Fine-needle aspiration biopsy: its Use in orbital tumors

Fifteen orbital tumors have been evaluated with the fine-needle aspiration biopsy (FNAB) technique. The best indication for FNAB is supposed malignant orbital disease. The technique has not been helpful in tumors or inflammatory disease with a high fibrous content. Lesions that are suspected of being pseudotumors are not recommended for FNAB since, even in histologic sections, they are notoriously difficult to distinguish from well-differentiated lymphocytic malignant lymphoma. Benign encapsulated tumors should not be subjected to FNAB

Immunocytologic Methods in the Diagnosis of Orbital Tumors

The pathologic diagnosis was supported or confirmed in three out of four cases that had an adequate cytologic specimen. The results demonstrate that adjunctive immunocytologic techniques can be used in combination with fine-needle aspiration for a variety of orbital tumors. The pathologic diagnosis was supported or confirmed in three out of four cases that had an adequate cytologic specimen. The results demonstrate that adjunctive immunocytologic techniques can be used in combination with fine-needle aspiration for a variety of orbital tumors

Subcellular Localization of Frizzled Receptors, Mediated by Their Cytoplasmic Tails, Regulates Signaling Pathway Specificity

The Frizzled (Fz; called here Fz1) and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/β-catenin pathway or Fz/planar cell polarity (PCP) signaling in Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/β-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways

siRNAs Induce Efficient RNAi Response in Bombyx mori Embryos

Short interference RNA (siRNA) is widely used in mammalian cells. In insects, however, reports concerning the suitablility of siRNA in vivo is very limited compared with that of long dsRNA, which is thought to be more effective. There is insufficient information on the essential rules of siRNA design in insects, as very few siRNAs have been tested in this context. To establish an effective method of gene silencing using siRNA in vivo in insects, we determined the effects of siRNA on seven target genes. We designed siRNAs according to a new guideline and injected them into eggs of Bombyx mori. At the mRNA level, the expression of most of these genes was successfully silenced, down to less than half the constitutive level, which in some cases led to the development of distinctive phenotypes. In addition, we observed stronger effect of siRNA both on the mRNA level and the phenotype than that of long dsRNA under comparable conditions. These results indicate that direct injection of siRNA is an effective reverse-genetics tool for the analysis of embryogenesis in vivo in insects

Pogostick: A New Versatile piggyBac Vector for Inducible Gene Over-Expression and Down-Regulation in Emerging Model Systems

Non-traditional model systems need new tools that will enable them to enter the field of functional genetics. These tools should enable the exploration of gene function, via knock-downs of endogenous genes, as well as over-expression and ectopic expression of transgenes.We constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ line transformation by the piggyBac transposable element. Pogostick can be found at www.addgene.org, a non-profit plasmid repository. The vector currently uses the heat-shock promoter Hsp70 from Drosophila to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. We detail how to clone candidate genes into this vector and test its functionality in Drosophila by targeting a gene coding for the fluorescent protein DsRed. By cloning a single DsRed copy into the vector, and generating transgenic lines, we show that DsRed mRNA and protein levels are elevated following heat-shock. When cloning a second copy of DsRed in reverse orientation into a flanking site, and transforming flies constitutively expressing DsRed in the eyes, we show that endogenous mRNA and protein levels drop following heat-shock. We then test the over-expression vector, containing the complete cDNA of Ultrabithorax (Ubx) gene, in an emerging model system, Bicyclus anynana. We produce a transgenic line and show that levels of Ubx mRNA expression rise significantly following a heat-shock. Finally, we show how to obtain genomic sequence adjacent to the Pogostick insertion site and to estimate transgene copy number in genomes of transformed individuals.This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles

Dissecting mitosis by RNAi in Drosophila tissue culture cells

Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated interference (RNAi) in cultured cells of Drosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique

Efficient Construction of an Inverted Minimal H1 Promoter Driven siRNA Expression Cassette: Facilitation of Promoter and siRNA Sequence Exchange

RNA interference (RNAi), mediated by small interfering RNA (siRNA), is an effective method used to silence gene expression at the post-transcriptional level. Upon introduction into target cells, siRNAs incorporate into the RNA-induced silencing complex (RISC). The antisense strand of the siRNA duplex then "guides" the RISC to the homologous mRNA, leading to target degradation and gene silencing. In recent years, various vector-based siRNA expression systems have been developed which utilize opposing polymerase III promoters to independently drive expression of the sense and antisense strands of the siRNA duplex from the same template.We show here the use of a ligase chain reaction (LCR) to develop a new vector system called pInv-H1 in which a DNA sequence encoding a specific siRNA is placed between two inverted minimal human H1 promoters (approximately 100 bp each). Expression of functional siRNAs from this construct has led to efficient silencing of both reporter and endogenous genes. Furthermore, the inverted H1 promoter-siRNA expression cassette was used to generate a retrovirus vector capable of transducing and silencing expression of the targeted protein by>80% in target cells.The unique design of this construct allows for the efficient exchange of siRNA sequences by the directional cloning of short oligonucleotides via asymmetric restriction sites. This provides a convenient way to test the functionality of different siRNA sequences. Delivery of the siRNA cassette by retroviral transduction suggests that a single copy of the siRNA expression cassette efficiently knocks down gene expression at the protein level. We note that this vector system can potentially be used to generate a random siRNA library. The flexibility of the ligase chain reaction suggests that additional control elements can easily be introduced into this siRNA expression cassette

piRNAs mediate posttranscriptional retroelement silencing and localization to pi-bodies in the Drosophila germline

Nuage, a well-conserved perinuclear organelle found in germline cells, is thought to mediate retroelement repression in Drosophila melanogaster by regulating the production of Piwi-interacting RNAs (piRNAs). In this study, we present evidence that the nuage–piRNA pathway components can be found in cytoplasmic foci that also contain retroelement transcripts, antisense piRNAs, and proteins involved in messenger RNA (mRNA) degradation. These mRNA degradation proteins, decapping protein 1/2 (DCP1/2), Me31B (maternal expression at 31B), and pacman (PCM), are normally thought of as components of processing bodies. In spindle-E (spn-E) and aubergine (aub) mutants that lack piRNA production, piRNA pathway proteins no longer overlap the mRNA degradation proteins. Concomitantly, spn-E and aub mutant ovaries show an accumulation of full-length retroelement transcripts and prolonged stabilization of HeT-A mRNA, supporting the role of piRNAs in mediating posttranscriptional retroelement silencing. HeT-A mRNA is derepressed in mRNA degradation mutants twin, dcp1, and ski3, indicating that these enzymes also aid in removing full-length transcripts and/or decay intermediates