Alexander Chong Shu-Chien

The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Δ4 activity and an Elovl5 elongase in striped snakehead (Channa striata).

The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.

Mitragynine attenuates withdrawal syndrome in morphine-withdrawn zebrafish.

A major obstacle in treating drug addiction is the severity of opiate withdrawal syndrome, which can lead to unwanted relapse. Mitragynine is the major alkaloid compound found in leaves of Mitragyna speciosa, a plant widely used by opiate addicts to mitigate the harshness of drug withdrawal. A series of experiments was conducted to investigate the effect of mitragynine on anxiety behavior, cortisol level and expression of stress pathway related genes in zebrafish undergoing morphine withdrawal phase. Adult zebrafish were subjected to two weeks chronic morphine exposure at 1.5 mg/L, followed by withdrawal for 24 hours prior to tests. Using the novel tank diving tests, we first showed that morphine-withdrawn zebrafish display anxiety-related swimming behaviors such as decreased exploratory behavior and increased erratic movement. Morphine withdrawal also elevated whole-body cortisol levels, which confirms the phenotypic stress-like behaviors. Exposing morphine-withdrawn fish to mitragynine however attenuates majority of the stress-related swimming behaviors and concomitantly lower whole-body cortisol level. Using real-time PCR gene expression analysis, we also showed that mitragynine reduces the mRNA expression of corticotropin releasing factor receptors and prodynorphin in zebrafish brain during morphine withdrawal phase, revealing for the first time a possible link between mitragynines ability to attenuate anxiety during opiate withdrawal with the stress-related corticotropin pathway.

The rubber tree genome shows expansion of gene family associated with rubber biosynthesis

Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55 Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis’s capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree.

Discovery of new nanomolar peroxisome proliferator-activated receptor γ activators via elaborate ligand-based modeling.

Peroxisome Proliferator-Activated Receptor γ (PPARγ) activators have drawn great recent attention in the clinical management of type 2 diabetes mellitus, prompting several attempts to discover and optimize new PPARγ activators. With this in mind, we explored the pharmacophoric space of PPARγ using seven diverse sets of activators. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select an optimal combination of pharmacophoric models and 2D physicochemical descriptors capable of accessing self-consistent and predictive quantitative structure-activity relationship (QSAR) (r2(71)=0.80, F=270.3, r2LOO=0.73, r2PRESS against 17 external test inhibitors=0.67). Three orthogonal pharmacophores emerged in the QSAR equation and were validated by receiver operating characteristic (ROC) curves analysis. The models were then used to screen the national cancer institute (NCI) list of compounds. The highest-ranking hits were tested in vitro. The most potent hits illustrated EC50 values of 15 and 224 nM.

Identification of Wap65, a human homologue of hemopexin as a copper-inducible gene in swordtail fish, Xiphophorus helleri.

Copper is one of the major heavy metal pollutants found in the aquatic environment. Therefore, it is important for determining the genes that play a key role in copper metabolism in aquatic organisms. This study, thus, aimed to identify a new copper-inducible gene in swordtail fish, Xiphophorus helleri. Using ACP-based RT-PCR coupled with RLM-RACE, we cloned Wap65, a mammalian homologue of hemopexin gene. The gene exhibits high identity at amino acid levels with the Wap65 gene of other fish species (42–68%) and mammalian hemopexin gene (35–37%). In addition, ten cysteine and two histidine residues are conserved in the swordtail fish Wap65 gene. These cysteine residues are vital for structural integrity, and histidine residues provide high binding affinity towards heme. As revealed by RT-PCR, the gene was upregulated in swordtail fish that were exposed to copper in a dose- and time-dependent manner. Therefore, the identification of Wap65, a mammalian homologue of hemopexin, as a new copper-inducible gene will provide greater insight into the role of this gene in copper metabolism.

The cytokine macrophage migration inhibitory factor (MIF) acts as a neurotrophin in the developing inner ear of the zebrafish, Danio rerio.

Macrophage migration inhibitory factor (MIF) plays versatile roles in the immune system. MIF is also widely expressed during embryonic development, particularly in the nervous system, although its roles in neural development are only beginning to be understood. Evidence from frogs, mice and zebrafish suggests that MIF has a major role as a neurotrophin in the early development of sensory systems, including the auditory system. Here we show that the zebrafish mif pathway is required for both sensory hair cell (HC) and sensory neuronal cell survival in the ear, for HC differentiation, semicircular canal formation, statoacoustic ganglion (SAG) development, and lateral line HC differentiation. This is consistent with our findings that MIF is expressed in the developing mammalian and avian auditory systems and promotes mouse and chick SAG neurite outgrowth and neuronal survival, demonstrating key instructional roles for MIF in vertebrate otic development.

Prolactin receptor mRNA is upregulated in discus fish (Symphysodon aequifasciata) skin during parental phase

Prolactin (PRL) has been shown to directly influence parental-care associated behavior in many vertebrate species. The discus fish (Symphysodon aequifasciata) displays extensive parental care behavior through utilization of epidermal mucosal secretion to raise free-swimming fry. Here, we cloned the full-length cDNA sequence of the S. aequifasciata prolactin receptor (dfPRLR) and investigated the mRNA expression pattern in several adult tissues. Bioinformatic analysis showed the dfPRLR shared rather high identity (79 and 67%) with the Nile tilapia PRLR 1 and black seabream PRLR 1, respectively. The presence of dfPRLR in several osmoregulatory tissues including kidney, gill and intestine is consistent with the known role of PRL in mediating hydromineral balance in teleosts. In addition, upregulated expression of PRLR mRNA was observed in skin of parental fish compared to non-parental fish, indicating possibility of a role of the PRL hormonal signaling in regulation of mucus production in relation to parental care behaviour.

Distinct developmental expression of two elongase family members in zebrafish.

Despite the known importance of long-chained polyunsaturated fatty acids (LC-PUFA) during development, very little is known about their utilization and biosynthesis during embryogenesis. Combining the advantages of the existence of a complete range of enzymes required for LC-PUFA biosynthesis and the well established developmental biology tools in zebrafish, we examined the expression patterns of three LC-PUFA biosynthesis genes, Elovl2-like elongase (elovl2), Elovl5-like elongase (elovl5) and fatty acyl desaturase (fad) in different zebrafish developmental stages. The presence of all three genes in the brain as early as 24 hours post fertilization (hpf) implies LC-PUFA synthesis activity in the embryonic brain. This expression eventually subsides from 72 hpf onwards, coinciding with the initiation of elovl2 and fad expression in the liver and intestine, 2 organs known to be involved in adult fish LC-PUFA biosynthesis. Collectively, these patterns strongly suggest the necessity for localized production of LC-PUFA in the brain during in early stage embryos prior to the maturation of the liver and intestine. Interestingly, we also showed a specific expression of elovl5 in the proximal convoluted tubule (PCT) of the zebrafish pronephros, suggesting a possible new role for LC-PUFA in kidney development and function.

Proteomics of Buccal Cavity Mucus in Female Tilapia Fish (Oreochromis spp.): A Comparison between Parental and Non-Parental Fish

Mouthbrooding is an elaborate form of parental care displayed by many teleost species. While the direct benefits of mouthbrooding such as protection and transportation of offsprings are known, it is unclear if mouthbrooding offers additional benefits to embryos during incubation. In addition, mouthbrooding could incur negative costs on parental fish, due to limited feeding opportunities. Parental tilapia fish (Oreochromis spp.) display an elaborated form of parental care by incubating newly hatched embryos in oral buccal cavity until the complete adsorption of yolk sac. In order to understand the functional aspects of mouthbrooding, we undertake a proteomics approach to compare oral mucus sampled from mouthbrooders and non-mouthbrooders, respectively. Majority of the identified proteins have also been previously identified in other biological fluids or mucus-rich organs in different organisms. We also showed the upregulation of 22 proteins and down regulation of 3 proteins in mucus collected from mouthbrooders. Anterior gradient protein, hemoglobin beta-A chain and alpha-2 globin levels were lower in mouthbrooder samples. Mouthbrooder oral mucus collectively showed increase levels of proteins related to cytoskeletal properties, glycolytic pathway and mediation of oxidative stress. Overall the findings suggest cellular stress response, probably to support production of mucus during mouthbrooding phase.

Molecular cloning and ontogenic mRNA expression of fatty acid desaturase in the carnivorous striped snakehead fish (Channa striata).

There is very little information on the capacity of freshwater carnivorous fish to biosynthesize highly unsaturated fatty acids (HUFA). The striped snakehead fish (Channa striata) is a carnivorous species cultured inland of several Southeast Asian countries due to its pharmaceutical properties in wound healing enhancement. We described here the full-length cDNA cloning of a striped snakehead fatty acid desaturase (fads), which is responsible for desaturation of unsaturated fatty acids in the HUFA biosynthesis. Bioinformatics analysis reveals a protein coding region with length of 445 amino acids containing all characteristic features of desaturase enzyme, including a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. The striped snakehead fads amino acid sequence shares high similarity with known fads of other teleosts. The mRNA expression of striped snakehead fads also showed an ontogenic-related increase in expression in 0-20 days after hatch larva. Using ISH, we localized the presence of fads in larva brain, liver and intestinal tissues.