Identification and Characterization of Genes Required for Compensatory Growth in Drosophila

To maintain tissue homeostasis, some organs are able to replace dying cells with additional proliferation of surviving cells. Such proliferation can be localized (e.g., a regeneration blastema) or diffuse (compensatory growth). The relationship between such growth and the growth that occurs during development has not been characterized in detail. Drosophila melanogaster larval imaginal discs can recover from extensive damage, producing normally sized adult organs. Here we describe a system using genetic mosaics to screen for recessive mutations that impair compensatory growth. By generating clones of cells that carry a temperature-sensitive cell-lethal mutation, we conditionally ablate patches of tissue in the imaginal disc and assess the ability of the surviving sister clones to replace the lost tissue. We have used this system together with a modified whole-genome resequencing (WGS) strategy to identify several mutations that selectively compromise compensatory growth. We find specific alleles of bunched (bun) and Ribonucleoside diphosphate reductase large subunit (RnrL) reduce compensatory growth in the imaginal disc. Other genes identified in the screen, including two alleles of Topoisomerase 3-alpha (Top3α), while also required for developmental growth, appear to have an enhanced requirement during compensatory growth. Compensatory growth occurs at a higher rate than normal growth and may therefore have features in common with some types of overgrowth. Indeed, the RnrL allele identified compromises both these types of altered growth and mammalian ribonucleotide reductase and topoisomerases are targets of anticancer drugs. Finally, the approach we describe is applicable to the study of compensatory growth in diverse tissues in Drosophila

A Buoyancy-Based Screen of Drosophila Larvae for Fat-Storage Mutants Reveals a Role for Sir2 in Coupling Fat Storage to Nutrient Availability

Obesity has a strong genetic component, but few of the genes that predispose to obesity are known. Genetic screens in invertebrates have the potential to identify genes and pathways that regulate the levels of stored fat, many of which are likely to be conserved in humans. To facilitate such screens, we have developed a simple buoyancy-based screening method for identifying mutant Drosophila larvae with increased levels of stored fat. Using this approach, we have identified 66 genes that when mutated increase organismal fat levels. Among these was a sirtuin family member, Sir2. Sirtuins regulate the storage and metabolism of carbohydrates and lipids by deacetylating key regulatory proteins. However, since mammalian sirtuins function in many tissues in different ways, it has been difficult to define their role in energy homeostasis accurately under normal feeding conditions. We show that knockdown of Sir2 in the larval fat body results in increased fat levels. Moreover, using genetic mosaics, we demonstrate that Sir2 restricts fat accumulation in individual cells of the fat body in a cell-autonomous manner. Consistent with this function, changes in the expression of metabolic enzymes in Sir2 mutants point to a shift away from catabolism. Surprisingly, although Sir2 is typically upregulated under conditions of starvation, Sir2 mutant larvae survive better than wild type under conditions of amino-acid starvation as long as sugars are provided. Our findings point to a Sir2-mediated pathway that activates a catabolic response to amino-acid starvation irrespective of the sugar content of the diet

Identification of anti-cyanobacterial leads targeting carbonic anhydrase from phytochemical database using <i>in silico</i> approach

In cyanobacteria, carbonic anhydrase (zinc metalloenzyme) is a major enzyme that converts CO2 to HCO3 maintaining the carbon concentration around the vicinity of RuBisCo, leading to cyanobacterial biomass generation. Anthropogenic activities, disposal of leached micro nutrients effluents from industries into the aquatic environment results in cyanobacterial blooms. The harmful cyanobacteria release cyanotoxins in open-water system which on ingression through oral route causes major health issues like hepatotoxicity and immunotoxicity. A database was prepared consisting of approximately 3k phytochemicals curated from previous literatures, earlier identified by GC-MS analysis. The phytochemicals were subjected to online servers to identify the novel lead molecules which followed ADMET and drug-like candidates. The identified leads were optimized by density functional theory method using B3YLP/G* level of theory. Carbonic anhydrase chosen as target to observe the binding interaction through molecular docking simulations. From the molecules included in the database the highest binding energy exhibited by alpha-tocopherol succinate and mycophenolic acid were found to be -9.23 kcal/mol and -14.41 kcal/mol and displayed interactions with GLY A102, GLN B30, ASP A41, LYS A105 including Zn2+ and their adjacent amino acids CYS 101, HIS 98, CYS 39 in both chain A and chain A-B of carbonic anhydrase. The identified molecular orbitals decipher computed global electrophilicity values (Energy gap, electrophilicity and Softness) of alpha tocopherol succinate and mycophenolic acid were found to be (5.262, 1.948, 0.380) eV and (4.710, 2.805, 0.424) eV demonstrates both molecules are effective and stable. The identified leads may serve as a better anti-carbonic anhydrase agent because they accommodate in the binding site and hampers the catalytic activity of Carbonic anhydrase thus inhibiting the generation of cyanobacterial biomass. This identified lead molecules may serve as a substructure to design novel phytochemicals against carbonic anhydrase present in cyanobacteria. Further in vitro study is necessary to evaluate the efficacy of these molecules

Identification of anti-cyanobacterial leads targeting carbonic anhydrase from phytochemical database using in silico approach

In cyanobacteria, carbonic anhydrase (zinc metalloenzyme) is a major enzyme that converts CO 2 to HCO 3- main¬taining the carbon concentration around the vicinity of RuBisCo, leading to cyanobacterial biomass generation. Anthropogenic activities, disposal of leached micro nutrients effluents from industries into the aquatic en¬viron¬ment results in cyanobacterial blooms. The harmful cyanobacteria release cyanotoxins in open-water system which on ingression through oral route causes major health issues like hepatotoxicity and immunotoxicity. A database was prepared consisting of approximately 3k phytochemicals curated from previous literatures, earlier identified by GC-MS analysis. The phytochemicals were subjected to online servers to identify the novel lead molecules which followed ADMET and drug-like candidates. The identified leads were optimized by density functional theory method using B3YLP/G* level of theory. Carbonic anhydrase chosen as target to observe the binding interaction through molecular docking simulations. From the molecules included in the database the highest binding energy ex¬hibited by alpha-tocopherol succinate and mycophenolic acid were found to be −9.23 kcal/mol and −14.41 kcal/mol and displayed interactions with GLY A102, GLN B30, ASP A41, LYS A105 including Zn 2+ and their adjacent amino acids CYS 101, HIS 98, CYS 39 in both chain A and chain A-B of carbonic anhydrase. The Identified molecular orbitals decipher computed global electrophilicity values (Energy gap, electrophilicity and Softness) of alpha-tocopherol succinate and mycophenolic acid were found to be (5.262, 1.948, 0.380) eV and (4.710, 2.805, 0.424) eV demonstrates both molecules are effective and stable. The identified leads may serve as a better anti-carbonic anhydrase agent because they accommodate in the binding site and hampers the catalytic activity of Carbonic anhydrase thus inhibiting the generation of cyanobacterial biomass. This identified lead mole¬cules may serve as a substructure to design novel phytochemicals against carbonic anhydrase present in cyano¬bacteria. Further in vitro study is necessary to evaluate the efficacy of these molecules