The impact of quantitative optimization of hybridization conditions on gene expression analysis.

BACKGROUND: With the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols. RESULTS: As shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of up to 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays.For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We can ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirmed an unbiased determination of generally optimal experimental conditions. CONCLUSIONS: Well calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive pro filing of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Multiple functionally divergent and conserved copies of alpha tubulin in bdelloid rotifers.

BACKGROUND: Bdelloid rotifers are microscopic animals that have apparently survived without sex for millions of years and are able to survive desiccation at all life stages through a process called anhydrobiosis. Both of these characteristics are believed to have played a role in shaping several unusual features of bdelloid genomes discovered in recent years. Studies into the impact of asexuality and anhydrobiosis on bdelloid genomes have focused on understanding gene copy number. Here we investigate copy number and sequence divergence in alpha tubulin. Alpha tubulin is conserved and normally present in low copy numbers in animals, but multiplication of alpha tubulin copies has occurred in animals adapted to extreme environments, such as cold-adapted Antarctic fish. Using cloning and sequencing we compared alpha tubulin copy variation in four species of bdelloid rotifers and four species of monogonont rotifers, which are facultatively sexual and cannot survive desiccation as adults. Results were verified using transcriptome data from one bdelloid species, Adineta ricciae. RESULTS: In common with the typical pattern for animals, monogonont rotifers contain either one or two copies of alpha tubulin, but bdelloid species contain between 11 and 13 different copies, distributed across five classes. Approximately half of the copies form a highly conserved group that vary by only 1.1% amino acid pairwise divergence with each other and with the monogonont copies. The other copies have divergent amino acid sequences that evolved significantly faster between classes than within them, relative to synonymous changes, and vary in predicted biochemical properties. Copies of each class were expressed under the laboratory conditions used to construct the transcriptome. CONCLUSIONS: Our findings are consistent with recent evidence that bdelloids are degenerate tetraploids and that functional divergence of ancestral copies of genes has occurred, but show how further duplication events in the ancestor of bdelloids led to proliferation in both conserved and functionally divergent copies of this gene.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Molecular Characterization and Patient Outcome of Melanoma Nodal Metastases and an Unknown Primary Site

Background Melanoma of unknown primary site (MUP) is not a completely understood entity with nodal metastases as the most common first clinical manifestation. The aim of this multicentric study was to assess frequency and type of oncogenic BRAF/NRAS/KIT mutations in MUP with clinically detected nodal metastases in relation to clinicopathologic features and outcome. Materials and Methods We analyzed series of 103 MUP patients (period: 1992-2010) after therapeutic lymphadenectomy (LND): 40 axillary, 47 groin, 16 cervical, none treated with BRAF inhibitors. We performed molecular characterization of BRAF/NRAS/KIT mutational status in nodal metastases using direct sequencing of respective coding sequences. Median follow-up time was 53 months. Results BRAF mutations were detected in 55 cases (53 %) (51 V600E, 93 %; 4 others, 7 %), and mutually exclusive NRAS mutations were found in 14 cases (14 %) (7 p.Q61R, 4 p.Q61K, 2 p.Q61H, 1 p.Q13R). We have not detected any mutations in KIT. The 5-year overall survival (OS) was 34 %; median was 24 months. We have not found significant correlation between mutational status (BRAF/NRAS) and OS; however, for BRAF or NRAS mutated melanomas we observed significantly shorter disease-free survival (DFS) when compared with wild-type melanoma patients (p = .04; 5-year DFS, 18 vs 19 vs 31 %, respectively). The most important factor influencing OS was number of metastatic lymph nodes >1 (p = .03). Conclusions Our large study on molecular characterization of MUP with nodal metastases showed that MUPs had molecular features similar to sporadic non-chronic-sun-damaged melanomas. BRAF/NRAS mutational status had negative impact on DFS in this group of patients. These observations might have potential implication for molecular-targeted therapy in MUPs

Strangelet search at RHIC

Two position sensitive Shower Maximum Detector (SMDs) for Zero-Degree Calorimeters (ZDCs) were installed by STAR before run 2004 at both upstream and downstream from the interaction point along the beam axis where particles with small rigidity are swept away by strong magnetic field. The ZDC-SMDs provides information about neutral energy deposition as a function of transverse position in ZDCs. We report the preliminary results of strangelet search from a triggered data-set sampling 100 million Au+Au collisions at top RHIC energy.Comment: Strange Quark Matter 2004 conference proceedin

Strangelet Search in AuAu Collisions at 200 GeV

We have searched for strangelets in a triggered sample of 61 million central (top 4%) Au+Au collisions at \sNN = 200 GeV near beam rapidities at the STAR detector. We have sensitivity to metastable strangelets with lifetimes of order $\geq 0.1 ns$, in contrast to limits over ten times longer in AGS studies and longer still at the SPS. Upper limits of a few 10^{-6} to 10^{-7} per central Au+Au collision are set for strangelets with mass ${}^{>}_{\sim}30$ GeV/c^{2}.Comment: As publishe

Longitudinal double-spin asymmetry for inclusive jet production in p+p collisions at sqrt(s)=200 GeV

We report a new STAR measurement of the longitudinal double-spin asymmetry A_LL for inclusive jet production at mid-rapidity in polarized p+p collisions at a center-of-mass energy of sqrt(s) = 200 GeV. The data, which cover jet transverse momenta 5 < p_T < 30 GeV/c, are substantially more precise than previous measurements. They provide significant new constraints on the gluon spin contribution to the nucleon spin through the comparison to predictions derived from one global fit of polarized deep-inelastic scattering measurements.Comment: 7 pages, 4 figures + 1 tabl

Multiplicity and Pseudorapidity Distributions of Charged Particles and Photons at Forward Pseudorapidity in Au + Au Collisions at sqrt{s_NN} = 62.4 GeV

We present the centrality dependent measurement of multiplicity and pseudorapidity distributions of charged particles and photons in Au + Au collisions at sqrt{s_NN} = 62.4 GeV. The charged particles and photons are measured in the pseudorapidity region 2.9 < eta < 3.9 and 2.3 < eta < 3.7, respectively. We have studied the scaling of particle production with the number of participating nucleons and the number of binary collisions. The photon and charged particle production in the measured pseudorapidity range has been shown to be consistent with energy independent limiting fragmentation behavior. The photons are observed to follow a centrality independent limiting fragmentation behavior while for the charged particles it is centrality dependent. We have carried out a comparative study of the pseudorapidity distributions of positively charged hadrons, negatively charged hadrons, photons, pions, net protons in nucleus--nucleus collisions and pseudorapidity distributions from p+p collisions. From these comparisons we conclude that baryons in the inclusive charged particle distribution are responsible for the observed centrality dependence of limiting fragmentation. The mesons are found to follow an energy independent behavior of limiting fragmentation while the behavior of baryons seems to be energy dependent.Comment: 17 pages and 20 figure

Genetics of human neural tube defects

Neural tube defects (NTDs) are common, severe congenital malformations whose causation involves multiple genes and environmental factors. Although more than 200 genes are known to cause NTDs in mice, there has been rather limited progress in delineating the molecular basis underlying most human NTDs. Numerous genetic studies have been carried out to investigate candidate genes in cohorts of patients, with particular reference to those that participate in folate one-carbon metabolism. Although the homocysteine remethylation gene MTHFR has emerged as a risk factor in some human populations, few other consistent findings have resulted from this approach. Similarly, attention focused on the human homologues of mouse NTD genes has contributed only limited positive findings to date, although an emerging association between genes of the non-canonical Wnt (planar cell polarity) pathway and NTDs provides candidates for future studies. Priorities for the next phase of this research include: (i) larger studies that are sufficiently powered to detect significant associations with relatively minor risk factors; (ii) analysis of multiple candidate genes in groups of well-genotyped individuals to detect possible gene–gene interactions; (iii) use of high throughput genomic technology to evaluate the role of copy number variants and to detect ‘private’ and regulatory mutations, neither of which have been studied to date; (iv) detailed analysis of patient samples stratified by phenotype to enable, for example, hypothesis-driven testing of candidates genes in groups of NTDs with specific defects of folate metabolism, or in groups of fetuses with well-defined phenotypes such as craniorachischisis

Identified baryon and meson distributions at large transverse momenta from Au+Au collisions at sNN=200\sqrt{s_{_{NN}}} = 200 GeV

Transverse momentum spectra of $\pi^{\pm}$, $p$ and $\bar{p}$ up to 12 GeV/c at mid-rapidity in centrality selected Au+Au collisions at $\sqrt{s_{_{NN}}} = 200$ GeV are presented. In central Au+Au collisions, both $\pi^{\pm}$ and $p(\bar{p})$ show significant suppression with respect to binary scaling at $p_T >$ 4 GeV/c. Protons and anti-protons are less suppressed than $\pi^{\pm}$, in the range 1.5 $< p_{T} <$6 GeV/c. The $\pi^-/\pi^+$ and $\bar{p}/p$ ratios show at most a weak $p_T$ dependence and no significant centrality dependence. The $p/\pi$ ratios in central Au+Au collisions approach the values in p+p and d+Au collisions at $p_T >$ 5 GeV/c. The results at high $p_T$ indicate that the partonic sources of $\pi^{\pm}$, $p$ and $\bar{p}$ have similar energy loss when traversing the nuclear medium.Comment: 6 pages, 4 figure

Forward Neutral Pion Transverse Single Spin Asymmetries in p+p Collisions at \sqrt{s}=200 GeV

We report precision measurements of the Feynman-x dependence, and first measurements of the transverse momentum dependence, of transverse single spin asymmetries for the production of \pi^0 mesons from polarized proton collisions at \sqrt{s}=200 GeV. The x_F dependence of the results is in fair agreement with perturbative QCD model calculations that identify orbital motion of quarks and gluons within the proton as the origin of the spin effects. Results for the p_T dependence at fixed x_F are not consistent with pQCD-based calculations.Comment: 6 pages, 4 figure