson et al., 2003.
Project description:This work evaluates the performance of the group contribution volume translated Peng-Robinson model when predicting the vapor-liquid equilibrium and single phase densities of 28 commercial refrigerant mixtures with low global warming potential and zero ozone depletion potential. Cubic equations of state, and particularly the Peng-Robinson equation of state, are widely used in the refrigeration industry due to their easy applicability for new substances, and their low computational time, although generally lower prediction accuracies must be expected compared to multiparameter equations of state. The group contribution volume translated Peng-Robinson equation of state combines the Peng-Robinson equation of state with a new attraction term, improved mixing rules using a group contribution approach, and volume translation. The results are compared with the estimates obtained using the non translated Peng-Robinson equation of state, and a multiparameter equation of state.
Project description:Congestive heart failure was done by artificial myocardial infarction. After extracting total RNA from control and infarcted ventricles with Triazol, messenger RNA was isolated with Qiagen mRNA isolation kit. About 1.5ug poly A+ RNA was taken for probe preparation. Probe was labeled with Alexa Fluor 546 and 657. Labeled probes were hybridized with Qiagen rat unigene oligo library. After 2 low and 1 high stringency washes Prolong Antifade was added to the slides to prevent bleaching effect. The data ratio was normalized using a location and intensity dependent Lowess formula.
Project description:Solubility of phenolics of mango ginger extract in supercritical carbon dioxide was studied at 40-60 °C and 100-350 bar. Critical temperature, critical pressure and critical volume of caffeic acid, the principal component of the extract were calculated using group contribution methods and compared with the values obtained by CHEMDRAW®. Vapor pressure of caffeic acid was predicted by Reidel method. Solubility prediction in supercritical carbon dioxide was studied using two different equation of states (EOS) models and eight empirical models. Peng-Robinson EOS predicted the solubility very well with average deviation of 0.68 % from the experimental solubility. Empirical equations based on the simple error minimization using non-linear regression method which do not require complex physiochemical properties was also found suitable to predict the solubility at different extraction conditions. Jouyban et al. model showed very less deviation (2.25 %) for predicted solubility values from the experiment.
Project description:The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.
Project description:This article presents the data on photovoltaic (PV) system used different perturb and observe (P&O) methods under fast multi-changing solar irradiances. The mathematical modeling of the PV system and tangent error P&O method was discussed in our previous study entitled "A novel tangent error maximum power point tracking algorithm for photovoltaic system under fast multi-changing solar irradiances" by Peng et al. (2018) . The data provided in this paper can be used directly without having to spend weeks to simulate the output performance. In addition, it is easy to apply the results for comparison with other algorithms (Kollimalla et al., 2014; Belkaid et al., 2016; Chenchen et al., 2015; Jubaer and Zainal, 2015) [2,3,4,5], and develop a new method for practical application.
Project description:adt12-01_uprt - plant rabt nÂ°1 - Impact of RNA labeling by Plant RABT on plant transcriptome - Determine the incidence of RNA marking by the RABT method Plantation on the plants transcriptome. In several studies Clearly et al. have described a method referred as "4TU tagging" which can be use to study mRNA synthesis and decay either in a mixed population of cells or in a specific cell type (Cleary, Meiering et al. 2005, Zeiner, Cleary et al. 2008, Miller, Robinson et al. 2009, Rabani et al., 2011). Through the specific cell type expression in drosophila and mammalian cells of theToxoplasma gondii uracil PhosphoribosylTransferase activity and the metabolization of the uracil analog 4-thiouracil (4 TU), mRNA were selectively tagged, purified and used in microarray based analysis. 6 dye-swap - treated vs untreated comparison
Project description:Background Array-based comparative genome hybridization (aCGH) is commonly used to determine the genomic content of bacterial strains. In aCGH data, systematic errors are comparable to those occurring in transcriptome data. However, especially for microbes, an additional source of variation exists: differences in hybridization due to gene sequence divergence between the strains hybridized. Current normalization methods do not take this source of variation into consideration. Results We present Supervised Lowess, or S-Lowess, an application of the subset Lowess normalization method that does take difference in genomic content into account. The performance of S-Lowess was assessed on aCGH experiments in which differentially labeled genomic DNA fragments of Lactococcus lactis IL1403 and L. lactis MG1363 strains were hybridized to IL1403 microarrays. Since both genome sequences are known (they have only on average 85 % sequence identity), the success rate in detecting deletions in the MG1363 genome of different aCGH normalization methods can be compared. S-Lowess detects 97% of the deletions, whereas other aCGH normalization methods detect up to only 60% of the deletions. Conclusions S-Lowess removes systematic errors from dual-dye aCGH data in two steps: (i) determination of likely homologous genes (LHG); and (ii) estimation of correction factors for systematic errors from spots of LHG and subset Lowess normalization of the remaining spots using these correction factors. It is implemented in a user-friendly web-tool accessible from http://bioinformatics.biol.rug.nl/websoftware/s-lowess. We demonstrate that it outperforms existing normalization methods and maximizes the number of detectable genomic deletions or duplications from microbial aCGH data. Keywords: comparative genome hybridisation In this study, 4 aCGH comparisons (slides) between L. lactis MG1363 and L. lactis IL1403 were performed (including dye swap with biological replicates; see also supplementary materials). The resulting aCGH slide signals were normalized using the different 'likely homologous gene' (LHG) sets (see above) yielding ratios of signals of labelled DNA of MG1363 over those of IL1403. A maximum of 8 ratios per amplicon (gene) were obtained for the 4 hybridized slides (each with 2 replicate spots per amplicon). Only genes with at least 5 measurements were used in this study. The normalization methods evaluated in this study are: a) no normalization, b) total signal normalization, c) grid-based Lowess (implemented in PreP; f = 0.7) [GarcM-CM--a de la Nava, J et al. 2003. Bioinformatics 19:2328-2329], and d) S-Lowess using different subsets of conserved lactococcal genes (for details see above). Results with the MANOR R package (spatial normalization; standard parameters) [Neuvial P, et al. 2006. BMC Bioinformatics 7:264; Liva S, et al. 2006. Nucleic Acids Res 34:W477-W481] are shown in our supplementary materials.
Project description:Three weeks after infarction, age matched rats with large infarction by ECG criteria were randomly assigned to one of the three groups: 1) Treatment with Captopril for 21 days and a daily subcutaneous dose of DITPA for the last 10 days of treatment, 2) Treatment for 10 days with DITPA alone, 3) Control heart failure and 4) Sham-operated. Keywords: Drug Effects on Heart Failure Total RNA was prepared from left ventricle using TRIzol and later Poly A+ RNA was isolated with Oligotex beads. About 0.5ug Poly A+ RNA was reverse transcribed and labeled with Alexa Fluor 546 and 647. Labeled probed were hybridized with Operon's rat Unigene oligo library printed on glass slides. After 2 lows and 1 high stringency washes, Prolong Antifade was added to the slides to prevent any bleaching effect. The experiments were repeated 2-4 times by dye-swaping. Slides were scanned in Array Worx scanner with dual wave length. The data ratio was normalized using a location and intensity dependent Lowess formula.
Project description:Legionella pneumophila is a gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to test the hypothesis that sRNAs play a similar role in L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which six were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed when the bacteria enter post exponential phase and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of Ï70-containing RNAP. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA encoded by the ssrS gene positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA as well as many genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media. A microarray was designed for the detection of 101 predicted sRNAs by using OligiWiz version 2.1.3 (Nielsen et al., 2003, Wernersson & Nielsen, 2005). The prokaryotic setting was used to design 50-mers probes located, when possible, at the 3â end of the predicted genes. Probes for 10 negative controls, representing 10 genes of the L. pneumophila Paris plasmid, were also designed. Probes (Illumina) were dissolved in 50% DMSO to a final concentration of 30 uM and printed in triplicate on UltraGAPS coated glass slides (Corning) using a PerkinElmer SpotArray microarray printer. Fifteen micrograms of total RNA was labelled during cDNA synthesis using Superscript II reverse transcriptase (Invitrogen) and amino-allyl dUTP (Sigma). Bacterial genomic DNA was used as the reference channel on each slide to allow comparison of each time point and different samples (Talaat et al., 2002). Five micrograms of genomic DNA (gDNA) was labelled with amino-allyl dUTP by using Klenow fragment and random primers (Invitrogen) at 37 Â°C for 18 h. DNA was subsequently coupled to the succinimidyl ester fluorescent dye AlexaFluor 546 (for cDNA) or Alexa Fluor 647 (for gDNA) (Invitrogen) following manufacturerâs protocols. Hybridization and data acquisition were performed as previously described (Hovel-Miner et al., 2009).Very low density array, like the sRNA microarray used here, cannot be normalised with common procedure like total intensity or lowess. Local background was removed from spot signal intensity and the noise signal was estimated by recording the average signal intensity of 10 negative controls printed on the chip. Normalisation of signal intensity was carried out by calculating the fold increase over the noise signal value. Correlation of replicates using this normalisation procedure was â¥ 0.95.