Comparison of pattern detection methods in microarray time series of the segmentation clock.

While genome-wide gene expression data are generated at an increasing rate, the repertoire of approaches for pattern discovery in these data is still limited. Identifying subtle patterns of interest in large amounts of data (tens of thousands of profiles) associated with a certain level of noise remains a challenge. A microarray time series was recently generated to study the transcriptional program of the mouse segmentation clock, a biological oscillator associated with the periodic formation of the segments of the body axis. A method related to Fourier analysis, the Lomb-Scargle periodogram, was used to detect periodic profiles in the dataset, leading to the identification of a novel set of cyclic genes associated with the segmentation clock. Here, we applied to the same microarray time series dataset four distinct mathematical methods to identify significant patterns in gene expression profiles. These methods are called: Phase consistency, Address reduction, Cyclohedron test and Stable persistence, and are based on different conceptual frameworks that are either hypothesis- or data-driven. Some of the methods, unlike Fourier transforms, are not dependent on the assumption of periodicity of the pattern of interest. Remarkably, these methods identified blindly the expression profiles of known cyclic genes as the most significant patterns in the dataset. Many candidate genes predicted by more than one approach appeared to be true positive cyclic genes and will be of particular interest for future research. In addition, these methods predicted novel candidate cyclic genes that were consistent with previous biological knowledge and experimental validation in mouse embryos. Our results demonstrate the utility of these novel pattern detection strategies, notably for detection of periodic profiles, and suggest that combining several distinct mathematical approaches to analyze microarray datasets is a valuable strategy for identifying genes that exhibit novel, interesting transcriptional patterns.

Specialized motor-driven dusp1 expression in the song systems of multiple lineages of vocal learning birds.

Mechanisms for the evolution of convergent behavioral traits are largely unknown. Vocal learning is one such trait that evolved multiple times and is necessary in humans for the acquisition of spoken language. Among birds, vocal learning is evolved in songbirds, parrots, and hummingbirds. Each time similar forebrain song nuclei specialized for vocal learning and production have evolved. This finding led to the hypothesis that the behavioral and neuroanatomical convergences for vocal learning could be associated with molecular convergence. We previously found that the neural activity-induced gene dual specificity phosphatase 1 (dusp1) was up-regulated in non-vocal circuits, specifically in sensory-input neurons of the thalamus and telencephalon; however, dusp1 was not up-regulated in higher order sensory neurons or motor circuits. Here we show that song motor nuclei are an exception to this pattern. The song nuclei of species from all known vocal learning avian lineages showed motor-driven up-regulation of dusp1 expression induced by singing. There was no detectable motor-driven dusp1 expression throughout the rest of the forebrain after non-vocal motor performance. This pattern contrasts with expression of the commonly studied activity-induced gene egr1, which shows motor-driven expression in song nuclei induced by singing, but also motor-driven expression in adjacent brain regions after non-vocal motor behaviors. In the vocal non-learning avian species, we found no detectable vocalizing-driven dusp1 expression in the forebrain. These findings suggest that independent evolutions of neural systems for vocal learning were accompanied by selection for specialized motor-driven expression of the dusp1 gene in those circuits. This specialized expression of dusp1 could potentially lead to differential regulation of dusp1-modulated molecular cascades in vocal learning circuits.

Immediate early gene expression in dog thyrocytes in response to growth, proliferation, and differentiation stimuli.

In dog thyroid cells, insulin or IGF-1 induces cell growth and is required for the mitogenic action of TSH through cyclic AMP, of EGF, and of phorbol esters. HGF per se stimulates cell proliferation and is thus the only full mitogenic agent. TSH and cAMP enhance, whereas EGF phorbol esters and HGF repress differentiation expression. In this study, we have investigated for each factor and regulatory cascade of the intermediate step of immediate early gene induction, that is, c-myc, c-jun, jun D, jun B, c-fos, fos B, fra-1, fra-2, and egr1; fra-1 and fra-2 expressions were very low. TSH or forskolin increased the levels of c-myc, jun B, jun D, c-fos, and fos B while decreasing those of c-jun and egr1. Phorbol myristate ester stimulated the expression of all the genes. EGF and HGF stimulated the expression of all the genes except jun D and for EGF fos B. All these effects were obtained in the presence and in the absence of insulin, which shows that insulin is not necessary for the effects of the mitogens on immediate early gene expression. The definition of the repertoire of early immediate genes inductible by the various growth cascades provides a framework for the analysis of gene expression in tumors. (1) Insulin was able to induce all the protooncogenes investigated except fos B. This suggests that fos B could be the factor missing for insulin to induce mitogenesis. (2) No characteristic pattern of immediate early gene expression has been observed for insulin, which induces cell hypertrophy and is permissive for the action of the other growth factors. These effects are therefore not accounted for by a specific immediate early gene expression. On the other hand, insulin clearly enhances the effects of TSH, phorbol ester, and EGF on c-myc, junB, and c-fos expression. This suggests that the effect of insulin on mitogenesis might result from quantitative differences in the transcription complexes formed. (3) c-myc, c-fos, and jun B mRNA induction by all stimulating agents, whether inducing cell hypertrophy, or growth and dedifferentiation, or growth and differentiation, suggests that, although these expressions are not sufficient, they may be necessary for the various growth responses of thyroid cells. (4) The inhibition of c-jun and egr1 mRNA expression, and the marked induction of jun D mRNA appear to be specific features of the TSH cAMP pathway. They might be related to its differentiating action. (5) fos B, which is induced by TSH, forskolin, phorbol ester, and HGF but not by insulin, could be involved in the mitogenic action of the former factors.

Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.

Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.

Cathepsin S expression: An independent prognostic factor in glioblastoma tumours - A pilot Study

Cysteine proteinases have been implicated in astrocytoma invasion. We recently demonstrated that cathepsin S (CatS) expression is up-regulated in astrocytomas and provided evidence for a potential role in astrocytoma invasion (Flannery et al., Am J Path 2003;163(1):175–82). We aimed to evaluate the significance of CatS in human astrocytoma progression and as a prognostic marker. Frozen tissue homogenates from 71 patients with astrocytomas and 3 normal brain specimens were subjected to ELISA analyses. Immunohistochemical analysis of CatS expression was performed on 126 paraffin-embedded tumour samples. Fifty-one astrocytoma cases were suitable for both frozen tissue and paraffin tissue analysis. ELISA revealed minimal expression of CatS in normal brain homogenates. CatS expression was increased in grade IV tumours whereas astrocytoma grades I–III exhibited lower values. Immunohistochemical analysis revealed a similar pattern of expression. Moreover, high-CatS immunohistochemical scores in glioblastomas were associated with significantly shorter survival (10 vs. 5 months, p = 0.014). With forced inclusion of patient age, radiation dose and Karnofsky score in the Cox multivariate model, CatS score was found to be an independent predictor of survival. CatS expression in astrocytomas is associated with tumour progression and poor outcome in glioblastomas. CatS may serve as a useful prognostic indicator and potential target for anti-invasive therapy.

Altered expression of the septin gene, SEPT9, in ovarian neoplasia.

The septin family of genes has been implicated in a variety of cellular processes including cytokinesis, membrane transport and fusion, exocytosis, and apoptosis. One member of the septin family maps to chromosome 17q25.3, a region commonly deleted in sporadic ovarian and breast tumours, and has also been identified as a fusion partner of MLL in acute myeloid leukaemias. The present study demonstrates that the pattern of expression of multiple splice variants of this septin gene is altered in ovarian tumours and cell lines. In particular, expression of the zeta transcript is detectable in the majority of tumours and cell lines, but not in a range of non-malignant adult and fetal tissues. Zeta expression is accompanied by loss of the ubiquitous beta transcript. Somatic mutations of the gene were not detected in ovarian tumours, but it was demonstrated that beta expression in tumour cell lines can be reactivated by 5-azacytidine treatment, suggesting a role for methylation in the control of expression of this gene. Copyright © 2003 John Wiley & Sons, Ltd.

Erythropoietin receptor expression in non-small cell lung carcinoma: a question of antibody specificity

Immunohistochemical studies on formalin-fixed, paraffin-embedded (FFPE) tissue utilizing polyclonal antibodies form the cornerstone of many reports claiming to demonstrate erythropoietin receptor (EPOR) expression in malignant tissue. Recently, Elliott et al. (Blood 2006;107:1892-1895) reported that the antibodies commonly used to detect EPOR expression also detect non-EPOR proteins, and that their binding to EPOR was severely abrogated by two synthetic peptides based on the sequence of heat shock protein (HSP) 70, HSP70-2, and HSP70-5. We have investigated the specificity of the C20 antibody for detecting EPOR expression in non-small cell lung carcinoma (NSCLC) utilizing tissue microarrays. A total of 34 cases were available for study. Antibody absorbed with peptide resulted in marked suppression of cytoplasmic staining compared with nonabsorbed antibody. Four tumors that initially showed a membranous pattern of staining retained this pattern with absorbed antibody. Positive membranous immunoreactivity was also observed in 6 of 30 tumors that originally showed a predominantly cytoplasmic pattern of staining. Using the C20 antibody for Western blots, we detected three main bands, at 100, 66, and 59 kDa. Preincubation with either peptide caused abolition of the 66-kDa band, which contains non-EPOR sequences including heat shock peptides. These results call into question the significance of previous immunohistochemical studies of EPOR expression in malignancy and emphasize the need for more specific anti-EPOR antibodies to define the true extent of EPOR expression in neoplastic tissue

PIM-1 kinase expression in adipocytic neoplasms: diagnostic and biological implications

The differential diagnosis of soft tissue tumours poses a considerable challenge for pathologists, especially adipocytic tumours, as these may show considerable overlap in clinical presentation and morphological features with many other mesenchymal neoplasms. Hence, a specific and reliable marker that identifies adipocytic differentiation is much sought. We investigated the immunohistochemical expression of PIM-1 kinase in 35 samples of soft tissue tumours using tissue microarray technology and 49 full sections of adipocytic (n = 26) and non-adipocytic tumours (n = 23). Benign and malignant adipocytic tumours showed strong expression of PIM-1 while the non-adipocytic tumours were either negative or showed only weak staining for the protein. In myxoid liposarcomas, PIM-1 showed a distinct, unique vacuolar staining pattern, clearly outlining fine cytoplasmic lipid vacuoles. By contrast, non-adipocytic myxoid tumours (myxoma, chordoma and myxoid chondrosarcoma) did not show this vacuolar pattern of PIM-1 staining, although vacuolated cells were present on H&E. This differential expression was confirmed at a gene expression level in selected cases. Our results indicate that the expression of PIM-1 in adipose tissue may be a useful marker of adipocytic differentiation, in particular if the staining is both of high intensity and present in a unique, vacuolar pattern.

Flow cytometric analysis of within-strain variation in polysaccharide expression by Bacteroides fragilis by use of murine monoclonal antibodies.

The reactivity of four different monoclonal antibodies (MAbs) with populations of Bacteroides fragilis NCTC 9343, enriched by density gradient centrifugation for a large capsule, small capsule and electron-dense layer (EDL) only visible by electronmicroscopy, was examined. The MAbs reacted strongly with polysaccharides present in both the large capsule- and EDL-enriched populations but not in the small capsule-enriched populations. The pattern of labelling was determined by immunoblotting, immunofluorescence and immuno-electronmicroscopy, and flow cytometry. The MAbs labelled cell membrane-associated epitopes in the large capsule- and EDL-enriched populations and cell-free material in the EDL population. By immunoblotting, ladders of repeating polysaccharide subunits were evident in the EDL population but not in the large capsule population. The proportion of cells labelled within each population was determined by flow cytometry. The reactivity of another MAb with the small capsule population was confirmed by flow cytometry. A qualitative indication of epitope expression was obtained by examination of the flow cytometric profiles. Differential expression of the same saccharide epitope was observed both between and within structurally distinct B. fragilis populations. The MAbs were species-specific and cross-reacted with several recent clinical isolates. These polysaccharides may be relevant to the virulence of B. fragilis.

Differential TERT promoter methylation and response to 5-aza-2'-deoxycytidine in acute myeloid leukemia cell lines:TERT expression, telomerase activity, telomere length, and cell death

The catalytic subunit of human telomerase (TERT) is highly expressed in cancer cells, and correlates with complex cytogenetics and disease severity in acute myeloid leukemia (AML). The TERT promoter is situated within a large CpG island, suggesting that expression is methylation-sensitive. Studies suggest a correlation between hypermethylation and TERT overexpression. We investigated the relationship between TERT promoter methylation and expression and telomerase activity in human leukemia and lymphoma cell lines. DAC-induced demethylation and cell death were observed in all three cell lines, as well as telomere shortening in HL-60 cells. DAC treatment reduced TERT expression and telomerase activity in OCI/AML3 and HL-60 cells, but not in U937 cells. Control U937 cells expressed lower levels of TERT mRNA, carried a highly methylated TERT core promoter, and proved more resistant to DAC-induced repression of TERT expression and cell death. AML patients had significantly lower methylation levels at several CpGs than "well elderly" individuals. This study, the first to investigate the relationship between TERT methylation and telomerase activity in leukemia cells, demonstrated a differential methylation pattern and response to DAC in three AML cell lines. We suggest that, although DAC treatment reduces TERT expression and telomerase activity, this is unlikely to occur via direct demethylation of the TERT promoter. However, further investigations on the regions spanning CpGs 7-12 and 14-16 may reveal valuable information regarding transcriptional regulation of TERT.

O-antigen expression in Salmonella enterica serovar Typhi is regulated by nitrogen availability through RpoN-mediated transcriptional control of the rfaH gene

The authors previously reported increased expression of the Salmonella enterica serovar Typhi (S. typhi) rfaH gene when the bacterial cells reach stationary phase. In this study, using a lacZ fusion to the rfaH promoter region, they demonstrate that growth-dependent regulation of rfaH expression occurs at the level of transcription initiation. It was also observed that production of the lipopolysaccharide (LPS) O-antigen by S. typhi Ty2 correlated with the differential expression of rfaH during bacterial growth. This was probably due to the increased cellular levels of RfaH, since expression of the distal gene in the O-antigen gene cluster of S. typhi Ty2, wbaP, was also increased during stationary growth, as demonstrated by RT-PCR analysis. Examination of the sequences upstream of the rfaH coding region revealed homologies to potential binding sites for the RcsB/RcsA dimer of the RcsC/YopJ/RcsB phosphorelay regulatory system and for the RpoN alternative sigma factor. The expression of the rfaH gene in rpoN and rcsB mutants of S. typhi Ty2 was measured. The results indicate that inactivation of rpoN, but not of rcsB, suppresses the growth-phase-dependent induction of rfaH expression. Furthermore, production of beta-galactosidase mediated by the rfaH-lacZ fusion increased approximately fourfold when bacteria were grown in a nitrogen-limited medium. Nitrogen limitation was also shown to increase the expression of the O-antigen by the wild-type S. typhi Ty2, as demonstrated by a similar electrophoretic profile to that observed during the stationary phase of growth in rich media. It is therefore concluded that the relationship between LPS production and nitrogen limitation parallels the pattern of rfaH regulation under the control of RpoN and is consistent with the idea that RpoN modulates LPS formation via its effect on rfaH gene expression during bacterial growth.

Parsing parallel evolution:Ecological divergence and differential gene expression in the adaptive radiations of thick-lipped Midas cichlid fishes from Nicaragua

The study of parallel evolution facilitates the discovery of common rules of diversification. Here, we examine the repeated evolution of thick lips in Midas cichlid fishes (the Amphilophus citrinellus species complex) - from two Great Lakes and two crater lakes in Nicaragua - to assess whether similar changes in ecology, phenotypic trophic traits and gene expression accompany parallel trait evolution. Using next-generation sequencing technology, we characterize transcriptome-wide differential gene expression in the lips of wild-caught sympatric thick- and thin-lipped cichlids from all four instances of repeated thick-lip evolution. Six genes (apolipoprotein D, myelin-associated glycoprotein precursor, four-and-a-half LIM domain protein 2, calpain-9, GTPase IMAP family member 8-like and one hypothetical protein) are significantly underexpressed in the thick-lipped morph across all four lakes. However, other aspects of lips' gene expression in sympatric morphs differ in a lake-specific pattern, including the magnitude of differentially expressed genes (97-510). Generally, fewer genes are differentially expressed among morphs in the younger crater lakes than in those from the older Great Lakes. Body shape, lower pharyngeal jaw size and shape, and stable isotopes (dC and dN) differ between all sympatric morphs, with the greatest differentiation in the Great Lake Nicaragua. Some ecological traits evolve in parallel (those related to foraging ecology; e.g. lip size, body and head shape) but others, somewhat surprisingly, do not (those related to diet and food processing; e.g. jaw size and shape, stable isotopes). Taken together, this case of parallelism among thick- and thin-lipped cichlids shows a mosaic pattern of parallel and nonparallel evolution. © 2012 Blackwell Publishing Ltd.

Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2)-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.

The role of secreted frizzled-related protein 2 expression in prostate cancer

AIMS: Improved prostate cancer (PCa)-specific biomarkers are urgently required to distinguish between indolent and aggressive disease, in order to avoid overtreatment. In this study, we investigated the prostatic tissue expression of secreted frizzled-related protein (SFRP)-2.

METHODS AND RESULTS: Following immunohistochemical analysis on PCa tissue microarrays with samples from 216 patients, strong/moderate SFRP-2 expression was observed in epithelial cells of benign prostatic hyperplasia, and negative/weak SFRP-2 expression was observed in the majority of tumour epithelia. However, among Gleason grade 5 carcinomas, 40% showed strong/moderate SFRP-2 expression and 60% showed negative SFRP-2 expression in epithelial cells. Further microscopic evaluation of Gleason grade 5 tumours revealed different morphological patterns, corresponding with differential SFRP-2 expression. The first subgroup (referred to as Type A) appeared to have a morphologically solid growth pattern, whereas the second subgroup (referred to as Type B) appeared to have a more diffuse pattern. Furthermore, 100% (4/4) of Type A patients experienced biochemical recurrence, as compared with 0% (0/6) of Type B patients.

CONCLUSIONS: These results imply: (i) that there is a loss of SFRP-2 expression from benign to malignant prostate glands; and (ii) differential SFRP-2 expression among two possible subgroups of Gleason grade 5 tumours.