Functional characterisation of the Sterile 20 like kinase Slik in tracheal morphogenesis in Drosophila melanogaster

The Drosophila Sterile20 like kinase Slik is involved in maintaining epithelial integrity and promotes tissue growth during development. It regulates activity of members of the band 4.1/Ezrin/Radixin/Moesin (ERM) superfamily proteins through phosphorylation. Apart from its kinase activity, Slik also interacts with Raf to promote cell survival and growth. Raf is an important downstream effector of the Bnl/Btl RTK pathway crucial for tracheal development. An immediate target of the RTK-MAPK signalling is SRF (serum response factor), a transcription factor known to be indispensible for terminal cell development. Here, I show that Slik contributes to tracheal terminal cell development through both its kinase-dependent and independent functions. Both Slik and activated Moesin (p-Moesin) are enriched at the apical membrane in terminal cells. slik mutant or knockdown terminal cells show branching defects and destabilised tubes similar to the phenotype of moesin mutants, suggesting that slik is an essential factor in terminal cell growth and development. This is further supported by the effect of expressing a kinase-dead form of slik, which causes a multilumen phenotype similar as the one seen in slik mutant cells. In addition, slik depletion results in the loss of p-Moesin at the apical membrane in the terminal cells indicating that Slik through its kinase dependent function towards Moesin regulates tracheal terminal cell development. This study also reports a novel regulator of Moesin; I have identified Btl as an important factor that post-translationally regulates the phosphorylation of Moesin. Apart from the luminal defects, slik depletion also resulted in reduced branching of terminal cells. The same phenotype is observed upon knockdown of raf. As Raf is thought not be a kinase substrate of Slik but rather a binding partner, the results suggest an additional, kinase independent function of Slik in tracheal development. The disruption of the downstream target of the Bnl/Btl signalling pathway srf, the signalling transducer Ras, or the receptor btl itself also resulted in similar branching defects. We propose that slik acts in the development of terminal cells through activation of Moesin at the apical membrane and a possible regulation of the Bnl/Btl RTK pathway through its interaction with Raf

A STUDY ON EFFECT OF INDOLE AS A SUBSTITUENT ON A KETO-ENOL TAUTOMER: A SYNTHETIC APPROACH ON β-DIKETONE

Objective: The existence of keto-enol tautomerism in β-diketones can typically study by a choice of analytical technique. The position of the keto-enol equilibrium depends on a number of factors like solvent, temperature, and substituents. Here an attempt was made to examine the effect of indole, a heterocyclic moiety with the moderately high polar surface area to examine its effect on ketonisation of β-diketone.Methods: The β-diketone studied and synthesized is a structural analog of magical drug curcumin. The structural influence of indole on ketonisation of β-diketone is studied to give a hypothesis on factors contributing towards ketonisation. This work involves the synthesis of 6-(1H-Indol-3-yl)-hex-5ene-2, 4-dione and the study on the single crystal structure of indole-3-carboxaldehyde, major functional component to result in the reaction. The tautomer was studied for its ability to bind with tetrahydrofolate reductase enzyme using Discovery Studio 3.5 version to differentiate the pharmacological significance of conformations.Results: The single crystal XRD structure of this compound was deposited in Cambridge crystallographic data center bearing CCDC No.1536311. The structural characterization of synthesized ligand was carried out by using IR, Mass, 1H NMR spectroscopic techniques. The docking study reveals that keto isomer found to exhibit more inhibition of the enzyme tetrahydrofolate reductase hence more pharmacologically active.Conclusion: The experimental evidence proves that indole substitution shifted the keto-enol equilibrium towards keto form of 6-(1H-Indol-3-yl)-hex-5ene-2, 4-dione

Chromyl chloride oxidation of cyclic alkenes and chemical constituents of Letharia Vulpina (L) HUE

Bibliography: p. 144-145

Slik and the Receptor Tyrosine Kinase Breathless Mediate Localized Activation of Moesin in Terminal Tracheal Cells

A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. Here we show that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. Our results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway

EOG Based Prosthetic Arm-Hand Control

ABSTRACT: Electrooculography (EOG) based HID (Human Interactive Device) has the potential to control a Robotic hand. The project presents a novel method to control robotic hand with the help of eye movements. Using this system, we can control the hand movements left, right, pick and place etc. As a result of road traffic accidents, stroke, etc., a lot of people have become disabled following which many of them have lost their ability to control their environment and communicate with others by conventional methods. Studies on such groups of persons with severe disabilities have shown that many of them retain the ability to control their eye movements which could be used to develop a new robotic hand control. This system can be used by persons with disabilities for environmental control, as a source of information as well as for entertainment. The device can be found most useful by handicapped people who can no longer control the robotic hand using their hands. As the device relies on user's eye movements, it can be used even by patients who are paralyzed from shoulder downwards. In recent years, EOG based HID is becoming the hotspot of bio-based HID research

Breathless-mediated regulation of Moesin is specific for terminal cells. (A–B″) total Moesin in control (A′) and <i>btl</i>-RNAi (B′) expressing terminal cells.

<p>(C–D″) Slik staining in control (C′) and <i>btl</i>-RNAi (D′) expressing terminal cells. Depleting Btl does not affect total Moesin (B′) or Slik (D′) in terminal cells. (E–F″) pMoesin staining in control and <i>btl</i>^LG19 mutant embryos. pMoesin localizes apically in control (E′) and <i>btl</i>^LG19 (F′) mutant embryonic tracheal branches. The insets in F′ and F″ are zoomed images from the boxed regions. (G–I″) pMoesin staining in control (G), <i>btl-</i>depleted (H) and <i>slik</i>-depleted (I) larval dorsal trunk, tracheoblasts and fusion cells (boxed regions in G′ and H′). pMoesin staining in <i>btl-</i>depleted cells is comparable to the control, whereas it is absent in <i>slik</i>-depleted cells. A-I″ are projections of confocal image stacks. Scale bars: (A–D″) 50 µm, (E–I″) 25 µm.</p

Breathless regulates pMoesin at the luminal membrane in terminal branches.

<p>(A–D″) pMoesin staining in control terminal cells (A–A″), and terminal cells expressing <i>btl</i>-RNAi (B-B″), <i>slik</i>-RNAi (C–C″) or <i>egfr</i>-RNAi (D–D″). pMoesin staining is absent in <i>btl</i>-RNAi (B′) and in <i>slik</i>-RNAi cells (C′). In <i>egfr</i>-RNAi cells (D′) pMoesin is present at levels comparable to control cells (A′). In both Btl and EGFR-depleted terminal cells branch numbers are significantly reduced (E, P value <0.0001 by two-tailed T test in both cases). In 63% of Btl-depleted cells (N = 19) pMoesin was absent, 27% showed pMoesin staining. Genotypes of crosses and number of terminal cells scored: Blue (control): <i>btl</i>-GAL4, UAS-GFP (N = 11). Red: <i>btl</i>-RNAi (N = 11). Violet: <i>egfr</i>-RNAi (n = 10). A–D″ are projections of confocal image stacks. Scale bars: (A–D″) 25 µm.</p

Overactivation of FGF signaling overrides requirement for Slik in terminal cell branching.

<p>(A–D) Moesin (A, B) and Slik (C–D) staining in larvae expressing constitutively active FGFR alone (λ<i>btl</i>, A′) or in combination with <i>slik</i>-RNAi (B′). λ<i>btl</i>, induces excessive branching; pMoesin and Slik staining are detectable in the λ<i>btl</i> expressing cells, (A′, C′) but absent if <i>slik</i>-RNAi is co-expressed(B′, D′). Depletion of Slik in λ<i>btl</i> expressed terminal cells does not affect expression or membrane localization of total Moesin (E and F′). A–E″ are projections of confocal image stacks. F–F″ are single focal planes from a image stack. Scale bars: (A–E″) 30 µm, (F–F″) 5 µm.</p

Slik is necessary for terminal cell development.

<p>(A–D) The terminal cells are labeled with cytoplasmic GFP (green). Examples of control (A), <i>slik</i>¹ mutant (B, C) and <i>slik</i>-RNAi (D) expressing terminal cells in third instar larvae. (E) <i>slik</i>¹ mutant and <i>slik</i>-RNAi expressing terminal cells have a reduced number of branches (P value <0.0001 by two tailed T test) compared to control cells. (G–J) Slik is expressed and is apically enriched in tracheal cells during different stages of embryonic development. Tracheal cells (H, I and J) are labeled by immunostaining for Dof. (J′) Slik expression in third instar larval terminal branch labeled with cytoplasmic GFP. Slik is distributed in the cytoplasm with an enrichment at the luminal membrane (J′). A–D and G–I″ are projections of confocal image stacks. (J–J″) is a single focal plane from a confocal image stack. All constructs (GFP, RNAi) are expressed under the control of the <i>btl</i>-GAL4, UAS-GFP transgene (control). Scale bars: (A–B) 50 µm, (G–I″) 10 µm, (J–J″) 5 µm.</p

Depletion Moesin does not suppress the FGF signaling overactivation phenotype in terminal cells.

<p>(A–B″) Total Moesin staining in larvae expressing constitutively active FGFR alone (λ<i>btl</i>, A′) or in combination with <i>moe</i>-RNAi (B′) Moesin staining is detectable in the control (A′) but not in <i>moe</i>-RNAi; λ<i>btl</i> cells(B′). (C–D″) pMoesin staining in larvae expressing constitutively active FGFR alone (λ<i>btl</i>, C) or in combination with <i>moe</i>-RNAi (D′). pMoesin is absent or reduced in the <i>slik</i>-RNAi; λ<i>btl</i> cells (D′). A–D″ are projections of confocal image stacks. Scale bars: (A–D″) 40 µm.</p