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

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

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

LIN-44/Wnt Directs Dendrite Outgrowth through LIN-17/Frizzled in C. elegans Neurons

Nervous system function requires proper development of two functional and morphological domains of neurons, axons and dendrites. Although both these domains are equally important for signal transmission, our understanding of dendrite development remains relatively poor. Here, we show that in C. elegans the Wnt ligand, LIN-44, and its Frizzled receptor, LIN-17, regulate dendrite development of the PQR oxygen sensory neuron. In lin-44 and lin-17 mutants, PQR dendrites fail to form, display stunted growth, or are misrouted. Manipulation of temporal and spatial expression of LIN-44, combined with cell-ablation experiments, indicates that this molecule is patterned during embryogenesis and acts as an attractive cue to define the site from which the dendrite emerges. Genetic interaction between lin-44 and lin-17 suggests that the LIN-44 signal is transmitted through the LIN-17 receptor, which acts cell autonomously in PQR. Furthermore, we provide evidence that LIN-17 interacts with another Wnt molecule, EGL-20, and functions in parallel to MIG-1/Frizzled in this process. Taken together, our results reveal a crucial role for Wnt and Frizzled molecules in regulating dendrite development in vivo

miRNA-Dependent Translational Repression in the Drosophila Ovary

Background: The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary. Methodology/Principal Findings: Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed. Conclusions/Significance: Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.This work was supported in part by NIH grant GM54409 and in part by Research Grant No. 1-FY08-445. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Cellular and Molecular Biolog

Silkworm Coatomers and Their Role in Tube Expansion of Posterior Silkgland

Background: Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgito-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation. Methodology/Principal Findings: Here, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (a,b,b9, d, e, and f-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of a-, b9- and c-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of a-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion. Conclusions/Significance: The initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and woul

Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

BACKGROUND: RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. METHODOLOGY/PRINCIPAL FINDINGS: The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. CONCLUSIONS: Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants

Idiopathic sclerosing inflammation of the orbit: A case of steroid-responsive disease in a patient with autoimmune hemolytic anemia

Purpose: Idiopathic sclerosing orbital inflammation (ISOI) has been categorized by some authors as a unique clinicopathologic entity, separate from the heterogenous group of disorders known collectively as nonspecific orbital inflammation. Histologic similarity and clinical association with other fibrosclerosing conditions has been shown. The authors present a case of ISOI in conjunction with autoimmune hemolytic anemia. Methods: A 59-year-old woman with a history of hemolytic anemia had left upper lid swelling, periocular pain, proptosis, and restriction of ocular motility. Magnetic resonance imaging (MRI) showed a homogenously enhancing lateral orbital mass. Biopsy revealed dense fibrous connective tissue with a paucicellular infiltrate, consistent with ISOI, and treatment with prednisolone 60 mg/day was instituted. Results: At 4-week review, the proptosis had settled and the patient regained full range of extraocular movements. At 14 months, the response was sustained and repeat MRI showed a 70% reduction in size of the mass. Conclusions. The first known case of ISOI and hemolytic anemia is presented. Despite dense fibrosis histologically, steroid responsiveness can be encountered in cases of ISOI. An early, aggressive approach to management is recommended, and corticosteroids should be considered as a treatment option.K.J. Billing, J. Hsuan and D. Selv