Weak purifying selection in allelic diversity of the ADSL gene in indigenous and local chicken breeds and red junglefowl in Thailand.

High levels of purine and uric acid, which are associated with health issues such as gout and cardiovascular disease, are found in the meat of fast-growing broiler chickens, which raises concerns about the quality of chicken meat and the health of the consumers who consume it. High genetic homogeneity and uniformity, particularly in genes involved in the synthesis of inosine monophosphate (IMP) and subsequent process of purine synthesis, which are associated with the meat quality, are exhibited in commercial broiler chickens owing to intensive inbreeding programs. Adenosine succinate lyase (ADSL) is a key enzyme involved in de novo purine biosynthetic pathway and its genetic polymorphisms affect IMP metabolism and purine content. In this study, we investigated the polymorphism of the ADSL gene in indigenous and local chicken breeds and red junglefowl in Thailand, using metabarcoding and genetic diversity analyses. Five alleles with 73 single nucleotide polymorphisms in exon 2, including missense and silent mutations, which may act on the synthesis efficiency of IMP and purine. Their protein structures revealed changes in amino acid composition that may affect ADSL enzyme activity. Weak purifying selection in these ADSL alleles was observed in the chicken population studied, implying that the variants have minor fitness impacts and a greater probability of fixation of beneficial mutations than strong purifying selection. A potential selective sweep was observed in Mae Hong Son chickens, whose purine content was lower than that in other breeds. This suggests a potential correlation between variations of the ADSL gene and reduced purine content and an impact of ADSL expression on the quality of chicken meat. However, further studies are required to validate its potential availability as a genetic marker for selecting useful traits that are beneficial to human health and well-being.

Novel Vaccine Development for Fish Culture Based on the Multiepitope Concept.

For the past several decades, aquaculture all around the world have been retarded by various disease outbreaks caused by many pathogens including parasites, bacteria, and viruses. Apart from being harmful to human health, the emerging diseases also dramatically affect the farm animals such as livestock and aquatic animals. To cope with this problem, one of the effective prophylactic measures is the application of vaccine. However, the traditional vaccines still have some limitations and several drawbacks; thus there is a need for the development of novel advanced vaccine such as chimeric multiepitope vaccine. Based on the current understanding of genomics and immunoproteomics together with the present bioinformatics tools, the researchers can identify the potential targeted epitopes being recognizable by the immune cells. Additionally, another critical point that should be considered for designing the chimeric multiepitope vaccine is the exposure of all those epitopes to the host organism. Thus, selecting an appropriate linker and joining each identified epitope in a suitable site can create the ideal protein structure protruding all the selected epitopes on its surface. Herein, our study would provide the fundamental platform to develop the multiepitope B-cell vaccine for the prevention and control of the aquatic animal disease starting with the epitope prediction until in vivo testing the multiepitope vaccine efficacy.

In-vitro Studies of Anti-EGFR Tyrosine Kinase Activity of Thai nutraceutical Plants.

Functional foods have emerged as a new approach to improve human health in term of nutraceutical to prevent people from illness rather than cure patients through medical treatment. In Asian society, particularly in Thailand, the utilizations of functional ingredients have been integrated in every parts of ordinary life. In this study, the tyrosine kinase activity of epidermal growth factor receptor (EGFR) inhibiting properties of 23 Thai's herbs-ethanol extracts have been examined. The crude extracts of only four species that inhibit the activity of EGFR-tyrosine kinase, Azadirachta indica (neem, Sa-dao), Brucea javanica (L.) Merr. (Rajadad), Hibiscus sabdariffa L. (Roselle, Krachiap daeng), and Saccharum chinensis Roxb. (Red sugar cane). Moreover, only ethanol extractions from A. indica and B. javanica were also showed antitumor effect to non-small cell lung cancer, A549 cells.

Novel Chimeric Multiepitope Vaccine for Streptococcosis Disease in Nile Tilapia (Oreochromis niloticus Linn.).

Streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including Nile tilapia (Oreochromis niloticus Linn.). Vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. Immunoproteomics analysis of S. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. Epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable RNA and protein structure for protein expression. 45F2 and 42E2 were identified as the best candidates for a chimeric multiepitope vaccine. Recombinant plasmids were constructed to produce a recombinant protein vaccine and DNA vaccine system. Overexpressed proteins were determined to be 30 kDa and 25 kDa in the E. coli and TK1 systems, respectively. The efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and DNA vaccine was evaluated in Nile tilapia, followed by S. agalactiae challenge at 1 × 107 CFU/mL. Relative percentage survival (RPS) and cumulative mortality were recorded at approximately 57-76% and 17-30%, respectively. These chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases.

FeptideDB: A web application for new bioactive peptides from food protein.

BACKGROUND: Bioactive peptides derived from food are important sources for alternative medicine and possess therapeutic activity. Several biochemical methods have been achieved to isolate bioactive peptides from food, which are tedious and time consuming. In silico methods are an alternative process to reduce cost and time with respect to bioactive peptide production. In this paper, FeptideDB was used to collect bioactive peptide (BP) data from both published research articles and available bioactive peptide databases. FeptideDB was developed to assist in forecasting bioactive peptides from food by combining peptide cleavage tools and database matching. Furthermore, this application was able to predict the potential of cleaved peptides from 'enzyme digestion module' to identify new ACE (angiotensin converting enzyme) inhibitors using an automatic molecular docking approach. RESULTS: The FeptideDB web application contains tools for generating all possible peptides cleaved from input protein by various available enzymes. This database was also used for analysis and visualization to assist in bioactive peptide discovery. One module of FeptideDB has the ability to create 3-dimensional peptide structures to further predict inhibitors for the target protein, ACE (angiotensin converting enzyme). CONCLUSIONS: FeptideDB is freely available to researchers who are interested in exploring bioactive peptides. The FeptideDB interface is easy to use, allowing users to rapidly retrieve data based on desired search criteria. FeptideDB is freely available at http://www4g.biotec.or.th/FeptideDB/. Ultimately, FeptideDB is a computational aid for assessing peptide bioactivities.

The potential peptides against angiotensin-I converting enzyme through a virtual tripeptide-constructing library.

Peptides derived from food proteins are promising bioactive source for inhibiting Angiotensin-I converting enzyme (ACE) activity. Bioactive peptides (BP) have received much attention, particularly from the pharmaceutical industry. As they not only own potent properties but also possess less side-effects than synthetic drugs. In this work, an 8000 possible tripeptides library was constructed to predict the potential ACE inhibitory peptides by using in silico tools. GOLD molecular docking was then applied to determine the binding mode of action between ACE and each of tripeptide from this in-house library. The first 662 high-ranking tripeptides by ChemScore were chosen to create association rules of tripeptides-ACE complexes. An orientation pattern of amino acid in the binding tunnel of ACE has been examined by frequency analysis. The association rules (confident values over 90%) illustrated that hydrophobic factor has been displayed as main components in the ACE tripeptides inhibitor from four factors in equation, hydrophobic, aromatic, polar, charged. According to in silico output, five tripeptides were chosen to test in vitro study of ACE-inhibitory activity. The half-maximal inhibitory concentration (IC50) of these selective five peptides, WCW, IWW, WWW, WWI and WLW for inhibiting ACE were 49.50 ±â€¯3.88 µM, 489.14 ±â€¯8.84 µM, 536.02 ±â€¯38.57 µM, 752.91 ±â€¯41.89 µM and 1783 ±â€¯0.113 µM, respectively. Molecular dynamics simulations approach was applied to study the interaction of WCW (Trp-Cys-Trp) within ACE pocket site. This ligand was stabilized by strong hydrogen bonding interactions with ACE active site, Tyr523-Trp'1 (99.76%) and His353-Trp'1 (95.68%). Our computational protocol could be considered as a new tool for identifying active peptide against ACE from hydrolysated peptides of natural sources.

Bioinformatics characterization of a cathepsin B transcript from the giant river prawn, Macrobrachium rosenbergii: Homology modeling and expression analysis after Aeromonas hydrophila infection.

Cathepsin B is a lysosomal proteolytic enzyme that has been suggested to play a role in pathological processes of immune system. In this study, the full-length cDNA sequence of cathepsin B transcript in the giant river prawn Macrobrachium rosenbergii (MrCTSB) was obtained from 454 pyrosequencing of cDNAs from hepatopancreas and muscle. It was 1158 bp in length, containing an open reading frame (ORF) of 987 bp corresponding to 328 amino acids. The predicted molecular mass and pI of MrCTSB protein was 36.04 kDa and 4.73. The major characteristics of MrCTSB protein consisted of a propeptide of C1 peptidase family at the N-terminus and a cysteine protease (Pept_C1) domain at the C-terminus. The 3-dimentional structure of MrCTSB was constructed by computer-assisted homology modeling. The folding of MrCTSB was highly conserved to human CTSB structure and the modeled MrCTSB displayed characteristics of cysteine proteinases superfamily. The docking study was performed to investigate binding interactions between known inhibitors against MrCTSB. Known inhibitors were oriented in the groove of catalytic site cleft. They bound to subsites from S2, S1, S1', and S2', respectively, with key residues in each subsite. Challenge of juvenile prawns with Aeromonas hydrophila revealed that the MrCTSB transcript in hepatopancreas significantly increased at 60-96 h post injection (hpi). This suggested that MrCTSB may play roles in innate immunity of M. rosenbergii. Our results provide useful information for a more comprehensive study in immune-related functions of MrCTSB.

cDNA sequence and protein bioinformatics analyses of MSTN in African catfish (Clarias gariepinus).

Myostatin, also known as growth differentiation factor 8, has been identified as a potent negative regulator of skeletal muscle growth. The purpose of this study was to characterize and predict function of the myostatin gene of the African catfish (Cg-MSTN). Expression of Cg-MSTN was determined at three growth stages to establish the relationship between the levels of MSTN transcript and skeletal muscle growth. The partial cDNA sequence of Cg-MSTN was cloned by using published information from its congener walking catfish (Cm-MSTN). The Cg-MSTN was 1194 bp in length encoding a protein of 397 amino acids. The deduced MSTN sequence exhibited key functional sites similar to those of other members of the TGF-ß superfamily, especially, the proteolytic processing site (RXXR motif) and nine conserved cysteines at the C-terminal. Expression of MSTN appeared to be correlated with muscle development and growth of African catfish. Protein bioinformatics revealed that the primary sequence of Cg-MSTN shared 98 % sequence identity with that of walking catfish Cm-MSTN with only two different residues, [Formula: see text]. and [Formula: see text]. The proposed model of Cg-MSTN revealed the key point mutation [Formula: see text] causing a 7.35 Å shorter distance between the N- and C-lobes and an approximately 11° narrow angle than those of Cm-MSTN. The substitution of a proline residue near the proteolytic processing site which altered the structure of myostatin may play a critical role in reducing proteolytic activity of this protein in African catfish.

cDNA structure and the effect of fasting on myostatin expression in walking catfish (Clarias macrocephalus, Günther 1864).

We cloned and sequenced the myostatin (MSTN) gene of walking catfish and characterized its expression under different conditions. The full cDNA sequence of MSTN was 1,784 bp, containing an open reading frame of 1,191 bp, which encoded 396 amino acids. The deduced MSTN sequence contained functional sites similar to other members of TGF-ß superfamily, including the proteolytic processing site and nine conserved cysteines in the C-terminal. Walking catfish MSTN mRNA was strongly expressed in skeletal muscle and brain tissues, consistent with the expression profiles of MSTN-1 isoform in other teleosts. Temporal expression analysis revealed that the MSTN was expressed at the highest levels in 1-week-old larvae and adults, but was lowest in early juveniles. A fasting-re-feeding experiment was used to evaluate the effects of starvation on growth and MSTN expression in juvenile walking catfish for 28 days. MSTN transcript levels increased significantly (threefold) after 7 days of fasting (P < 0.05) compared with the fed control. Subsequently, MSTN expression levels decreased 1.6-fold when fasting was extended to 14 days. Although re-feeding decreased the MSTN expression relative to the levels of the fed control, the period was not long enough for growth recovery of the juveniles. Our results supported a role of MSTN as a negative regulator of muscle growth and, possibly, a role in energy conservation in fish.

Computational analyses of curcuminoid analogs against kinase domain of HER2.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) has an important role in cancer aggressiveness and poor prognosis. HER2 has been used as a drug target for cancers. In particular, to effectively treat HER2-positive cancer, small molecule inhibitors were developed to target HER2 kinase. Knowing that curcumin has been used as food to inhibit cancer activity, this study evaluated the efficacy of natural curcumins and curcumin analogs as HER2 inhibitors using in vitro and in silico studies. The curcumin analogs considered in this study composed of 4 groups classified by their core structure, ß-diketone, monoketone, pyrazole, and isoxazole. RESULTS: In the present study, both computational and experimental studies were performed. The specificity of curcumin analogs selected from the docked results was examined against human breast cancer cell lines. The screened curcumin compounds were then subjected to molecular dynamics simulation study. By modifying curcumin analogs, we found that protein-ligand affinity increases. The benzene ring with a hydroxyl group could enhance affinity by forming hydrophobic interactions and the hydrogen bond with the hydrophobic pocket. Hydroxyl, carbonyl or methoxy group also formed hydrogen bonds with residues in the adenine pocket and sugar pocket of HER2-TK. These modifications could suggest the new drug design for potentially effective HER2-TK inhibitors. Two outstanding compounds, bisdemethylcurcumin (AS-KTC006) and 3,5-bis((E)-3,4-dimethoxystyryl)isoxazole (AS-KTC021 ),were well oriented in the binding pocket almost in the simulation time, 30 ns. This evidence confirmed the results of cell-based assays and the docking studies. They possessed more distinguished interactions than known HER2-TK inhibitors, considering them as a promising drug in the near future. CONCLUSIONS: The series of curcumin compounds were screened using a computational molecular docking and followed by human breast cancer cell lines assay. Both AS-KTC006 and AS-KTC021 could inhibit breast cancer cell lines though inhibiting of HER2-TK. The intermolecular interactions were confirmed by molecular dynamics simulation studies. This information would explore more understanding of curcuminoid structures and HER2-TK.

Homology modeling and virtual screening for antagonists of protease from yellow head virus.

Yellow head virus (YHV) is one of the causative agents of shrimp viral disease. The prevention of YHV infection in shrimp has been developed by various methods, but it is still insufficient to protect the mass mortality in shrimp. New approaches for the antiviral drug development for viral infection have been focused on the inhibition of several potent viral enzymes, and thus the YHV protease is one of the interesting targets for developing antiviral drugs according to the pivotal roles of the enzyme in an early stage of viral propagation. In this study, a theoretical modeling of the YHV protease was constructed based on the folds of several known crystal structures of other viral proteases, and was subsequently used as a target for virtual screening-molecular docking against approximately 1364 NCI structurally diversity compounds. A complex between the protease and the hit compounds was investigated for intermolecular interactions by molecular dynamics simulations. Five best predicted compounds (NSC122819, NSC345647, NSC319990, NSC50650, and NSC5069) were tested against bacterial expressed YHV. The NSC122819 showed the best inhibitory characteristic among the candidates, while others showed more than 50 % of inhibition in the assay condition. These compounds could potentially be inhibitors for curing YHV infection.

In silico screening of epidermal growth factor receptor (EGFR) in the tyrosine kinase domain through a medicinal plant compound database.

The unregulated epidermal growth factor receptor tyrosine kinase (ErbB1-TK or EGFR-TK) protein is involved in the proliferation of more than 50% of all cancer types. The reduction of EGFR-TK activity by small or medium-sized molecules has been proven to be an effective treatment for cancer. There is a widespread belief that Chinese medicinal herbs are active against several diseases, including various types of cancer. In this study, 29,960 compounds from the Chemiebase medicinal compound database were virtually screened against the EGFR-TK using AutoDock4.0, GOLD and GLIDE (XP). The results revealed eight potential hits: CAS nos. 104096-45-9, 112649-21-5, 113866-89-0, 142608-98-8, 142608-99-9, 144761-33-1, 155233-17-3 and 80510-05-0. These compounds have been reported to show anticancer activities in the literature. With the help of SiMMap and MOE interaction analysis, the protein-ligand interaction patterns between the functional groups of these compounds and the binding pocket residues were analyzed. Hydrogen bonding and hydrophobic forces are the main components of the interactions of these hits, similar to those observed for the known inhibitors erlotinib, gefitinib and AEE. The physicochemical filter indicates that compounds CAS nos. 104096-45-9 and 144761-33-1 are likely to be potential leads in the drug discovery process.

Receptor-based virtual screening of EGFR kinase inhibitors from the NCI diversity database.

Epidermal growth factor receptor (EGFR) abnormalities have been associated with several types of human cancer. The crystal structures of its tyrosine kinase domain (EGFR-TK) complexed with small molecule inhibitors revealed the kinase inhibition modes, prompting us to search for novel anti-cancer drugs. A total of 1,990 compounds from the National Cancer Institute (NCI) diversity set with nonredundant structures have been tested to inhibit cancer cell lines with unknown mechanism. Cancer inhibition through EGFR-TK is one of the mechanisms of these compounds. In this work, we performed receptor-based virtual screening against the NCI diversity database. Using two different docking algorithms, AutoDock and Gold, combined with subsequent post-docking analyses, we found eight candidate compounds with high scoring functions that all bind to the ATP-competitive site of the kinase. None of these compounds belongs to the main group of the currently known EGFR-TK inhibitors. Binding mode analyses revealed that the way these compounds complexed with EGFR-TK differs from quinazoline inhibitor binding and the interaction mainly involves hydrophobic interactions. Also, the common kinase-inhibitor (NH---N and CO---HC) hydrogen bonds between the hinge region and the hit compounds are rarely observed. Our results suggest that these molecules could be developed as novel lead compounds in anti-cancer drug design.