Phaik-Eem Lim

Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals.

BackgroundSpirulina is a commercial alga well known to contain various antioxidants, especially phycocyanin. Apart from being sold as a nutraceutical, Spirulina is incorporated as a functional ingredient in food products and beverages. Most of the previous reports on antioxidant activity of Spirulina were based on chemical rather than cell-based assays. The primary objective of this study was to assess the antioxidant activity of aqueous extract from Spirulina based on its protective effect against cell death induced by free radicals.MethodsThe antioxidant activity of the cold water extract from food-grade Spirulina platensis was assessed using both chemical and cell-based assays. In the cell-based assay, mouse fibroblast cells (3T3) cells were incubated for 1 h in medium containing aqueous extract of Spirulina or vitamin C (positive control) at 25, 125 and 250 μg/mL before the addition of 50 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH) or 3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The cells were incubated for another 24 h before being assessed for cell death due to apoptosis using the Cell Death Detection ELISA Kit. Spectrophotometric assays based on DPPH and ABTS were also used to assess the antioxidant activity of the extract compared to vitamin C and vitamin E (positive controls).ResultsSpirulina extract did not cause cytotoxic effect on 3T3 cells within the range of concentrations tested (0 - 250 μg/mL). The extract reduced significantly (p < 0.05) apoptotic cell death due to DPPH and ABTS by 4 to 5-fold although the activity was less than vitamin C. Based on the DPPH assay, the radical scavenging activity of the extract was higher than phycocyanin and was at least 50% of vitamin C and vitamin E. Based on the ABTS assay, the antioxidant activity of the extract at 50 μmug/mL was as good as vitamin C and vitamin E.ConclusionsThe results showed that aqueous extract of Spirulina has a protective effect against apoptotic cell death due to free radicals. The potential application of incorporating Spirulina into food products and beverages to enhance their antioxidant capacity is worth exploring.

DNA metabarcoding of insects and allies: an evaluation of primers and pipelines

Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80-90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

A new species of Burkholderia isolated from sugarcane roots promotes plant growth.

Sugarcane is a globally important food, biofuel and biomaterials crop. High nitrogen (N) fertilizer rates aimed at increasing yield often result in environmental damage because of excess and inefficient application. Inoculation with diazotrophic bacteria is an attractive option for reducing N fertilizer needs. However, the efficacy of bacterial inoculants is variable, and their effective formulation remains a knowledge frontier. Here, we take a new approach to investigating diazotrophic bacteria associated with roots using culture‐independent microbial community profiling of a commercial sugarcane variety (Q208A) in a field setting. We first identified bacteria that were markedly enriched in the rhizosphere to guide isolation and then tested putative diazotrophs for the ability to colonize axenic sugarcane plantlets (Q208A) and promote growth in suboptimal N supply. One isolate readily colonized roots, fixed N2 and stimulated growth of plantlets, and was classified as a new species, Burkholderia australis sp. nov. Draft genome sequencing of the isolate confirmed the presence of nitrogen fixation. We propose that culture‐independent identification and isolation of bacteria that are enriched in rhizosphere and roots, followed by systematic testing and confirming their growth‐promoting capacity, is a necessary step towards designing effective microbial inoculants.

Molecular differentiation of Angiostrongylus taxa (Nematoda: Angiostrongylidae) by cytochrome c oxidase subunit I (COI) gene sequences.

Nematodes of the genus Angiostrongylus are parasites of rodents and carnivores. They reside in the pulmonary or mesenteric arteries of their hosts. Two species are pathogenic in humans -Angiostrongylus cantonensis causes eosinophilic meningitis or meningoencephalitis, and Angiostrongylus costaricensis produces abdominal angiostrongyliasis. In addition Angiostrongylus malaysiensis may have the potential of being pathogenic in humans. The mitochondrial gene cytochrome c oxidase subunit I (COI) of these Angiostrongylus species and three geographical isolates (China, Hawaii and Thailand) of A. cantonensis were studied by polymerase chain reaction amplification and DNA sequencing. COI sequences of A. cantonensis, A. costaricensis and Angiostrongylus vasorum in the GenBank were included for comparison. Phylogenetic analysis by maximum-likelihood (ML), maximum-parsimony (MP), neighbour-joining (NJ) and Bayesian inference (BI) produced similar tree topology except variation in the bootstrap support values. There were two major clades - (1) A. cantonensis and A. malaysiensis, and (2) A. costaricensis and A. vasorum. The three geographical isolates of A. cantonensis formed a clade with low to high bootstrap values, and consisted of two subclades: (a) China and Hawaii isolates, and (b) monophyletic Thailand isolate. The individuals of each isolate formed a distinct cluster. In the second major clade, the Europe isolates of A. vasorum were distinctly different from the Brazil isolates. For A. costaricensis, the Costa Rica isolate was distinct from the Brazil isolate with an uncorrected (p) distance of 11.39%, indicating the possible occurrence of cryptic species. The present results indicate that COI sequences might be a useful marker for differentiating geographical isolates of A. cantonensis and in uncovering cryptic species. Efforts are being made to carry out an extensive collaborative study to cover a wide range of Angiostrongylus species and geographical isolates.

Phylogenetic relationship of Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta) in Malaysia

The genera Kappaphycus Doty and Eucheuma J. Agardh are important sources of carrageenan in Malaysia, offering lucrative revenues to the carrageenan industry, economy, and the local community. The extensive range of morphotypes and the lack of distinct morphological characteristics led to the application of molecular systematics in elucidating this taxonomic confusion. Local varieties of Kappaphycus and Eucheuma, identified using putative external morphology, were analyzed using the mitochondrial cox2–3 spacer and plastid RuBisCO spacer molecular markers. Phylogenetic analysis of these and non-local specimens indicate that Kappaphycus and Eucheuma are genetically distinct. Three main genotypes of Kappaphycus alvarezii were identified, of which two are extant in Hawaii. Morphological and color variations are not supported by molecular data, indicating that most of the local names are not genetically based. Both the cox2–3 spacer and RuBisCO spacer generated phylogenetic trees with similar topology except in variation of nodal supports. The two markers showed clear separation between Kappaphycus and Eucheuma and the existence of three Malaysian Kappaphycus cultivars. Cox2–3 spacer data is more variable and provides better resolution than the RuBisCO spacer, showing that Kappaphycus is more diversified with a larger number of genotypes, strains, and species which are unique to Southeast Asia. Kappaphycus sp. “Aring-aring” appeared to be phenotypically and genotypically different from other Kappaphycus congeners, whereas Kappaphycus striatum exhibited two different genotypes. Our data indicate that Eucheuma denticulatum is the dominant species in Malaysian waters and also suggested paraphyly in Eucheuma which will require further studies. The application of molecular taxonomy on Malaysian Kappaphycus and Eucheuma proves useful, offering valuable insights into the taxonomy and distribution of these commercially important Rhodophytes.

Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae)

The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nematodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-likelihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades - Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonensis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of bootstrap values. The published nucleotide sequences of A. cantonensis adult-specific native 66kDa protein mRNA, clone L5-400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%.

Potential Bioactive Compounds from Seaweed for Diabetes Management

Diabetes mellitus is a group of metabolic disorders of the endocrine system characterised by hyperglycaemia. Type II diabetes mellitus (T2DM) constitutes the majority of diabetes cases around the world and are due to unhealthy diet, sedentary lifestyle, as well as rise of obesity in the population, which warrants the search for new preventive and treatment strategies. Improved comprehension of T2DM pathophysiology provided various new agents and approaches against T2DM including via nutritional and lifestyle interventions. Seaweeds are rich in dietary fibres, unsaturated fatty acids, and polyphenolic compounds. Many of these seaweed compositions have been reported to be beneficial to human health including in managing diabetes. In this review, we discussed the diversity of seaweed composition and bioactive compounds which are potentially useful in preventing or managing T2DM by targeting various pharmacologically relevant routes including inhibition of enzymes such as α-glucosidase, α-amylase, lipase, aldose reductase, protein tyrosine phosphatase 1B (PTP1B) and dipeptidyl-peptidase-4 (DPP-4). Other mechanisms of action identified, such as anti-inflammatory, induction of hepatic antioxidant enzymes’ activities, stimulation of glucose transport and incretin hormones release, as well as β-cell cytoprotection, were also discussed by taking into consideration numerous in vitro, in vivo, and human studies involving seaweed and seaweed-derived agents.

Assessment of Four Molecular Markers as Potential DNA Barcodes for Red Algae Kappaphycus Doty and Eucheuma J. Agardh (Solieriaceae, Rhodophyta)

DNA barcoding has been a major advancement in the field of taxonomy, seeing much effort put into the barcoding of wide taxa of organisms, macro and microalgae included. The mitochondrial-encoded cox1 and plastid-encoded rbcL has been proposed as potential DNA barcodes for rhodophytes, but are yet to be tested on the commercially important carrageenophytes Kappaphycus and Eucheuma. This study gauges the effectiveness of four markers, namely the mitochondrial cox1, cox2, cox2-3 spacer and the plastid rbcL in DNA barcoding on selected Kappaphycus and Eucheuma from Southeast Asia. Marker assessments were performed using established distance and tree-based identification criteria from earlier studies. Barcoding patterns on a larger scale were simulated by empirically testing on the commonly used cox2-3 spacer. The phylogeny of these rhodophytes was also briefly described. In this study, the cox2 marker which satisfies the prerequisites of DNA barcodes was found to exhibit moderately high interspecific divergences with no intraspecific variations, thus a promising marker for the DNA barcoding of Kappaphycus and Eucheuma. However, the already extensively used cox2-3 spacer was deemed to be in overall more appropriate as a DNA barcode for these two genera. On a wider scale, cox1 and rbcL were still better DNA barcodes across the rhodophyte taxa when practicality and cost-efficiency were taken into account. The phylogeny of Kappaphycus and Eucheuma were generally similar to those earlier reported. Still, the application of DNA barcoding has demonstrated our relatively poor taxonomic comprehension of these seaweeds, thus suggesting more in-depth efforts in taxonomic restructuring as well as establishment.

Molecular differentiation of two morphological variants of Gracilaria salicornia

Xia in 1986 combined Gracilaria salicornia, G. canaliculata (G. crassa), G. cacalia and G. minor into one species: G.salicornia. Two morphological variants of G. salicornia were collectedfrom different localities in Malaysia. Variant A collected from Morib,Selangor grew on the roots of Avicennia. The samples showed absenceof main axis; segmented constrictions throughout; cylindrical or slightlycompressed thalli. Variant B was collected from the mudflats of TanjungTuan, growing on rocks, coral or forming mats on the mud. Plants showedabsence of main axis; segments were not constricted throughout the plant(if present only slightly articulated at the upper part), branching wasdichotomous or irregular; cylindrical or slightly compressed thalli. Thetechnique of Random Amplified Polymorphic DNA analysis (RAPD) wasused to investigate molecular characteristics of the two variants. Out ofsixty Operon primers that were screened, four primers, OPA 1, OPA 10,OPA 11 and OPK 7 were able to give polymorphism. The fingerprintsgenerated were stable and reproducible on repeated analysis. The DNAfingerprints generated were visually analysed and clustering analysis wascarried out using GelCompar 4.0. The matrix of similarities was based onthe Dice coefficients (SD) and the cluster analysis was carried outusing the unweighted pair group method using arithmetic averages(UPGMA). DNA analysis showed that two primers (OPA 01, CAGGCCCTTC and OPK 07, AGCGAGCAAG) were able to differentiate the two variants.

Molecular phylogeny of crustose brown algae (Ralfsiales, Phaeophyceae) inferred from rbcL sequences resulting in the proposal for Neoralfsiaceae fam. nov.

P.-E. Lim, M. Sakaguchi, T. Hanyuda, K. Kogame, S.-M. Phang and H. Kawai. 2007. Molecular phylogeny of crustose brown algae (Ralfsiales, Phaeophyceae) inferred from rbcL sequences resulting in the proposal for Neoralfsiaceae fam. nov. Phycologia 46: 456–466. DOI: 10.2116/06-90.1 The order Ralfsiales was established to accommodate the brown algal taxa having a crustose thallus, an isomorphic life history, discoid early development of the thallus and containing a single, plate-shaped chloroplast without pyrenoids in each cell. However, the validity of the order has been questioned by many researchers because several exceptions to these criteria have been found within the order. Molecular phylogenetic analysis of the taxa assigned to the order, using rbcL DNA sequences, reveals that Ralfsiales is not a monophyletic group but is separated into two major groups, excluding Lithodermataceae, which were not included in the present analysis: clade I, comprising the members of Ralfsiaceae, Mesosporaceae, Analipus japonicus and Heteroralfsia saxicola; and clade II, consisting of Diplura species, sister to the Ishigeales clade. On the basis of these results, we propose emendment of the Ralfsiales to contain only species having (1) discoidal early development of the thallus; (2) intercalary plurilocular gametangia with terminal cells and terminal unilocular zoidangia; and (3) a crustose phase in the life history. Furthermore, we propose the establishment of the new family Neoralfsiaceae to accommodate the new genus Neoralfsia, on the basis of Ralfsia expansa.