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Mitosis and endoreplication are suppressed in <i>DBHD^−/−</i> larvae.

<p>PH3 marks the mitotic cells. EdU marks the cells undergoing DNA synthesis. DAPI marks the nuclei. (A and B): Eye imaginal discs. (C, D, K, L): Brains. (E, F, I, J): Fat bodies. (G and H): Salivary glands. The sibling heterozygotes (−/+) were taken as the wild-type controls. Note all the <i>DBHD^−/−</i> samples (−/−) are reduced in size, the polyploidy are also reduced in cells from fat body (F) and salivary gland (H).</p

Generating a <i>DBHD</i> null allele.

<p>(A) The <i>DBHD</i> genomic locus and the targeting strategy. P1–P4 represent the PCR primers. (B) PCR analysis of the genomic DNA. In <i>DBHD^−/−</i> larvae (−/−), a 4.8 kb fragment and a 5 kb fragment could be amplified by the corresponding primer pairs. (C) rtPCR analysis of the <i>DBHD</i> transcripts in various tissues. LB: larval brain; disc: mixtures of larval imaginal discs. α-tubulin (at 84B) was used as the positive control. (D) A DBHD antibody recognizes a band at about 55 kDa (arrow) of the whole larval extracts, which is absent in the <i>DBHD^−/−</i> larvae. <i>w¹¹¹⁸</i> was used as the wild-type control (WT). (E) Statistical analysis of the developmental profiles of two strains. The +/TM3 flies contains a healthy 3rd chromosome and a GFP-marked third chromosome balancer (<i>TM3, Kr::GFP</i>). The −/TM3 flies contains the same balancer and the <i>DBHD⁻</i> allele. Animals survived to different stages were counted. Numbers in the parenthesis are the theoretical values according to the Mendel rules. *: all the survived adults are heterozygotes. (F) Comparison of <i>DBHD^−/−</i> (−/−) and the sibling heterozygotes (−/+) at different days after egg laying. Embryos collected within three hours and cultured in the same food vials were picked for images at each time point. All heterozygotes have eclosed by 14 days after egg laying and thus were not pictured.</p

Clonal analyses of <i>DBHD^−/−</i> cells in the larval imaginal discs.

<p>(A–C) GFP signals. (C’) PH3. Random clones were generated in the wing (a) and eye (b) imaginal discs. <i>eyeless-flipase</i> induced large clones in the eye disc (C, C’). The <i>DBHD^−/−</i> cells are absent of GFP (green) and circled with solid lines. The wild-type twin spot cells are marked with double GFP signals and enclosed with dashed lines. Note the <i>DBHD^−/−</i> and the twin spots are similar in clone size. The amount of PH3-positive cells is not clearly declined or increased in the <i>DBHD⁻</i> clones.</p

Autophagy is elevated in the <i>DBHD^−/−</i> larvae.

<p>(A–C) LysoTracker staining (red) of unfixed larval fat bodies. The GFP-positive tissues (green) are from heterozygotes (<i>Kr::GFP</i>). (A) The LysoTracker signal is much stronger in the <i>DBHD^−/−</i> (−/−) fat bodies than in the heterozygotes (−/+). The 4-day-old larvae were picked form the same food vial, processed in the same staining tube and imagined in one optical field. (B) LysoTracker signal became strong in the heterozygotes starved for 3 hours by supplying with distilled water only. The same staining of fat body from starved <i>DBHD^−/−</i> is also shown. (C) 3-Methyladenine (3-MA) suppresses autophagy in <i>DBHD^−/−</i> fat body. (D) Foraging assay by feeding larvae with colored food (the baker’s yeast powder mixed with black ink). Green: GFP (A–C); Blue: DAPI (C).</p

Rescue of the <i>DBHD^−/−</i> growth defects by nutrients.

<p>(A) <i>DBHD^−/−</i> larvae were sensitive to yeast supply. NF: normal food; Star: starvation, normal food without yeast. All samples were picked at Day 8 after hatching from eggs. (B) The developmental profiles of flies cultured on two kinds of nutritious foods. yeast paste: the baker’s yeast powder was mixed with water and supplied on agar plate. leucine: normal food supplemented with 100 mM leucine. Fifty newly hatched larvae for each genotype were picked. Numbers of the heterozygotes (left) and <i>DBHD^−/−</i> were separated by slashes. (C) Examples of four dead <i>DBHD^−/−</i> pharates cultured on yeast pastes. (D) Rescue effects of leucine. Animals aged for 7 days after hatching were imaged. NF: normal food; leu: normal food with 100 mM leucine; rapa: normal food with 1 µM rapamycin; leu+rapa: normal food with 100 mM leucine and 1 µM rapamycin. (D) Free amino acids analysis of the larvae. The amino acids levels are displayed as milligram per gram of body weight (mg/g). The amounts of larvae for each experiment are listed in the parenthesis.</p

Expression patterns of <i>DBHD-res</i>.

<p>The expression of <i>DBHD-res</i> was detected by the EGFP staining (green). Genotype in all panels: <i>DBHD-res</i>; <i>DBHD^−/−</i>. (A) Larval fat body. (B) Larval salivary gland. (C) eye-antennal disc. (D) Magnified view of eye disc. (E) Larval brain. (F) Ring glands. (G) Epithelium of larval midgut. (H) Epithelium of adult midgut. (I) Magnified view of adult midgut. (J) Testis. (K) Germarium. (L) Egg follicles. Arm (red) marks the cell borders; Prospero (Pros, red, in nuclei) marks the intestinal EE cells; Dpn marks the neuroblasts in the brains; DAPI (blue) marks the nuclei in G–I, L. Note the <i>DBHD-res</i> was also expressed in some EC cells in the gut (E–G, marked by the DAPI-labeled polyploid cells).</p

The developmental profiles of flies cultured on various yeast foods.

<p>Note: The developmental profiles were displayed by listing the last stages that they survived and the time point when they started to enter (days after hatching). Pure yeast paste had the best rescue effects. Diluted food means the food nutrients (sugar, corn flour and baker’s yeast) were 50% of the normal food recipe. Yeast-free food has normal food recipe without yeast. In all test, we picked the newly hatched larvae at the same time point. No more than twenty larvae were cultured within each food chamber. At least 100 larvae in total were counted for each experiment.</p

The human FLCN could partially rescue the <i>DBHD^−/−</i> larvae.

<p>(A) Dorsal view of pupae. The heterozygote (−/+) is revealed by <i>Sb</i> (marked with short and thick bristles on the notum, arrow), see materials and methods for the cross scheme. (B) The genotypes of the pupae were confirmed by PCR analysis of genomic DNA. The fly CG10414 gene was used as a positive control.</p

Communicating Functional Agents and their Application to Graphical User Interfaces

We demonstrate how concepts of communicating agents can be integrated into purely functional languages by an orthogonal extension of the usual I/O monad. These agents communicate via so-called service access points and support programming in the style of client-server architectures. We then show the feasibility of the approach by applying it to the example of graphical user interfaces, which we consider to be a typical instance of reactive systems. For this purpose we develop the concept of so-called gates, which serve as a mediator between user events and the application logic. It turns out that the combination of functional expressiveness and concurrency yields a powerful framework for the realization of reactive systems such as graphical user interfaces. All concepts discussed in this paper are represented in the functional language Opal and have been implemented in the Opal programming environment