maSigPro: a method to identify significantly differential expression profiles in time-course microarray experiments

MOTIVATION: Multi-series time-course microarray experiments are useful approaches for exploring biological processes. In this type of experiments, the researcher is frequently interested in studying gene expression changes along time and in evaluating trend differences between the various experimental groups. The large amount of data, multiplicity of experimental conditions and the dynamic nature of the experiments poses great challenges to data analysis. RESULTS: In this work, we propose a statistical procedure to identify genes that show different gene expression profiles across analytical groups in time-course experiments. The method is a two-regression step approach where the experimental groups are identified by dummy variables. The procedure first adjusts a global regression model with all the defined variables to identify differentially expressed genes, and in second a variable selection strategy is applied to study differences between groups and to find statistically significant different profiles. The methodology is illustrated on both a real and a simulated microarray dataset.     

Gene Vector Analysis (Geneva): A unified method to detect differentially-regulated gene sets and similar microarray experiments

Background  

Microarray experiments measure changes in the expression of thousands of genes. The resulting lists of genes with changes in expression are then searched for biologically related sets using several divergent methods such as the Fisher Exact Test (as used in multiple GO enrichment tools), Parametric Analysis of Gene Expression (PAGE), Gene Set Enrichment Analysis (GSEA), and the connectivity map.     

Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: a novel probe for core fucose

The alpha1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the alpha1,6-fucosylated chain among alpha1,2-, alpha1,3-, alpha1,4-, and alpha1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the alpha1,2-fucosylated PA-sugar chain (H antigen) relative to the alpha1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with alpha1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the alpha1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from alpha1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA.     

Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments

One of the main objectives in the analysis of microarray experiments is the identification of genes that are differentially expressed under two experimental conditions. This task is complicated by the noisiness of the data and the large number of genes that are examined simultaneously. Here, we present a novel technique for identifying differentially expressed genes that does not originate from a sophisticated statistical model but rather from an analysis of biological reasoning. The new technique, which is based on calculating rank products (RP) from replicate experiments, is fast and simple. At the same time, it provides a straightforward and statistically stringent way to determine the significance level for each gene and allows for the flexible control of the false-detection rate and familywise error rate in the multiple testing situation of a microarray experiment. We use the RP technique on three biological data sets and show that in each case it performs more reliably and consistently than the non-parametric t-test variant implemented in Tusher et al.'s significance analysis of microarrays (SAM). We also show that the RP results are reliable in highly noisy data. An analysis of the physiological function of the identified genes indicates that the RP approach is powerful for identifying biologically relevant expression changes. In addition, using RP can lead to a sharp reduction in the number of replicate experiments needed to obtain reproducible results.     

Iterative Group Analysis (iGA): A simple tool to enhance sensitivity and facilitate interpretation of microarray experiments

Background  

The biological interpretation of even a simple microarray experiment can be a challenging and highly complex task. Here we present a new method (Iterative Group Analysis) to facilitate, improve, and accelerate this process.     

A novel fluorescent probe based on naphthalimide for imaging nitroreductase (NTR) in bacteria and cells

A nitroreductase (NTR) responsive fluorescent probe, Na-NO₂, comprising p-nitrobenzyl as the unique recognition group and 1,8-naphthalimide as fluorophore, was synthesized. Na-NO₂ showed remarkable fluorescence “turn-on” signal in the presence of NTR under DMSO/H₂O (1:19, v/v) buffered with PBS (pH = 7) solution in the presence of NADH (300 µM). Furthermore, the probe has a low detection limit down to 3.4 ng/mL and it is very sensitive towards the NTR in Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), normal and tumor cells such as HL-7702, HepG-2 and MCF-7.     

A novel molecular fingerprint probe based on the endogenous avian retroviral element (EAV) of chickens

We have developed a novel molecular probe that is useful for DNA fingerprint analysis in chickens. The probe is based on the middle-repetitive, chicken endogenous retroviral (EAV) element. It consists of 1503 bp of the 3′ portion of the EAV element, extending from the downstream end of the envelope gene to the beginning of the downstream long terminal repeat (LTR). Unlike other probes that are currently in use for fingerprint analysis with chicken DNA, the EAV-based probe works well at normal levels of stringency, and with standard hybridization buffers. Digestion of chicken genomic DNA with a variety of restriction enzymes routinely yields up to 30 resolvable bands per bird in the 500 bp to 20kbp range. In order to test the efficacy of the EAV-based fingerprint probe, we have used it to estimate the degree of inbreeding in the inbred WG strain of White Leghorns. We find that the estimates derived with the EAV probe are very similar to those reported previously for the WG strain. These results suggest that molecular probes based on endogenous retroviruses and other middle-repetitive DNA elements should be useful for fingerprint analysis in chickens, and in vertebrates in general.     

The use of a novel quantitation strategy based on Reductive Isotopic Di-Ethylation (RIDE) to evaluate the effect of glufosinate on the unicellular algae Ostreococcus tauri

We report a novel stable-isotope labeling strategy for quantitative proteomics analysis. The method consists of labeling N-termini and lysine ε-amino groups through reductive amination using acetaldehyde. This allows isotope labeling using pairs of either 2H/1H or 13C/12C without mass spectrum overlap. Our labeling procedure, which is significantly different than that developed for dimethylation, can be completed with little trace of partial ethylation; non-labeled peptides represent less than 0.05% of all peptides. Co-elution of both isotopic 13C/12C peptide pairs was observed in all cases, simplifying data analysis, which can be performed using standard commercial software such as Mascot Distiller. A 13C/12C labeled mix in a 1:1 ratio from a complex extract digest of the unicellular algae Ostreococcus tauri, showed a relative standard deviation of less than 14%. This quantitative method was used to characterize O. tauri in the presence of glufosinate, an herbicide which inhibits glutamine synthetase. Blocking glutamine synthetase significantly reduced the expression of several enzymes and transporters involved in nitrogen assimilation and the expression of a number of proteins involved in various stresses including oxidative damage response were up-regulated.     

A novel method using an acedan-based Zn(DPA) probe to monitor ATP localization in an in vivo system

In in vitro and in vivo systems, understanding localization and the functional role of ATP is essential, but effective methods to monitor ATP in cells and tissues are limited. Although quinacrine dihydrochloride is a well-known fluorescent dye used to detect ATP, it is limited in its use because it shows non-specific nuclear staining both in vitro and in vivo. A commercial luciferin-luciferase bioluminescence assay has also been used to detect ATP, but it can not be easily used in vivo. Thus, to effectively monitor ATP in vivo, we employed a novel two-photon ATP fluorescent probe, acedan-based Zn(DPA). Using the acedan-based Zn(DPA) probe, we show that this probe produces high quality images of ATP in lung, spleen, liver and spinal cord tissues.     

Field experiments on a new method for determining age in tsetse flies (Diptera: Glossinidae)

ABSTRACT.

• 1 Data are presented which suggests that the accurate determination of the age of tsetse flies (Glossina morsitans morsitans Westwood and G. pallidipes Austen) in the field can be achieved by measuring the fluorescence content of the head capsule.
• 2 The way in which this can be achieved and further work which would improve the accuracy of the technique are discussed.

     

Radioiodinated 1-(2-fluoro-4-iodo-2,4-dideoxy-beta-L-xylopyranosyl)-2-nitroimidazole: a novel probe for the noninvasive assessment of tumor hypoxia.

1-(2-Fluoro-4-iodo-2,4-dideoxy-beta-L-xylopyranosyl)-2-nitroimidazole (FIAZP) has been synthesized and labeled with radioiodine (125I). Radioiodinated FIAZP is one of a series of sugar-coupled 2-nitroimidazoles developed in our laboratory as probes for noninvasive scintigraphic assessment of tumor hypoxia. An in vivo biodistribution study with [125I]FIAZP in the murine BALB/c EMT-6 tumor model showed a tumor-to-blood ratio of 6, 24 h after injection, with 0.5% of the injected dose present per gram of tumor. These values are several times higher than the respective ratios and distribution values in any of the organs, with the exception of liver. Radioactivity from tissues other than tumor and liver declined with time, following the decline of blood radioactivity. Rapid whole-body elimination of radioactivity was observed (> 96% in 24 h). The thyroid showed little uptake of radioactivity, indicating minimal in vivo deiodination. 1-(2-Fluoro-4-iodo-2,4-dideoxy-beta-L-xylopranosyl)-2-nitroimidazo le appears to undergo hypoxia-dependent binding in tumor tissue at levels comparable to those of other sugar-coupled 2-nitroimidazoles. The potential for imaging with this compound is discussed.     

Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution

Sequence saturation mutagenesis (SeSaM) is a conceptually novel and practically simple method that truly randomizes a target sequence at every single nucleotide position. A SeSaM experiment can be accomplished within 2–3 days and comprises four steps: generating a pool of DNA fragments with random length, ‘tailing’ the DNA fragments with universal base using terminal transferase at 3′-termini, elongating DNA fragments in a PCR to the full-length genes using a single-stranded template and replacing the universal bases by standard nucleotides. Random mutations are created at universal sites due to the promiscuous base-pairing property of universal bases. Using enhanced green fluorescence protein as the model system and deoxyinosine as the universal base, we proved by sequencing 100 genes the concept of the SeSaM method and achieved a random distribution of mutations with the mutational bias expected for deoxyinosine.     

Protonation changes upon ligand binding to trypsin and thrombin: structural interpretation based on pK(a) calculations and ITC experiments

The protonation states of a protein and a ligand can be altered upon complex formation. Such changes can be detected experimentally by isothermal titration calorimetry (ITC). For a series of ligands binding to the serine proteases trypsin and thrombin, we previously performed an extensive ITC and crystallographic study and were able to identify protonation changes for four complexes. However, since ITC measures only the overall proton exchange, it does not provide structural insights into the functional groups involved in the proton transfer. Using Poisson-Boltzmann calculations based on our recently developed PEOE_PB charges, we compute pK(a) values for all complexes of our former study in order to reveal the residues with altered protonation states. The results indicate that His57, a member of the catalytic triad, is responsible for the most relevant pK(a) shifts leading to the experimentally detected protonation changes. This finding is in contrast to our previous assumption that the observed protonation changes occur at the carboxylic group of the ligands. The newly detected proton acceptor is used for a revised factorization of the ITC data, which is necessary whenever the protonation inventory changes upon complexation. The pK(a) values of complexes showing no protonation change in the ITC experiment are reliably predicted in most cases, whereas predictions of strongly coupled systems remain problematic.     

Host specificity assessment and potential impact of Megamelus scutellaris (Hemiptera: Delphacidae) on waterhyacinth Eichhornia crassipes (Pontederiales: Pontederiaceae)

The delphacid Megamelus scutellaris Berg was evaluated for host specificity and potential impact as part of a biological control program targeting Eichhornia. crassipes. Survival and development of adults and nymphs were used as metrics with no-choice, two-choice, nymph transfer, and sustainability tests conducted under quarantine conditions. A total of 69 plant species were tested including 12 from the Pontederiaceae (including E. crassipes). Additionally, 27 native and 5 exotic associated wetland species and 11 economic species were tested. Megamelus scutellaris exhibited a high level of oviposition and developmental fidelity to E. crassipes by failing to sustain populations on any non-target test plant past the F₁ generation. Nymph transfer tests which simulated potential spill-over events found that survival was virtually non-existent on associated wetland plants, regardless of taxonomic relatedness, including on Pontederia cordata, an important and widespread native species. Eichhornia crassipes plants exposed to two consecutive generations of feeding produced 66.9% less biomass and 73.4% fewer leaves than those in the controls. We conclude that Megamelus scutellaris is safe to release on E. crassipes in the United States.     

A novel intravital method to evaluate cerebral vasospasm in rat models of subarachnoid hemorrhage: a study with synchrotron radiation angiography

Precise in vivo evaluation of cerebral vasospasm caused by subarachnoid hemorrhage has remained a critical but unsolved issue in experimental small animal models. In this study, we used synchrotron radiation angiography to study the vasospasm of anterior circulation arteries in two subarachnoid hemorrhage models in rats. Synchrotron radiation angiography, laser Doppler flowmetry-cerebral blood flow measurement, [(125)I]N-isopropyl-p-iodoamphetamine cerebral blood flow measurement and terminal examinations were applied to evaluate the changes of anterior circulation arteries in two subarachnoid hemorrhage models made by blood injection into cisterna magna and prechiasmatic cistern. Using synchrotron radiation angiography technique, we detected cerebral vasospasm in subarachnoid hemorrhage rats compared to the controls (p<0.05). We also identified two interesting findings: 1) both middle cerebral artery and anterior cerebral artery shrunk the most at day 3 after subarachnoid hemorrhage; 2) the diameter of anterior cerebral artery in the prechiasmatic cistern injection group was smaller than that in the cisterna magna injection group (p<0.05), but not for middle cerebral artery. We concluded that synchrotron radiation angiography provided a novel technique, which could directly evaluate cerebral vasospasm in small animal experimental subarachnoid hemorrhage models. The courses of vasospasm in these two injection models are similar; however, the model produced by prechiasmatic cistern injection is more suitable for study of anterior circulation vasospasm.