Chai Ling Ho

Development of Multicellular Tumor Spheroid (MCTS) Culture from Breast Cancer Cell and a High Throughput Screening Method Using the MTT Assay

In comparison to monolayer cells, MCTS has been claimed as more suitable candidate for studying drug penetration due to the high resemblance to solid tumors. However, the cultivation of MCTS is cumbersome, time consuming, and most technique fail to generate spheroids with uniform sizes. Therefore, the application of spheroid cultures in high throughput screening has been rather limiting. Besides, the lack of a well established screening protocol method that is applicable to spheroid could also be attributed to this limitation. Here we report a simple way of cultivating homogenous MCTS cultures with compact and rigid structure from the MCF-7 cells. Besides, we had also made some modifications to the standard MTT assay to realize high throughput screening of these spheroids. Using the modified protocol, tamoxifen showed cytotoxicity effect towards MCTS cultures from MCF-7 with high consistency. The results correlated well with the cultures’ response assessed by LDH release assay but the latter assay was not ideal for detecting a wide range of cytotoxicity due to high basal background reading. The MTT assay emerged as a better indicator to apoptosis event in comparison to the LDH release assay. Therefore, the method for spheroid generation and the modified MTT assay we reported here could be potentially applied to high throughput screening for response of spheroid cultures generated from MCF-7 as well as other cancer cell lines towards cytotoxic stimuli.

Analysis and functional annotation of expressed sequence tags (ESTs) from multiple tissues of oil palm (Elaeis guineensis Jacq.).

BackgroundOil palm is the second largest source of edible oil which contributes to approximately 20% of the worlds production of oils and fats. In order to understand the molecular biology involved in in vitro propagation, flowering, efficient utilization of nitrogen sources and root diseases, we have initiated an expressed sequence tag (EST) analysis on oil palm.ResultsIn this study, six cDNA libraries from oil palm zygotic embryos, suspension cells, shoot apical meristems, young flowers, mature flowers and roots, were constructed. We have generated a total of 14537 expressed sequence tags (ESTs) from these libraries, from which 6464 tentative unique contigs (TUCs) and 2129 singletons were obtained. Approximately 6008 of these tentative unique genes (TUGs) have significant matches to the non-redundant protein database, from which 2361 were assigned to one or more Gene Ontology categories. Predominant transcripts and differentially expressed genes were identified in multiple oil palm tissues. Homologues of genes involved in many aspects of flower development were also identified among the EST collection, such as CONSTANS-like, AGAMOUS-like (AGL)2, AGL20, LFY-like, SQUAMOSA, SQUAMOSA binding protein (SBP) etc. Majority of them are the first representatives in oil palm, providing opportunities to explore the cause of epigenetic homeotic flowering abnormality in oil palm, given the importance of flowering in fruit production. The transcript levels of two flowering-related genes, EgSBP and EgSEP were analysed in the flower tissues of various developmental stages. Gene homologues for enzymes involved in oil biosynthesis, utilization of nitrogen sources, and scavenging of oxygen radicals, were also uncovered among the oil palm ESTs.ConclusionThe EST sequences generated will allow comparative genomic studies between oil palm and other monocotyledonous and dicotyledonous plants, development of gene-targeted markers for the reference genetic map, design and fabrication of DNA array for future studies of oil palm. The outcomes of such studies will contribute to oil palm improvements through the establishment of breeding program using marker-assisted selection, development of diagnostic assays using gene targeted markers, and discovery of candidate genes related to important agronomic traits of oil palm.

Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65–1.14 μg g−1 fresh weight) with high quality (A260:280 ratio 1.80–2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies.

Overexpression of monodehydroascorbate reductase from a mangrove plant (AeMDHAR) confers salt tolerance on rice.

Monodehydroascorbate reductase (MDHAR), an important enzyme of the ascorbate-glutathione cycle, is involved in salt tolerance of plants through scavenging of reactive oxygen species (ROS). In this study, a cDNA encoding MDHAR from the mangrove plant Acanthus ebracteatus was introduced into rice to examine its role in salt tolerance. Three stable transgenic lines (MT22, MT24 and MT25) overexpressing AeMDHAR were selected in vitro using hygromycin and confirmed by PCR, quantitative reverse-transcription (qRT) PCR and enzyme assay. The transgenic line MT24 was predicted to possess a single copy of the transgene while the other two transgenic lines were predicted to have multiple transgene integrations. The AeMDHAR transcripts were detected only in transgenic rice lines but not in untransformed rice. The abundance of AeMDHAR transcripts in transgenic lines MT22 and MT25 was approximately 2.75 times the amount found in MT24. The transgenic rice lines overexpressing AeMDHAR showed a significant increase in MDHAR enzyme activity compared to untransformed plants under both NaCl and control conditions. All transgenic lines showed better yield attributes such as a higher tiller number and increased 1000-grain weight compared to non-transgenics. They also showed tolerance to salt at germination and seedling stages. The transgenic line MT24, which harbors a single copy of AeMDHAR, displayed a lower rate of sterility, a higher number of tillers and longer panicle compared to untransformed plants when subjected to salt stress.

Analyses of expressed sequence tags from an agarophyte, Gracilaria changii (Gracilariales, Rhodophyta)

Red seaweeds of the genus Gracilaria are agarophytes that produce more than 60% of the worlds agar supply. Despite the importance of this genus in agar production, the potential of Gracilaria as a candidate for genomic research has been almost unexplored. In this study, a total of 8,088 expressed sequence tags (ESTs) were generated from Gracilaria changii, and clustered into 4922 tentative unique genes (TUGs), of which approximately 35% showed significant matches (bit scores greater than 50 and E-values less than 10−5) to other genes in the databases. Among the TUGs that have significant similarity to the genes in the databases are ESTs corresponding to diverse functional groups such as metabolism, transcription, signalling, translation, transportation, protein folding, sorting, destination and degradation, cell division, cellular processes, replication and repair, cell structure, and miscellaneous. cDNAs involved in diverse metabolic pathways were identified among the EST collection. The presence and frequency of the transcripts allow us to survey the transcriptomic activities of this tropical agarophyte.

A predicted protein interactome for rice.

BackgroundProtein-protein interactions (PPIs) create the steps in signaling and regulatory networks central to most fundamental biological processes. It is possible to predict these interactions by making use of experimentally determined orthologous interactions in other species.ResultsIn this study, prediction of PPIs in rice was carried out by the interolog method of mapping deduced orthologous genes to protein interactions supported by experimental evidence from reference organisms. We predicted 37112 interactions for 4567 rice proteins, including 1671 predicted self interactions (homo-interactions) and 35441 predicted interactions between different proteins (hetero-interactions). These matched 168 of 675 experimentally-determined interactions in rice. Interacting proteins were significantly more co-expressed than expected by chance, which is typical of experimentally-determined interactomes. The rice interacting proteins were divided topologically into 981 free ends (proteins with single interactions), 499 pipes (proteins with two interactions) and 3087 hubs of different sizes ranging from three to more than 100 interactions.ConclusionsThis predicted rice interactome extends known pathways and improves functional annotation of unknown rice proteins and networks in rice, and is easily explored with software tools presented here.

Hepatoprotective Activity of Elephantopus scaber on Alcohol-Induced Liver Damage in Mice

Elephantopus scaber has been traditionally used as liver tonic. However, the protective effect of E. scaber on ethanol-induced liver damage is still unclear. In this study, we have compared the in vivo hepatoprotective effect of E. scaber with Phyllanthus niruri on the ethanol-induced liver damage in mice. The total phenolic and total flavanoid content of E. scaber ethanol extract were determined in this study. Accelerating serum biochemical profiles (including AST, ALT, ALP, triglyceride, and total bilirubin) associated with fat drop and necrotic body in the liver section were observed in the mice treated with ethanol. Low concentration of E. scaber was able to reduce serum biochemical profiles and the fat accumulation in the liver. Furthermore, high concentration of E. scaber and positive control P. niruri were able to revert the liver damage, which is comparable to the normal control. Added to this, E. scaber did not possess any oral acute toxicity on mice. These results suggest the potential effect of this extract as a hepatoprotective agent towards-ethanol induced liver damage without any oral acute toxicity effect. These activities might be contributed, or at least in part, by its high total phenolic and flavonoid contents.

TRANSCRIPTOMIC ANALYSIS OF GRACILARIA CHANGII (RHODOPHYTA) IN RESPONSE TO HYPER‐ AND HYPOOSMOTIC STRESSES

Osmotic stress is one of the most significant natural abiotic stresses that occur in the intertidal zones. Seaweeds may physiologically acclimate to changing osmolarity by altering their transcriptome. Here, we investigated the transcriptomic changes of Gracilaria changii (B. M. Xia et I. A. Abbott) I. A. Abbott, J. Zhang et B. M. Xia in response to hyper‐ and hypoosmotic stresses using a cDNA microarray approach. Microarray analysis revealed that 199 and 200 genes from ∼3,300 genes examined were up‐ and down‐regulated by >2‐fold in seaweed samples treated at 50 parts per thousand (ppt) artificial seawater (ASW) compared with those at 30 ppt ASW, respectively. The number of genes that were up‐ and down‐regulated by >2‐fold in seaweed samples treated at 10 ppt ASW compared with those at 30 ppt ASW were 154 and 187, respectively. A majority of these genes were only differentially expressed under hyper‐ or hypoosmotic conditions, whereas 67 transcripts were affected by both stresses. The findings of this study have shed light on the expression profiles of many transcripts during the acclimation of G. changii to hyperosmotic and hypoosmotic conditions. This information may assist in the prioritization of genes to be examined in future studies.

ANALYSES OF EXPRESSED SEQUENCE TAGS FROM SARGASSUM BINDERI (PHAEOPHYTA)1

Sargassum is a source for many metabolites, including alginic acid, alginates, sulfated fucoidans, pigments, oils, sterols, and mannitols. The present study is the first attempt to examine Sargassum binderi (Sonder) J. Agardh, a tropical brown seaweed, using an expressed sequence tag (EST) approach. Single‐pass 5′ sequencing of 2304 cDNA clones generated 1876 ESTs of good quality, from which 1270 tentative unique genes (TUGs) consisting of 991 singletons and 279 contigs were obtained. Approximately 39.5% of the TUGs from S. binderi showed significant matches to the existing data bases and were further divided into nine subcategories: metabolism (24.9%); transcription and translation (22.5%); cellular processes (7.2%); protein folding, sorting, and degradation (5.6%); signaling (2.8%); membrane and transport (3%); receptor (1.1%); other functions (4.2%); and unknown proteins (28.7%). The sequences were compared with ESTs from Laminaria digitata (Hudson) J. V. Lamour.

Generation and analysis of expressed sequence tags from the mangrove plant, Acanthus ebracteatus Vahl

Salinity is a major abiotic stress that greatly affects plant growth and crop production. Sodium ions in saline soil are toxic to plants because of their adverse effects on potassium nutrition, cytosolic enzyme activities, photosynthesis, and metabolism. It is important to identify genes involved in salinity tolerance from mangrove plants that survive under saline conditions. In this study, a total of 864 randomly selected cDNA clones were isolated and sequenced from the primary cDNA library of Acanthusebracteatus. Among the 521 readable sequences, 138 of them were assembled into 43 contigs, whereas 383 were singletons. Sequence analyses demonstrated that 349 of these expressed sequence tags showed significant homology to functional proteins, of which 18% are particularly interesting as they correspond to genes involved in stress response. Some of these clones, including putative mannitol dehydrogenase, plastidic aldolase, secretory peroxidase, ascorbate peroxidase, and vacuolar H+-ATPase, may be related to osmotic homeostasis, ionic homeostasis, and detoxification.