org/tools/protparam.html) [29]. Secondary structure of Fein-Penaeidin was predicted using GOR IV [30]. GOR IV uses all possible pair frequencies within a window of 17 amino acid residues and cross-validates on a data base of 267 proteins. The protein sequence of Fein-Penaeidin was submitted to the full-chain structure prediction server ROBETTA [31], [32] and [33] and visualized using PyMOL. Robetta provides both ab initio and comparative models of protein domains. It uses the ROBETTA fragment insertion method [34]. Domains Palbociclib without a detectable PDB homolog are modeled with the Rosetta

de novo protocol [35]. Comparative models are built from Parent PDBs detected by UW-PDB-BLAST or HHSEARCH and aligned by various methods which include HHSEARCH, Compass, and Promals. Loop regions are assembled from fragments and optimized to fit the aligned template structure [36]. Models so produced were ranked on Structural Analysis and Verification Server (SAVES). Models were evaluated on basis of the geometrical and stereo chemical constraints using

ProCheck [37] and factors such as unfavorable atomic contacts, side chain planarity problems; connections to aromatic rings out of plane etc. GW786034 were assessed using What Check (WhatIf) [38]. Ramachandran plot statistics [39] were used to evaluate the best model. The root mean square deviation (RMSd) values were calculated using the modeler by fitting the carbon backbone of the predicted. Five models were predicted using different templates among those that showed the good resolution factor and R-factor was used. In order to study the Fein-Penaeidin

expression in different organs Tolmetin of F. indicus, total RNA was isolated from the hemocyte, heart, gills, muscles, hepatopancreas, intestine and eyestalk using TRIzol reagent according to manufacturer instructions. The first-strand cDNAs were synthesized from 2 μg of the total RNA using reverse transcriptase enzymes. Comparative analysis of Fein-Penaeidin gene expression was achieved by qRT-PCR using an ABI PRISM 7500 Sequence Detection System (Model 7500, Applied Biosystems, Foster City, CA, USA), and QuantiTect® SYBR Green qRT-PCR reagents (Qiagen). Total RNA was purified and a 3 μl aliquot from each sample was used as template for a 25 μl one-step RT-PCR reaction with final a primer concentration of 0.4 μM. The reaction conditions were set-up as follows: initial denature step for 5 min at 94 °C, 40 cycles of denaturing (94 °C for 5 s), annealing (60 °C for 10 s), and extending (72 °C for 10 s). Fluorescent detection was performed after each extension step. All samples were run in triplicate with a standard curve of serially diluted F. indicus pooled RNA. Ct values were calculated for experimental samples and compared to the standard curve to determine the amount of RNA for each gene.