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Multicolor fluorescence in situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Often, the intensity distributions between different images are found to be considerably different and the difference becomes the source of misclassifications of the pixels. Improved pixel classification accuracy is the most important task to ensure the success of the M-FISH technique. In this paper, we introduce a new feature normalization method for M-FISH images that reduces the difference in the feature distributions among different images using the expectation maximization (EM) algorithm. We also introduce a new unsupervised, nonparametric classification method for M-FISH images. The performance of the classifier is as accurate as the maximum-likelihood classifier, whose accuracy also significantly improved after the EM normalization. We would expect that any classifier will likely produce an improved classification accuracy following the EM normalization. Since the developed classification method does not require training data, it is highly convenient when ground truth does not exist. A significant improvement was achieved on the pixel classification accuracy after the new feature normalization. Indeed, the overall pixel classification accuracy improved by 20% after EM normalization. Index Terms--Chromosome, classification, expectation maximization (EM), maximum-likelihood, multicolor fluorescence in situ hybridization (M-FISH), normalization, unsupervised.

Here, we report a high-efficiency single-cell entrapment system with a poly(dimethylsiloxane) (PDMS) microfluidic device integrated with a micromesh, and its application to single-cell fluorescence in situ hybridization (FISH) analysis. A micromesh comprising of 10 x 10 microcavities was fabricated on a black poly(ethylene terephthalate) (PET) substrate by laser ablation. The cavity was approximately 2 [micro]m in diameter. Mammalian cells were driven and trapped onto the microcavities by applying negative pressure. Trapped cells were uniformly arrayed on the micromesh, enabling high-throughput microscopic analysis. Furthermore, we developed a method of PDMS surface modification by using air plasma and the copolymer Pluronic F-127 to prevent nonspecific adsorption on the PDMS microchannel. This method decreased the nonspecific adsorption of cells onto the microchannel to less than 1%. When cells were introduced into the microfluidic device integrated with the black PET micromesh, approximately 70-80% of the introduced cells were successfully trapped. Moreover, for mRNA expression analysis, on-chip fluorescence in situ hybridization (e.g., membrane permeabilization, hybridization, washing) can be performed in a microfluidic assay on an integrated device. This microfluidic device has been employed for the detection of [beta]-actin mRNA expression in individual Raji cells. Differences in the levels of [beta]-actin mRNA expression were observed in serum-supplied or serum-starved cell populations.

Abstract: To study genes that are specifically expressed in the eyes, we employed microarray and in situ hybridization analyses to identify and characterize differentially expressed ocular genes in eyeless masterblind [...]

An automated method for analyzing fluorescence in situ hybridization (FISH) images with fuzzy c-means clustering (FCM) which was developed by using swine manure and soil as representative complex environmental samples is presented.

The bacterial symbionts of Watersiporia arcuata are analyzed for their phylogeny. The symbionts of different Watersiporia species were found to be closely related species of [alpha]-proteobacteria.

We studied the effects of photoperiod on metabolic profiles, adiposity, and gene expression of hypothalamic appetite-regulating peptides in gonad-intact and castrated Soay rams. Groups of five to six animals were studied 6, 18, or 30 wk after switching from long photoperiod (LP: 16 h of light) to short photoperiod (SP: 8 h of light). Reproductive and metabolic indexes were measured in blood plasma. Expression of neuropeptide Y (NPY), proopiomelanocortin (POMC), and leptin receptor (ObRb) in the arcuate nucleus was measured using in situ hybridization. Testosterone levels of intact animals were low under LP, increased to a peak at 16 wk under SP, and then declined. Voluntary food intake (VFI) was high under LP in both intact and castrated animals, decreased to a nadir at 12-16 wk under SP, and then recovered, but only in intact rams as the reproductive axis became photorefractory to SP. NPY gene expression varied positively and POMC expression varied negatively with the cycle in VFI, with differences between intact and castrate rams in the refractory phase. ObRb expression decreased under SP, unrelated to changes in VFI. Visceral fat weight also varied between the intact and castrated animals across the cycle. We conclude that 1) photoperiodic changes in VFI reflect changes in NPY and POMC gene expression, 2) changes in ObRb gene expression are not necessarily determinants of changes in VFI, 3) gonadal status affects the pattern of VFI that changes with photoperiod, and 4) in the absence of gonadal factors, animals can eat less but gain adiposity. Soay ram; neuropeptide Y; pro-opiomelanocortin; leptin receptor; sex steroids

Abstract: Recent studies, using cytometric techniques based on fluorescence microscopy, have provided new information on how linker histones interact with chromatin in vivo or in situ. In particular, the use [...]

Abstract Antagonists at neurokinin 1 (NK1) receptors are attracting attention as potential treatments for depressive states in light of their actions in behavioural models predictive of antidepressant properties, their modulation [...]

Magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) show considerable plasticity during pregnancy and lactation. Prolactin receptors (PRL-R) have been identified in both these nuclei. The aim of this study was to investigate the cell type(s) expressing mRNA for the long form of prolactin receptor (PRL-[R.sub.L]) and to determine whether patterns of expression change during pregnancy and lactation. In addition, we examined effects of prolactin on excitability of oxytocin and vasopressin neurons. Sections from brains of nonpregnant, pregnant, and lactating rats were hybridized with an 35S-labeled probe to label PRL-[R.sub.L] mRNA together with digoxigenin-labeled probes to detect either oxytocin or vasopressin mRNA. In the SON, PRL-[R.sub.L] mRNA was predominantly colocalized with oxytocin mRNA, with over 80% of oxytocin neurons positive for PRL-RL mRNA. Very few ( pregnancy; lactation; magnocellular neurons; in situ hybridization

The acetate-utilizing microbial consortium in a full-scale activates sludge process is determined without prior enrichment using stable isotope probing (SIP), full-cycle rRNA analysis and fluorescent in situ hybridization (FISH)-microautoradiography. A combination of molecular methods have indicated that bacteria related to the genera Acidovorax, Dechloromonas and Thauera belonging to the families Comamonadaceae and Rhodocyclaceae are capable of utilizing acetate under anoxic conditions.

A novel microcultivation method for soil bacteria that mimics natural conditions is described. By combining microcultivation with fluorescent in situ hybridization, previously unculturable organisms belonging to cultivated and noncultivated divisions, including candidate division TM7 can be identified by fluorescence microscopy.

The role of multiple gonadotropin-releasing hormone receptor (GnRH-R) types in the regulation of gonadotropic and nongonadotropic cells remains speculative. To address this issue, we developed a technology integrating laser-captured microdissection of single digoxigenin-labeled pituitary cells coupled with real-time quantitative PCR to examine the expression profiles of three endogenous GnRH-R types (R1, R2, and R3) in immature and mature males of tilapia Oreochromis niloticus. Here, in addition to gonadotropes (luteinizing and folicle-stimulating hormone, FSH), we show GnRH-Rs are also present in lactotropes, somatotropes, thyrotropes, melanotropes (melanocyte-stimulating hormone, MSH), corticotropes and somatolactin cells. Subpopulations of pituitary cells express single (42.9%), multiple (32.4%) or lack (24.7%) GnRH-Rs. In immature males, the percentage of FSH cells containing combinations of GnRH-Rs was significantly higher (R1+R2: 24%, P < 0.05; R1+R2+R3: 25%, P < 0.01) than in mature males, whereas the percentage showing only R1 and R1 and R3 transcripts (P < 0.05) was higher in mature males. Significantly greater copies of R1 and R3 transcripts were found in MSH cells of immature and mature males, respectively (P < 0.05). GnRH-R transcripts in other pituitary cells (lactotropes, R1 and R2, soma-tolactin cells/thyrotropes/corticotropes, R1, R2, and R3) were significantly higher in mature males (P < 0.05) but were unaltered in somatotropes and luteinizing hormone cells. Thus, FSH and MSH cells are required for both reproductive states, whereas other pituitary cells are recruited only during testicular maturation. The differential expression of GnRH-Rs in gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity of mechanisms controlling normal sexual development. G protein | in situ hybridization | tilapia

Byline: Nino Guarino (1), Valeria Solari (1), Hideki Shima (1), Prem Puri (1) Keywords: Newborn; Lung; Immunohistochemistry; In situ hybridization; Growth factors Abstract: Newborn infants with congenital diaphragmatic hernia (CDH) still have a high mortality rate. Epidermal growth factor (EGF) and transforming growth factor-[alpha] (TGF-[alpha]) are peptide growth factors involved in the fetal lung growth and development. The EGF and TGF-[alpha] have been reported to promote pulmonary branching activity and alveolar type-II pneumocyte proliferation. Epidermal growth factor and TGF-[alpha] immunoreactivity and mRNA expression in the bronchial and bronchiolar epithelium is maximal during early fetal life and barely detectable in the proximal airways of neonatal lung. The purpose of this study was to determine protein and gene expression of EGF and TGF-[alpha] in CDH lung in order to elucidate the potential role of these growth factors in the pathogenesis of pulmonary hypoplasia in CDH. Lung tissue specimens were obtained from archival lung tissue from 11 patients with CDH and 5 controls. Indirect immunohistochemistry was performed using ABC method with anti-EGF and anti-TGF-[alpha] antibodies. In situ hybridization was performed using EGF and TGF-[alpha] specific digoxigenin-labeled oligonucleotide probes. The most striking difference between hypoplastic CDH lung and control lung was the strong EGF and TGF-[alpha] mRNA expression and immunoreactivity in the bronchial and bronchiolar epithelium in CDH lung. The upregulated protein and gene expression of EGF and TGF-[alpha] in the proximal airways in the CDH hypoplastic lung suggests persistence of fetal stage of pulmonary airway development in CDH. Author Affiliation: (1) Children's Research Centre, Our Lady's Hospital for Sick Children, University College, Crumlin, Dublin 12, Ireland Article History: Registration Date: 21/05/2003 Online Date: 09/01/2004

Two clones of Miscanthus, grown under the names M. Xgiganteus and M. saccharifiorus, have been used in biomass trials in Europe, but neither the identity of these clones nor their origin has been established. DNA sequencing, amplified fragment length polymorphism (AFLP), and chromosome studies confirm that M. Xgiganteus is an allotriploid (2n = 3x = 57) combining genomes from M. sinensis (2n = 2x = 38) and M. sacchariflorus (2n = 38 or 76). Two alleles of the internal transcribed spacer of 18S-25S nuclear ribosomal DNA (ITS) were discovered in polymerase chain reaction products of M. Xgiganteus. Cloning of these revealed that one matched M. sinensis and the other M. sacchariflorus. Plastid trnL intron and trnL-F spacer sequences showed that the maternal lineage of M. Xgiganteus was M. sacchariflorus. Fluorescent in situ hybridization, FISH, was used to investigate genome organization in Miscanthus but was unable to differentiate between the different parental genomes present in M. Xgiganteus, indicating that two parental genomes are still extremely similar at the repetitive DNA level. This study is an example in which rDNA sequences and AFLP fingerprints permit identification of the parental genomes in a hybrid, but FISH methods, at the repetitive DNA level (including genomic in sim hybridization, GISH), were unable to do so because their sequences remain too similar. Key words: AFLP; in situ hybridization; ITS; FISH; GISH; Miscanthus; Poaceae; polyploidy; trnL-F.

Abstract Background/aims--Uveal melanoma is the commonest malignancy of the eye, with a high proportion of patients dying of metastatic disease. Tumours showing a loss of chromosome 3 and gains of [...]

Researchers have constructed an accessibility map for the 23S rRNA of E. coli that can be used to develop oligonucleotide probes for fluorescence in situ hybridization. Accessibility to probes varied substantially, with the best signal generated from a probe that was complementary to positions 2018 to 2035.

Inaccessible regions of ribosomal RNA can be made accessible to FISH probes by using oligonucleotides that are complementary to regions close to the probe target site. These so-called helper oligonucleotides increased the fluorescence of several FISH probes of E. coli 16S rRNA.

The Ran-GTPase cycle and certain nucleoporins necessary for export from the nucleus of the small ribosomal subunit are discussed. Information is based on a study using the cytoplasmic localization of the 5' ITS1 fragment as indicator for the export of the small ribosomal subunit to identify necessary genes. Mutations in Ran or its regulators cause 5' ITS1 to accumulate in the nucleoplasm. Accumulation of 5' ITS1 in the nucleus was not found in 28 yeast strains with mutations in other genes that affect traffic in the nucleus.