Fruit Flies (<em>Drosophila spp.</em>) Collection, Handling, and Maintenance: Field to Laboratory

As drosophilids are versatile, low maintenance and non-harming model organisms, they can be easily used in all fields of life sciences like Genetics, Biotechnology, Cancer biology, Genomics, Reproductive biology, Developmental biology, Micro chemical studies, ecology and much more. For using such a model organism, we need to learn capturing, rearing and culturing their progeny along with basic identification and differentiation between males and females. This chapter is being emphasized on techniques of capturing these flies with different and effective techniques. Along with it, most species-specific baits are discussed to catch more yield. Culture food media, a set measurement of different ingredients is used to rear the collected sample. The reasons for using each ingredient are also discussed in this chapter. At last, this chapter highlights the basic clues to identify different species in the field and lab along with learning distinguishing characteristics of males and females easily and effectively

Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application

Plasmodium vivax Tryptophan-Rich Antigen PvTRAg33.5 Contains Alpha Helical Structure and Multidomain Architecture

Tryptophan-rich proteins from several malarial parasites have been identified where they play an important role in host-parasite interaction. Structural characterization of these proteins is needed to develop them as therapeutic targets. Here, we describe a novel Plasmodium vivax tryptophan-rich protein named PvTRAg33.5. It is expressed by blood stage(s) of the parasite and its gene contains two exons. The exon 1 encodes for a 23 amino acids long putative signal peptide which is likely to be cleaved off whereas the exon 2 encodes for the mature protein of 252 amino acids. The mature protein contains B-cell epitopes which were recognized by the human immune system during P.vivax infection. The PvTRAg33.5 contains 24 (9.5%) tryptophan residues and six motifs whose patterns were similar among tryptophan-rich proteins. The modeled structure of the PvTRAg33.5 consists of a multidomain architecture which is stabilized by the presence of large number of tryptophan residues. The recombinant PvTRAg33.5 showed predominantly α helical structure and alpha helix to beta sheet transition at pH below 4.5. Protein acquires an irreversible non-native state at temperature more than 50°C at neutral pH. Its secondary and tertiary structures remain stable in the presence of 35% alcohol but these structures are destabilized at higher alcohol concentrations due to the disturbance of hydrophobic interactions between tryptophanyl residues. These structural changes in the protein might occur during its translocation to interact with other proteins at its final destination for biological function such as erythrocyte invasion

Conservation and Role of Electrostatics in Thymidylate Synthase

International audienceConservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome. The electrostatic potential of a protein plays a crucial role in steering ligands to their binding sites, and orienting them correctly for binding 1. In enzymes, the active site electrostatic potential is important for stabilizing the transition state and thereby catalyzing the reaction 2. Therefore, conservation of protein function across a protein family is often accompanied by conservation of the electrostatic potential in the active site region, even though the rest of the protein may lack a conserved electrostatic potential 3,4. Consequently, comparison of protein electrostatic potentials has been employed as a tool to predict protein function and to derive similarities in protein function across protein families 5–7. Optimizing the electrostatic complementarity between a ligand and the binding site of a protein is also an important aspect in drug design 8,9 and may provide a route to gain target selectivity 10

Synthesis, biological evaluation and molecular docking of aryl hydrazines and hydrazides for anticancer activity

265-268Aryl hydrazine and hydrazide analogues were synthesized based on p-tolyl hydrazine, isolated as a breakdown product of a secondary metabolite from the mushroom, Agaricus bisporus, and tested to be highly active molecule than 5-fluorouracil in in vitro anticancer studies. The synthesized analogues were tested for anticancer activity using NCI protocol. Anolgues 12 and 15 emerged as molecules with significant in vitro anticancer activity. Molecular docking study revealed the binding orientations of aryl hydrazines and hydrazides analogues in the active sites of thymidylate synthase

Structure-Based Optimization of the Terminal Tripeptide in Glycopeptide Dendrimer Inhibitors of Pseudomonas aeruginosa Biofilms Targeting LecA

The galactopeptide dendrimer GalAG2 ((-Gal-OC6H4CO-Lys-Pro-Leu)(4)(Lys-Phe-Lys-Ile)(2)Lys-His-Ile-NH2) binds strongly to the Pseudomonas aeruginosa (PA) lectin LecA, and it inhibits PA biofilms, as well as disperses already established ones. By starting with the crystal structure of the terminal tripeptide moiety GalA-KPL in complex with LecA, a computational mutagenesis study was carried out on the galactotripeptide to optimize the peptide-lectin interactions. 25 mutants were experimentally evaluated by a hemagglutination inhibition assay, 17 by isothermal titration calorimetry, and 3 by X-ray crystallography. Two of these tripeptides, GalA-KPY (dissociation constant (K-D)=2.7M) and GalA-KRL (K-D=2.7M), are among the most potent monovalent LecA ligands reported to date. Dendrimers based on these tripeptide ligands showed improved PA biofilm inhibition and dispersal compared to those of GalAG2, particularly G2KPY ((-Gal-OC6H4CO-Lys-Pro-Tyr)(4)(Lys-Phe-Lys-Ile)(2)Lys-His-Ile-NH2). The possibility to retain and even improve the biofilm inhibition in several analogues of GalAG2 suggests that it should be possible to fine-tune this dendrimer towards therapeutic use by adjusting the pharmacokinetic parameters in addition to the biofilm inhibition through amino acid substitutions

Novel approaches for targeting thymidylate synthase to overcome the resistance and toxicity of anticancer drugs

International audienc

CH−π “T-Shape” Interaction with Histidine Explains Binding of Aromatic Galactosides to Pseudomonas aeruginosa Lectin LecA

The galactose specific lectin LecA mediates biofilm formation in the opportunistic pathogen P. aeruginosa . The interaction between LecA and aromatic &beta;-galactoside biofilm inhibitors involves an intermolecular CH-&pi; T-shape interaction between C(&epsilon;1)-H of residue His50 in LecA and the aromatic ring of the galactoside aglycone. The generality of this interaction was tested in a diverse family of &beta;-galactosides. LecA binding to aromatic &beta;-galactosides (KD &sim; 8 &mu;M) was consistently stronger than to aliphatic &beta;-galactosides (KD &sim; 36 &mu;M). The CH-&pi; interaction was observed in the X-ray crystal structures of six different LecA complexes, with shorter than the van der Waals distances indicating productive binding. Related XH/cation/&pi;-&pi; interactions involving other residues were identified in complexes of aromatic glycosides with a variety of carbohydrate binding proteins such as concanavalin A. Exploiting such interactions might be generally useful in drug design against these targets