Perbandingan filogenetik protein antigen-I yang berpotensi sebagai calon diagnostik dan vaksin terhadap parasit cryptocaryon irritans

Protein antigen-i parasit ikan C. irritans berpotensi tinggi digunakan sebagai calon dalam pembangunan vaksin komersial terhadap C. irritans. Walau bagaimanapun, kewujudan variasi pada antigen-i serotip C. irritans yang berbeza mempengaruhi tahap perlindungan yang bakal diberikan terhadap varians C. irritans yang berbeza apabila antigen-i digunakan sebagai vaksin. Kajian ini dijalankan untuk membandingkan jujukan pelbagai antigen-i pencilan C. irritans di Malaysia berbanding antigen-i pencilan C. irritans yang pernah dilaporkan. Perbandingan filogenetik dijalankan untuk meramalkan potensi protein tersebut dalam usaha membangunkan calon serodiagnostik dan pemvaksinan terhadap pencilan C. irritans yang berlainan. Penjajaran jujukan berbilang bagi jujukan asid amino antigen-i dilakukan dengan perisian CLUSTALX dan analisis filogenetik antigen-i dilakukan menggunakan kaedah parsimoni maksimum (MP) dan kaedah Bayes. Sembilan transkrip unik (TU) C. irritans yang mempunyai padanan signifikan dengan antigen-i di pangkalan data protein NCBI didapati mempunyai peratus kesamaan antara 41% hingga 71%. Kedua-dua pohon MP dan Bayesian yang dijana menunjukkan varians antigen-i cn56 and cn57 terkelompok bersama dalam satu kumpulan manakala varians antigen-i yang lain terbahagi kepada dua kumpulan berasingan dan pengkelompokan ini disokong oleh kehadiran asid amino yang terpulihara dalam kumpulan masing-masing. Kajian lanjutan boleh dilakukan untuk mengenal pasti varians antigen-i yang sesuai sebagai calon serodiagnosis dan juga dapat memberi perlindungan silang terhadap pelbagai pencilan C. irritans di serata dunia

Characterization of the Functional Domain of β2-Microglobulin from the Asian Seabass, Lates calcarifer

BACKGROUND: β2-Microglobulin (β(2)M) is the light chain of major histocompatibility class I (MHC I) that binds non-covalently with the α heavy chain. Both proteins attach to the antigen peptide, presenting a complex to the T cell to be destroyed via the immune mechanism. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a cDNA sequence encoding β(2)M in the Asian seabass (Lates calcarifer) was identified and analyzed using in silico approaches to predict and characterize its functional domain. The β(2)M cDNA contains an open reading frame (ORF) of 351 bases with a coding capacity of 116 amino acids. A large portion of the protein consists of the IG constant domain (IGc1), similar to β(2)M sequences from other species studied thus far. Alignment of the IGc1 domains of β(2)M from L. calcarifer and other species shows a high degree of overall conservation. Seven amino acids were found to be conserved across taxa whereas conservation between L. calcarifer and other fish species was restricted to 14 amino acids at identical conserved positions. CONCLUSION/SIGNIFICANCE: As the L. calcarifer β(2)M protein analyzed in this study contains a functional domain similar to that of β(2)M proteins in other species, it can be postulated that the β(2)M proteins from L. calcarifer and other organisms are derived from a common ancestor and thus have a similar immune function. Interestingly, fish β(2)M genes could also be classified according to the ecological habitat of the species, i.e. whether it is from a freshwater, marine or euryhaline environment

Cloning and characterisation of multiple ferritin isoforms in the Atlantic salmon (Salmo salar)

Peer reviewedPublisher PD

Fungal and bacterial species in degrading carbamazepine: a metabolite perspective: Mini-review

Carbamazepine (CBZ) is a ubiquitous pharmaceutical pollutant found in various water environments. This is due to the ineffective CBZ removal, despite employing advanced physiochemical treatment technologies in the current conventional wastewater treatment plants. Thus, bioremediation that utilizes enzymes in microorganisms' systems to bio-mineralize CBZ is suggested as an alternative or complementary technique to remove CBZ more effectively. However, information from published research on the biodegradation of CBZ, the toxicity of metabolites, or toxicity testing was rarely evaluated or assessed cohesively. This aspect is important because if bioremediation of CBZ produces toxic metabolites, it will defeat the main purpose of bioremediation. Thus, the focus of this review is to assess the effectiveness of fungi and bacteria in the biodegradation of CBZ, particularly by looking at the type of enzymes expressed, and the metabolites produced. In this review, information related to the fungal and bacterial species that were reported to degrade CBZ was collated from the published literature and analyzed. Results of the analysis showed that cytochrome P450, laccase, and manganese peroxidase were the common enzymes responsible to degrade CBZ. However, such enzymatic activities can sometimes produce epoxy-CBZ, which is a more toxic compound than the parent compound. Only the fungus Pleurotus ostreatus was able to oxidize epoxy-CBZ via the acridine pathway into acridone, the latter a metabolite that is susceptible to further biodegradation into nontoxic metabolites. However, the identity of the end metabolites is not reported nor characterized. Further, Pseudomonas spp. is the most promising bioremediating agent since it can metabolize CBZ into catechol, the latter can enter the carbon central pathways to generate energy for the bacterial cells

Molecular characterisation and expression analysis of cathepsin D from the Asian seabass lates calcarifer

The lysosomal aspartic proteinase cathepsin D is an acute phase protein involved in various physiological processes, including vitellogenesis, yolk processing and immune responses. In this study, we characterised the cathepsin D from the Asian seabass Lates calcarifer and examined its expression profile during infection. The complete coding sequence of L. calcarifer cathepsin D consists of 1191 nucleotides, encoding a 396 amino acid protein molecule that is made up of a putative signal peptide, a leader peptide and a mature peptide. Phylogenetic analyses showed that two types of cathepsin D are present in the teleost lineage i.e. cathepsin D1 and D2, whereas higher vertebrates possess only one type of cathepsin D. L. calcarifer cathepsin D was clustered together with cathepsin D1 from other teleosts. Compared to mammalian sequences, L. calcarifer cathepsin D lacks the β-hairpin loop that forms the double chain and is present as a single chain peptide with conserved aspartic active sites like other fish. Both multiple sequence alignment and phylogenetic analysis indicated that the L. calcarifer cathepsin D sequence codes for cathepsin D1 and suggested that it shares the same functions with cathepsin D from other fish. Expression profiling analysis of cathepsin D in L. calcarifer infected with Aeromonas hydrophila showed that it is up-regulated in immune-related tissues such as gills, spleen and liver, suggesting that cathepsin D plays an important role in the innate immune response of L. calcarifer against pathogens

Characterisation of simple sequence repeats in the Asian Seabass,Lates calcarifer by random sequencing

In recent years, there has been considerable interest in simple sequence repeats (SSRs) particularly as molecular markers with applications in many different fields. We have carried out an effort to identify and analyse SSRs in the genome of the Asian seabass, Lates calcarifer by random sequencing. Genomic DNA was isolated from the muscle tissue of L. calcarifer, sheared by nebulisation and ligated into plasmid vector. Recombinant clones were selected randomly from the genomic libraries constructed. Subsequently, plasmid DNA was extracted and subjected to one-pass sequencing. A total of 4175 random sequences, also known as genome survey sequences (GSSs), with a total length of 1.7 Mb was generated. Screening of the whole L. calcarifer GSS data set allowed for the identification of a total of 151 perfect (100% similarity) SSRs. These SSR consensus patterns spread over a wide range of size (1 to 226 bp). The most frequent consensus pattern is dinucleotide, which represents 60% of all SSRs identified. The dinucleotides (AC)n, (AT)n and (AG)n were also found to occur frequently in the L. calcarifer genome. Sequence comparison between L. calcarifer and other fish species showed variation in repeat content, indicating the different ways in which repeats may evolve in the genome of these species. Data generated from this random sequencing of the L. calcarifer genome should serve as a valuable resource for further studies of this organis

Pembinaan dan pencirian perpustakaan genom siakap (Lates calcarifer)

Siakap (Lates calcarifer) merupakan spesies ikan yang penting dari segi ekonomi di Malaysia. Dalam kajian ini, satu perpustakaan genom L. calcarifer telah dibina. DNA genom telah dipencil daripada tisu otot L. calcarifer, diserpih secara nebulisasi, dibaiki hujung tumpul dan diligasi ke dalam vektor plasmid pCR®4Blunt-TOP®. Campuran ligasi seterusnya telah ditransformasikan ke dalam Escherichia coli. Bagi mencirikan perpustakaan genom yang terhasil, DNA plasmid telah diekstrak daripada klon-klon rekombinan yang dipilih secara rawak. Elektroforesis gel agaros menunjukkan klon-klon rekombinan ini membawa selitan DNA yang bersaiz di antara 0.5 kb - 3.5 kb, dengan purata saiz 1.7 kb. Seterusnya, penjujukan DNA telah dijalankan dengan menggunakan DNA plasmid yang diperoleh sebagai templat. Sebanyak 121 jujukan rawak telah dihasilkan daripada perpustakaan genom L. calcarifer ini. Analisis jujukan rawak memberikan implikasi yang perpustakaan ini adalah representatif bagi genom L. calcarifer. Berdasarkan persamaan dengan jujukan dalam pangkalan data, 17% jujukan rawak ini didapati mempunyai keputusan BLAST yang bermakna, yang menunjukkan mereka mewakili gen L. calcarifer. Analisis dengan menggunakan perisian RepeatMasker menunjukkan sebanyak 25% jujukan rawak ini dikenal pasti sebagai unsur berulang. Pencirian awal terhadap perpustakaan genom ini menunjukkan ia berpotensi sebagai sumber yang berguna dalam kajian genom L. calcarifer

Phylogenetic analyses uncover a novel clade of transferrin in nonmammalian vertebrates

Transferrin is a protein super-family involved in iron transport, a central process in cellular homeostasis. Throughout the evolution of vertebrates, transferrin members have diversified into distinct subfamilies including serotransferrin, ovotransferrin, lactoferrin, melanotransferrin, the inhibitor of carbonic anhydrase, pacifastin, and the major yolk protein in sea urchin. Previous phylogenetic analyses have established the branching order of the diverse transferrin subfamilies but were mostly focused on the transferrin repertoire present in mammals. Here, we conduct a comprehensive phylogenetic analysis of transferrin protein sequences in sequenced vertebrates, placing a special focus on the less-studied nonmammalian vertebrates. Our analyses uncover a novel transferrin clade present across fish, sauropsid, and amphibian genomes but strikingly absent from mammals. Our reconstructed scenario implies that this novel class emerged through a duplication event at the vertebrate ancestor, and that it was subsequently lost in the lineage leading to mammals. We detect footprints of accelerated evolution following the duplication event, which suggest positive selection and early functional divergence of this novel clade. Interestingly, the loss of this novel class of transferrin in mammals coincided with the divergence by duplication of lactoferrin and serotransferrin in this lineage. Altogether, our results provide novel insights on the evolution of iron-binding proteins in the various vertebrate groups.This work was supported by grants from the Malaysian Ministry of Science, Technology and Innovation (07-05-MGI-GMB009), the Spanish Ministry of Science and Innovation (BFU2009-09168), and Universiti Kebangsaan Malaysia (UKM-DLP-2011-027

Transcriptome analysis of the Cryptocaryon irritans tomont stage identifies potential genes for the detection and control of cryptocaryonosis

BACKGROUND: Cryptocaryon irritans is a parasitic ciliate that causes cryptocaryonosis (white spot disease) in marine fish. Diagnosis of cryptocaryonosis often depends on the appearance of white spots on the surface of the fish, which are usually visible only during later stages of the disease. Identifying suitable biomarkers of this parasite would aid the development of diagnostic tools and control strategies for C. irritans. The C. irritans genome is virtually unexplored; therefore, we generated and analyzed expressed sequence tags (ESTs) of the parasite to identify genes that encode for surface proteins, excretory/secretory proteins and repeat-containing proteins. RESULTS: ESTs were generated from a cDNA library of C. irritans tomonts isolated from infected Asian sea bass, Lates calcarifer. Clustering of the 5356 ESTs produced 2659 unique transcripts (UTs) containing 1989 singletons and 670 consensi. BLAST analysis showed that 74% of the UTs had significant similarity (E-value < 10(-5)) to sequences that are currently available in the GenBank database, with more than 15% of the significant hits showing unknown function. Forty percent of the UTs had significant similarity to ciliates from the genera Tetrahymena and Paramecium. Comparative gene family analysis with related taxa showed that many protein families are conserved among the protozoans. Based on gene ontology annotation, functional groups were successfully assigned to 790 UTs. Genes encoding excretory/secretory proteins and membrane and membrane-associated proteins were identified because these proteins often function as antigens and are good antibody targets. A total of 481 UTs were classified as encoding membrane proteins, 54 were classified as encoding for membrane-bound proteins, and 155 were found to contain excretory/secretory protein-coding sequences. Amino acid repeat-containing proteins and GPI-anchored proteins were also identified as potential candidates for the development of diagnostic and control strategies for C. irritans. CONCLUSIONS: We successfully discovered and examined a large portion of the previously unexplored C. irritans transcriptome and identified potential genes for the development and validation of diagnostic and control strategies for cryptocaryonosis

NJ phylogenetic tree of β₂M protein sequences representing whole organisms.

<p>The phylogenetic tree shown is the collapsed tree of 55 sets of sequence data. This tree shows that β₂M sequences are clustered together according to their taxons. β₂M sequences from Eutheria are clustered together and consist of sequences from Primates, Equine, Rodents, Ruminants, SscQ07717, OcuP01885 and FcaQ5MGS7. Marsupials, Monotremes and Avians are the intermediate taxons between Eutheria and Fish. Amphibian Xla protein Q9IA97 is clustered together with Actinopterygii fishes while the outgroup in this tree is a cartilaginous fish Reg Q8AXA0. The divergence of fish and mammalian β₂M received a high bootstrap value (89) to support the reliability of this phylogenetic tree.</p