GeneTrailExpress: a web-based pipeline for the statistical evaluation of microarray experiments

Background  

High-throughput methods that allow for measuring the expression of thousands of genes or proteins simultaneously have opened new avenues for studying biochemical processes. While the noisiness of the data necessitates an extensive pre-processing of the raw data, the high dimensionality requires effective statistical analysis methods that facilitate the identification of crucial biological features and relations. For these reasons, the evaluation and interpretation of expression data is a complex, labor-intensive multi-step process. While a variety of tools for normalizing, analysing, or visualizing expression profiles has been developed in the last years, most of these tools offer only functionality for accomplishing certain steps of the evaluation pipeline.     

Protein evolution of ANTP and PRD homeobox genes

Background  

Although homeobox genes have been the subject of many studies, little is known about the main amino acid changes that occurred early in the evolution of genes belonging to different classes.     

Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping

The majority of verified plant disease resistance genes isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. We took advantage of the sequence conservation in the NBS motif to clone, by PCR, gene fragments from barley representing putative disease resistance genes of this class. Over 30 different resistance gene analogs (RGAs) were isolated from the barley cultivar Regatta. These were grouped into 13 classes based on DNA sequence similarity. Actively transcribed genes were identified from all classes but one, and cDNA clones were isolated to derive the complete NBS-LRR protein sequences. Some of the NBS-LRR genes exhibited variation with respect to whether and where particular introns were spliced, as well as frequent premature polyadenylation. DNA sequences related to the majority of the barley RGAs were identified in the recently expanded public rice genomic sequence database, indicating that the rice sequence can be used to extract a large proportion of the RGAs from barley and other cereals. Using a combination of RFLP and PCR marker techniques, representatives of all barley RGA gene classes were mapped in the barley genome, to all chromosomes except 4H. A number of the RGA loci map in the vicinity of known disease resistance loci, and the association between RGA S-120 and the nematode resistance locus Ha2 on chromosome 2H was further tested by co-segregation analysis. Most of the RGA sequences reported here have not been described previously, and represent a useful resource as candidates or molecular markers for disease resistance genes in barley and other cereals.     

Biochemical characterization of prostasin, a channel activating protease

Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1', P2', and P3' displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4' had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC alpha- and beta-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1' explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate.     

HPLC based method for the measurement of the reduction of aflatoxin B<Subscript>1</Subscript> by bacterial cultures isolated from different African foods

The consumption of fermented foods contaminated with aflatoxin B₁ is linked to aflatoxicosis. Aflatoxicosis is a serious problem in developing countries with environmental conditions appropriate for the biosynthesis of AFB₁ byAspergillus flavus andAspergillus parasiticus. In Africa, especially in Ghana and Nigeria, there is a very high risk of liver cancer which is caused by the consumption of AFB₁-intoxicated, traditionally fermented maize and sorghum products. It is suggested that one way to diminish this health risk might be the reduction of the AFB₁ concentration in foods by bacteria. Especially bacteria used for food fermentation processes are of great importance, with a special emphasis on lactic acid bacteria which are involved in traditionally fermented African foods based on maize and sorghum.Most publications dealing with aflatoxin degradation by microorganisms describe a phosphate buffer test system for the performance of degradation experiments. In contrast to that, a test system based on physiological active bacterial and yeast cells has been developed, to assess food fermentation organisms for their ability to reduce the AFB₁ concentration in vitro. The aflatoxin B₁ concentration in test samples was quatitatively determined by HPLC.The assessment of lactic acid bacteria originating from different German and other European culture collections only showed a very slight reduction of the AFB₁ concentration from 3% to 12%. Screening experiments in which other bacterial genera and lactic acid bacteria, isolated from different African foods have been assessed, in most cases showed the same results. However, some bacterial strains, e.g. strains of the genusBacillus derived from European culture collections and strains of the genusLactobacillus isolated from African foods, caused a release of AFB₁ which was chemically bound before to components of the test medium and which therefore could not be extracted with chloroform.A process quite similar to that may happen during food fermentations. Different experiments showed that e.g. cellulose can bind AFB₁ very effectively. Cellulose and different other food components are well known to absorb AFB₁. During fermentation the cellulose and other AFB₁-absorbing components may be degraded and the AFB₁ will be released again.The only bacterial strain known as yet which is able to reduce the AFB₁ concentration in vitro and in different food comodities isNocardia corynebacteroides (formerFlavobacterium aurantiacum). Nevertheless the mechanism of this AFB₁ reduction is actually not well understood, it still has to be investigated. In the meantime several other bacterial strains, presumably from the taxonomic group of theActinomycetes could be proved to be effective reducers of the AFB₁ concentration in our in vitro test system. Because as yet no food relevant microorganism could be found, which is able to degrade AFB₁, these new strains in general offer the possibility for a genetic modification of food relevant microorganisms. This seems to be the way to come to starter cultures which are able to degrade AFB₁ during food fermentations.     

Effects of sex and dietary phosphorus level on the apparent metabolizable energy and nutrient digestibility in broiler chickens

The influence of sex of broilers and dietary phosphorus (P) level on energy utilization and apparent ileal digestibility of N, P and phytate-P were investigated. The AME_N was determined using 3- and 6-week old broilers, while the apparent ileal digestibility were determined only with 6-week old birds. Sex of broilers had no effect on the AME_N values determined during week 3. During week 6, the AME_N values for male broilers were higher (P?0.01) than those for the females. An interaction (P?0.05) between sex and dietary P level was also observed. AME_N values determined with male broilers were lower in the adequate-P diet compared to those in the low-P diet, whereas no effect of P level was observed in females. Female broilers tended (P?0.10) to have a higher ileal N digestibility than the males, but a significant (P?0.01) sex ×?P level interaction was observed. Males receiving the adequate-P diet had a lower N digestibility than those receiving the low-P diet, whereas the opposite was true in the females. Ileal phytate P degradation in males was higher than in females (0.282 vs. 0.234), but the differences were not significant. Increasing dietary P level increased ileal P digestibility in both the males and females, but the improvements were greater in the females, as indicated by a significant sex × P level interaction.     

Duodenal mucosal risk markers in patients with familial adenomatous polyposis: effects of celecoxib/ursodeoxycholic acid co-treatment and comparison with patient controls

Background

Familial adenomatous polyposis (FAP) is a disease characterized by the development of hundreds to thousands of adenomatous polyps in the colorectum early in life. Virtually all patients with FAP will develop colorectal cancer before the age of 40 to 50 years, unless prophylactic colectomy is performed, which significantly improves their prognosis. The mortality pattern has changed and duodenal cancer now is one of the main cancer-related causes of death in these patients. Practically all patients with FAP develop premalignant duodenal adenomas, which may develop to duodenal cancer in approximately 3-7% of patients. Duodenal cancer in patients with FAP has a poor prognosis. The clinical challenge is to identify patients at high-risk for duodenal carcinoma. Chemoprevention would be desirable to avoid duodenectomy. The main goal of this study is to identify risk markers in normal duodenal mucosa of patients with FAP, that could help identify patients at increased risk for malignant transformation.

Methods

Messenger RNA (mRNA) levels of glutathione S-transferase A1 (GSTA1), glutathione S-transferase P1 (GSTP1), KIAA1199, E-cadherin, peroxisome proliferative activated receptor δ (PPARδ), caspase-3, cyclin D1, β-catenin, and cyclooxygenase-2 (COX-2) were measured in duodenal mucosa, using the QuantiGene 2.0 Plex assay. Levels in normal appearing mucosa of patients with FAP (n?=?37) were compared with levels in non-FAP patient controls (n?=?16). In addition, levels before and after treatment with either celecoxib & ursodeoxycholic acid (UDCA, n?=?14) or celecoxib & placebo (n?=?13) were evaluated in patients with FAP.

Results

mRNA levels of glutathione S-transferase A1 (28.16% vs. 38.24%, p?=?0.008) and caspase-3 (3.30% vs. 5.31%, p?=?0.001) were significantly lower in patients with FAP vs. non-FAP patient controls, respectively. COX-2 mRNA levels in normal duodenal mucosa of patients with FAP were found to be unexpectedly low. None of the potential risk markers was influenced by celecoxib or celecoxib & UDCA.

Conclusions

Protection against toxins and carcinogens (GSTA1) and apoptosis (caspase-3) is low in patients with FAP, which could contribute to increased susceptibility for malignant transformation of duodenal mucosa.

Trial registration

http://ClinicalTrials.gov number NCT00808743

     

Mannose-Binding Lectin 2 Gene and Risk of Adult Glioma

Background and Aims

The immune system is likely to play a key role in the etiology of gliomas. Genetic polymorphisms in the mannose-binding lectin gene, a key activator in the lectin complement pathway, have been associated with risk of several cancers.

Methods

To examine the role of the lectin complement pathway, we combined data from prospectively collected cohorts with available DNA specimens. Using a nested case-control design, we genotyped 85 single nucleotide polymorphisms (SNPs) in 9 genes in the lectin complement pathway and 3 additional SNPs in MBL2 were tested post hoc). Initial SNPs were selected using tagging SNPs for haplotypes; the second group of SNPs for MBL2 was selected based on functional SNPs related to phenotype. Associations were examined using logistic regression analysis. All statistical tests were two-sided. Nominal p-values are presented and are not corrected for multiple comparisons.

Results

A total of 143 glioma cases and 419 controls were available for this analysis. Statistically significant associations were observed for two SNPs in the mannose-binding lectin 2 (ML2) gene and risk of glioma (rs1982266 and rs1800450, test for trend p = 0.003 and p = 0.04, respectively, using the additive model). One of these SNPs, rs1800450, was associated with a 58% increase in glioma risk among those carrying one or two mutated alleles (odds ratio = 1.58, 95% confidence interval = 0.99–2.54), compared to those homozygous for the wild type allele.

Conclusions

Overall, our findings suggest that MBL may play a role in the etiology of glioma. Future studies are needed to confirm these findings which may be due to chance, and if reproduced, to determine mechanisms that link glioma pathogenesis with the MBL complement pathway.     

Heteromeric complex formation between CYP2E1 and CYP1A2: evidence for the involvement of electrostatic interactions

Mixed reconstituted systems containing CYP2B4, CYP1A2, and NADPH-cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin O-dealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2-CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin O-deethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1-CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450-P450 complexes. The synergistic stimulation observed with the reductase-CYP1A2-CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2-CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2-CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.     

Whole miRNome-wide Differential Co-expression of MicroRNAs

Co-regulation of genes has been extensively analyzed, however, rather limited knowledge is available on co-regulations within the miRNome. We investigated differential co-expression of microRNAs (miRNAs) based on miRNome profiles of whole blood from 540 individuals. These include patients suffering from different cancer and non-cancer diseases, and unaffected controls. Using hierarchi-cal clustering, we found 9 significant clusters of co-expressed miRNAs containing 2-36 individual miRNAs. Through analyzing multiple sequencing alignments in the clusters, we found that co-expression of miRNAs is associated with both sequence similarity and genomic co-localization. We calculated correlations for all 371,953 pairs of miRNAs for all 540 individuals and identified 184 pairs of miRNAs with high correlation values. Out of these 184 pairs of miRNAs, 16 pairs (8.7%) were differentially co-expressed in unaffected controls, cancer patients and patients with non-cancer diseases. By computing correlated and anti-correlated miRNA pairs, we constructed a network with 184 putative co-regulations as edges and 100 miRNAs as nodes. Thereby, we detected specific clusters of miRNAs with high and low correlation values. Our approach represents the most comprehensive co-regulation analysis based on whole miRNome-wide expression profiling. Our findings further decrypt the interactions of miRNAs in normal and human pathological processes.