Identification of Novel Pax8 Targets in FRTL-5 Thyroid Cells by Gene Silencing and Expression Microarray Analysis

The differentiation program of thyroid follicular cells (TFCs), by far the most abundant cell population of the thyroid gland, relies on the interplay between sequence-specific transcription factors and transcriptional coregulators with the basal transcriptional machinery of the cell. However, the molecular mechanisms leading to the fully differentiated thyrocyte are still the object of intense study. The transcription factor Pax8, a member of the Paired-box gene family, has been demonstrated to be a critical regulator required for proper development and differentiation of thyroid follicular cells. Despite being Pax8 well-characterized with respect to its role in regulating genes involved in thyroid differentiation, genomics approaches aiming at the identification of additional Pax8 targets are lacking and the biological pathways controlled by this transcription factor are largely unknown.To identify unique downstream targets of Pax8, we investigated the genome-wide effect of Pax8 silencing comparing the transcriptome of silenced versus normal differentiated FRTL-5 thyroid cells. In total, 2815 genes were found modulated 72 h after Pax8 RNAi, induced or repressed. Genes previously reported to be regulated by Pax8 in FRTL-5 cells were confirmed. In addition, novel targets genes involved in functional processes such as DNA replication, anion transport, kinase activity, apoptosis and cellular processes were newly identified. Transcriptome analysis highlighted that Pax8 is a key molecule for thyroid morphogenesis and differentiation.This is the first large-scale study aimed at the identification of new genes regulated by Pax8, a master regulator of thyroid development and differentiation. The biological pathways and target genes controlled by Pax8 will have considerable importance to understand thyroid disease progression as well as to set up novel therapeutic strategies

Mutations in protocadherin 15 and cadherin 23 affect tip links and mechanotransduction in mammalian sensory hair cells

Immunocytochemical studies have shown that protocadherin-15 (PCDH15) and cadherin-23 (CDH23) are associated with tip links, structures thought to gate the mechanotransducer channels of hair cells in the sensory epithelia of the inner ear. The present report describes functional and structural analyses of hair cells from Pcdh15av3J (av3J), Pcdh15av6J (av6J) and Cdh23v2J (v2J) mice. The av3J and v2J mice carry point mutations that are predicted to introduce premature stop codons in the transcripts for Pcdh15 and Cdh23, respectively, and av6J mice have an in-frame deletion predicted to remove most of the 9th cadherin ectodomain from PCDH15. Severe disruption of hair-bundle morphology is observed throughout the early-postnatal cochlea in av3J/av3J and v2J/v2J mice. In contrast, only mild-to-moderate bundle disruption is evident in the av6J/av6J mice. Hair cells from av3J/av3J mice are unaffected by aminoglycosides and fail to load with [3H]-gentamicin or FM1-43, compounds that permeate the hair cell's mechanotransducer channels. In contrast, hair cells from av6J/av6J mice load with both FM1-43 and [3H]-gentamicin, and are aminoglycoside sensitive. Transducer currents can be recorded from hair cells of all three mutants but are reduced in amplitude in all mutants and have abnormal directional sensitivity in the av3J/av3J and v2J/v2J mutants. Scanning electron microscopy of early postnatal cochlear hair cells reveals tip-link like links in av6J/av6J mice, substantially reduced numbers of links in the av3J/av3J mice and virtually none in the v2J/v2J mice. Analysis of mature vestibular hair bundles reveals an absence of tip links in the av3J/av3J and v2J/v2J mice and a reduction in av6J/av6J mice. These results therefore provide genetic evidence consistent with PCDH15 and CDH23 being part of the tip-link complex and necessary for normal mechanotransduction

Automated array-CGH optimized for archival formalin-fixed, paraffin-embedded tumor material

BACKGROUND: Array Comparative Genomic Hybridization (aCGH) is a rapidly evolving technology that still lacks complete standardization. Yet, it is of great importance to obtain robust and reproducible data to enable meaningful multiple hybridization comparisons. Special difficulties arise when aCGH is performed on archival formalin-fixed, paraffin-embedded (FFPE) tissue due to its variable DNA quality. Recently, we have developed an effective DNA quality test that predicts suitability of archival samples for BAC aCGH. METHODS: In this report, we first used DNA from a cancer cell-line (SKBR3) to optimize the aCGH protocol for automated hybridization, and subsequently optimized and validated the procedure for FFPE breast cancer samples. We aimed for highest throughput, accuracy, and reproducibility applicable to FFPE samples, which can also be important in future diagnostic use. RESULTS: Our protocol of automated array-CGH on archival FFPE ULS-labeled DNA showed very similar results compared with published data and our previous manual hybridization method. CONCLUSION: This report combines automated aCGH on unamplified archival FFPE DNA using non-enzymatic ULS labeling, and describes an optimized protocol for this combination resulting in improved quality and reproducibility

The role of dendritic cell precursors in tumour vasculogenesis

In this review, we discuss the recent identification in vivo of a population of CD11c+ cells exhibiting simultaneous expression of both endothelial and dendritic cell markers, termed vascular leukocytes (VLCs). VLCs are highly represented in human ovarian carcinomas and, depending on the milieu, can assemble into functional blood vessels or act as antigen-presenting cells. The identification of dendritic cell precursors as bipotent cells has important implications for the physiopathology and therapy of tumours. VLCs emerge as a novel therapeutic target against tumour vascularisation

Comparative physical maps derived from BAC end sequences of tilapia (Oreochromis niloticus)

Background: The Nile tilapia is the second most important fish in aquaculture. It is an excellent laboratory model, and is closely related to the African lake cichlids famous for their rapid rates of speciation. A suite of genomic resources has been developed for this species, including genetic maps and ESTs. Here we analyze BAC endsequences to develop comparative physical maps, and estimate the number of genome rearrangements, between tilapia and other model fish species. Results: We obtained sequence from one or both ends of 106,259 tilapia BACs. BLAST analysis against the genome assemblies of stickleback, medaka and pufferfish allowed identification of homologies for approximately 25,000 BACs for each species. We calculate that rearrangement breakpoints between tilapia and these species occur about every 3 Mb across the genome. Analysis of 35,000 clones previously assembled into contigs by restriction fingerprints allowed identification of longer-range syntenies. Conclusions: Our data suggest that chromosomal evolution in recent teleosts is dominated by alternate loss of gene duplicates, and by intra-chromosomal rearrangements (~one per million years). These physical maps are a useful resource for comparative positional cloning of traits in cichlid fishes. The paired BAC end sequences from these clones will be an important resource for scaffolding forthcoming shotgun sequence assemblies of the tilapia genome. (Résumé d'auteur

Minireview: Nonsteroidal anti-inflammatory drugs in colorectal cancer: from prevention to therapy

In this review, we discuss the available experimental evidences supporting the chemopreventive efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) on colorectal cancer and the biological basis for their possible role as anticancer agents. Although the comprehension of the mechanisms underlying the effects of these drugs on colon cancer cells is incomplete, research efforts in identifying the biochemical pathway by which NSAIDs exert their chemopreventive effect have provided a rationale for the potential use of NSAIDs alone or in combination with conventional and experimental anticancer agents in the treatment of colorectal cancer. In this paper, we review three main issues: (i) the role of COX-2 in colon cancer, (ii) the common death pathways between NSAIDs and anticancer drugs; and (iii) the biological basis for the combination therapy with COX-2 selective inhibitors and new selective inhibitors of growth factor signal transduction pathways. (C) 2003 Cancer Research UK

Parallel Evolution of Auditory Genes for Echolocation in Bats and Toothed Whales

The ability of bats and toothed whales to echolocate is a remarkable case of convergent evolution. Previous genetic studies have documented parallel evolution of nucleotide sequences in Prestin and KCNQ4, both of which are associated with voltage motility during the cochlear amplification of signals. Echolocation involves complex mechanisms. The most important factors include cochlear amplification, nerve transmission, and signal re-coding. Herein, we screen three genes that play different roles in this auditory system. Cadherin 23 (Cdh23) and its ligand, protocadherin 15 (Pcdh15), are essential for bundling motility in the sensory hair. Otoferlin (Otof) responds to nerve signal transmission in the auditory inner hair cell. Signals of parallel evolution occur in all three genes in the three groups of echolocators—two groups of bats (Yangochiroptera and Rhinolophoidea) plus the dolphin. Significant signals of positive selection also occur in Cdh23 in the Rhinolophoidea and dolphin, and Pcdh15 in Yangochiroptera. In addition, adult echolocating bats have higher levels of Otof expression in the auditory cortex than do their embryos and non-echolocation bats. Cdh23 and Pcdh15 encode the upper and lower parts of tip-links, and both genes show signals of convergent evolution and positive selection in echolocators, implying that they may co-evolve to optimize cochlear amplification. Convergent evolution and expression patterns of Otof suggest the potential role of nerve and brain in echolocation. Our synthesis of gene sequence and gene expression analyses reveals that positive selection, parallel evolution, and perhaps co-evolution and gene expression affect multiple hearing genes that play different roles in audition, including voltage and bundle motility in cochlear amplification, nerve transmission, and brain function

A change in the optical polarization associated with a gamma-ray flare in the blazar 3C 279

It is widely accepted that strong and variable radiation detected over all accessible energy bands in a number of active galaxies arises from a relativistic, Doppler-boosted jet pointing close to our line of sight. The size of the emitting zone and the location of this region relative to the central supermassive black hole are, however, poorly known, with estimates ranging from light-hours to a light-year or more. Here we report the coincidence of a gamma-ray flare with a dramatic change of optical polarization angle. This provides evidence for co-spatiality of optical and gamma-ray emission regions and indicates a highly ordered jet magnetic field. The results also require a non-axisymmetric structure of the emission zone, implying a curved trajectory for the emitting material within the jet, with the dissipation region located at a considerable distance from the black hole, at about 10^5 gravitational radii.Comment: Published in Nature issued on 18 February 2010. Corresponding authors: Masaaki Hayashida and Greg Madejsk

Horizontal Branch Stars: The Interplay between Observations and Theory, and Insights into the Formation of the Galaxy

We review HB stars in a broad astrophysical context, including both variable and non-variable stars. A reassessment of the Oosterhoff dichotomy is presented, which provides unprecedented detail regarding its origin and systematics. We show that the Oosterhoff dichotomy and the distribution of globular clusters (GCs) in the HB morphology-metallicity plane both exclude, with high statistical significance, the possibility that the Galactic halo may have formed from the accretion of dwarf galaxies resembling present-day Milky Way satellites such as Fornax, Sagittarius, and the LMC. A rediscussion of the second-parameter problem is presented. A technique is proposed to estimate the HB types of extragalactic GCs on the basis of integrated far-UV photometry. The relationship between the absolute V magnitude of the HB at the RR Lyrae level and metallicity, as obtained on the basis of trigonometric parallax measurements for the star RR Lyrae, is also revisited, giving a distance modulus to the LMC of (m-M)_0 = 18.44+/-0.11. RR Lyrae period change rates are studied. Finally, the conductive opacities used in evolutionary calculations of low-mass stars are investigated. [ABRIDGED]Comment: 56 pages, 22 figures. Invited review, to appear in Astrophysics and Space Scienc