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Kevin S W Tan

Publications and source records attributed to Kevin S W Tan.

16 recordsLinked to original sources
PubMed2006

MalariaCount: an image analysis-based program for the accurate determination of parasitemia.

Malaria is a serious global health problem and rapid, precise determination of parasitemia is necessary for malaria research and in clinical settings. Manual counting by light microscopy is the most widely used technique for parasitemia determination but it is a time-consuming and laborious process. The aim of our study was to develop an automated image analysis-based system for the rapid and accurate determination of parasitemia. We have developed, for the first time, a software, MalariaCount, that automatically generates parasitemias from images of Giemsa-stained blood smears. The potential application and robustness of MalariaCount was tested in normal and drug-treated in vitro cultures of Plasmodium falciparum. The results showed a tight correlation between MalariaCount and manual count parasitemia values. These findings suggest that MalariaCount can potentially be used as a tool to provide rapid and accurate determination of parasitemia in research laboratories where frequent, large-scale, efficient determination of parasitemia is required.

Animals↗
PubMed2006

Protease activity of Blastocystis hominis.

Parasite-derived proteases are important for the parasite life cycle and the pathogenesis of the disease they produce. Proteases of intestinal protozoan parasite Blastocystis hominis were studied for the first time with azocasein assays and gelatin SDS-PAGE analysis. Parasitic lysates were found to have high protease activity and nine protease bands of low (20-33 kDa) and high (44-75 kDa) molecular weights were reported. Proteases were found to be pH-dependent and highest proteolytic activity was observed at neutral pH. Inhibition studies showed that B. hominis isolate B, like many other protozoan parasites, contains mainly cysteine proteases.

Animals↗
PubMed2006

Blastocystis ratti induces contact-independent apoptosis, F-actin rearrangement, and barrier function disruption in IEC-6 cells.

Blastocystis is an enteric protozoan purportedly associated with numerous clinical cases of diarrhea, flatulence, vomiting, and other gastrointestinal symptoms. Despite new knowledge of Blastocystis cell biology, genetic diversity, and epidemiology, its pathogenic potential remains controversial. Numerous clinical and epidemiological studies either implicate or exonerate the parasite as a cause of intestinal disease. Therefore, the aim of this study was to investigate the pathogenic potential of Blastocystis by studying the interactions of Blastocystis ratti WR1, an isolate of zoonotic potential, with a nontransformed rat intestinal epithelial cell line, IEC-6. Here, we report that B. ratti WR1 induces apoptosis in IEC-6 cells in a contact-independent manner. Furthermore, we found that B. ratti WR1 rearranges F-actin distribution, decreases transepithelial resistance, and increases epithelial permeability in IEC-6 cell monolayers. In addition, we found that the effects of B. ratti on transepithelial electrical resistance and epithelial permeability were significantly abrogated by treatment with metronidazole, an antiprotozoal drug. Our results suggest for the first time that Blastocystis-induced apoptosis in host cells and altered epithelial barrier function might play an important role in the pathogenesis of Blastocystis infections and that metronidazole has therapeutic potential in alleviating symptoms associated with Blastocystis.

Actins↗
PubMed2005

Protozoan programmed cell death--insights from Blastocystis deathstyles.

Programmed cell death (PCD) is an essential process in the growth and development of multicellular organisms. However, accumulating evidence indicates that unicellular eukaryotes can also undergo PCD with apoptosis-like features. The protozoan parasite Blastocystis hominis has been reported to exhibit both apoptotic and non-apoptotic features of PCD when exposed to a variety of stimuli. Recent observations of PCD pathways in Blastocystis suggest that this protozoan, as is the case with its multicellular counterparts, possesses complex cell-death mechanisms.

Animals↗
PubMed2005

Degradation of human secretory immunoglobulin A by Blastocystis.

Microbial immunoglobulin A (IgA) proteases cleave human secretory IgA, promoting the mucosal adhesion of pathogens. To investigate if the enteric protozoan Blastocystis degrades human secretory IgA, cell lysate and conditioned medium from two species were exposed to immunoglobulin A. Secretory IgA was cleaved by both cell lysate and conditioned medium with mainly cysteine proteinase activity in B. hominis B isolate and aspartic proteinase activity in B. ratii WR1 isolate. These findings suggest that Blastocystis proteases may play a role in parasite survival in vivo.

Animals↗
PubMed2005

Programmed cell death in Blastocystis hominis occurs independently of caspase and mitochondrial pathways.

We demonstrated previously that a cytotoxic monoclonal antibody (MAb) 1D5 elicits a programmed cell death (PCD) response in Blastocystis hominis and showed that caspase-3-like protease influences but is not essential for PCD in MAb 1D5-treated B. hominis. We also showed that mitochondrial dysregulation played a role in cell death. In the current study, we further analyzed the signaling pathways involved in PCD mediated by MAb 1D5. B. hominis cells were treated with MAb 1D5 or control MAb 5, either with or without pretreatment with a pan-caspase inhibitor, zVAD.fmk, and/or a mitochondrial transition pore blocker, cyclosporine A (CA). Flow cytometric examination of cell size, mitochondrial membrane potential (delta psi(m)), caspase activation and in situ DNA fragmentation showed that zVAD.fmk and CA, used independently or in combination, failed to inhibit MAb 1D5-mediated PCD. Interestingly, cell exposure to either inhibitor resulted in partial inhibition of DNA fragmentation while combined exposure of cells to inhibitors abolished DNA fragmentation completely. This study sheds new light on the conserved nature of PCD pathways in parasitic protozoa and is also the first report describing caspase- and mitochondria-independent cell death pathways in a protozoan parasite.

Amino Acid Chloromethyl Ketones↗
PubMed2005

Systematic study of sequence motifs for RNA trans splicing in Trypanosoma brucei.

mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5' splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3' splice sites (3'SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3'SS, we constructed a luciferase-beta-galactosidase double-reporter system. By testing approximately 90 sequences, we demonstrated that the optimum poly(Y) tract length is approximately 25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3'SS is important, and an AC dinucleotide at positions -3 and -4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3'SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.

Animals↗
PubMed2004

Plasmodium falciparum: a simplified technique for obtaining singly infected erythrocytes.

We report the development of a simple technique involving 15 ml polypropylene tubes and a rotatory incubator for obtaining erythrocytes singly infected with Plasmodium falciparum. This technique will be useful for cloning of the parasite. Our finding that P. falciparum merozoite invasion is inhibited during rotation suggests that this method may also be useful for the study of parasite-erythrocyte interactions under dynamic circulatory conditions.

Animals↗
PubMed2004

Blastocystis in humans and animals: new insights using modern methodologies.

Among the waterborne protozoan parasites of medical and veterinary importance, Blastocystis is perhaps one of the less well-understood. However, in recent years, there has been a surge of interest in the organism, fueled in part by the possible association of Blastocystis infection with intestinal disorders, and its unusual taxonomic affiliations. Although there is information on the parasite's morphology, taxonomy and mode of transmission, its pathogenicity, life cycle, and function of certain organelles continue to baffle investigators. The clinical relevance of Blastocystis will be better answered once an animal model is found. Blastocystis infections have a worldwide distribution but prevalence is highest in areas with poor hygiene and deficient sanitation services and facilities. Application of modern molecular tools has advanced knowledge of the organism's genetic diversity, taxonomy and zoonotic potential.

Animals↗
PubMed2004

Isolation and characterization of the mitochondrion-like organelle from Blastocystis hominis.

Blastocystis hominis in an unusual protozoan parasite of the human intestinal tract. Previous studies have described the presence of mitochondrial-like structures despite the anaerobic nature of the organism. In this study, we describe a simple and rapid technique to isolate and characterize mitochondrion-like organelles (MLO) from B. hominis. The parasite was disrupted using glass beads and the MLO were collected and purified using a sucrose gradient. Negative staining and transmission electron microscopy of the isolated organelles showed mitochondrial-like structures. B. hominis cells were stained with rhodamine 123 and MitoLight to show the presence of transmembrane potential of the MLO. DAPI staining of the cells suggested the presence of DNA in the MLO. Though brief reports have been made in literature, this study is the first to describe a technique for the isolation of the MLO from this organism. Using this technique of isolation, major metabolic functions of the organelle, their associated macromolecules and intra-mitochondrial location can be extensively studied. The role of MLO in this anaerobic protozoan can be widely investigated using this protocol.

Animals↗
PubMed2004

Caspase-3-like protease influences but is not essential for DNA fragmentation in Blastocystis undergoing apoptosis.

Blastocystis hominis undergo apoptosis after treatment with a cytotoxic monoclonal antibody (MAb), 1D5, by mechanisms that are not fully understood, although our previous study demonstrated that caspase-3-like protease activity is involved. To elucidate the mechanism of MAb 1D5-induced apoptosis, we inhibited Blastocystis caspase-3-like protease to investigate if there would be a concomitant decrease in in situ DNA fragmentation. However, MAb 1D5-induced apoptosis, evidenced by DNA fragmentation, was not completely blocked by pretreating with specific caspase-3 inhibitor, Ac-DEVD-CHO, indicating that caspase-independent apoptotic pathways might also be involved. Our results also revealed that the treatment with MAb 1D5 resulted in the loss of mitochondrial membrane potential (deltapsim), independent of Ac-DEVD-CHO pretreatment. In conclusion, this study demonstrates that MAb 1D5-induced apoptosis in B. hominis is not wholly dependent on caspase-3-like protease activity and is associated with mitochondrial dysregulation. This is the first report showing evidence for complex apoptotic pathways in a unicellular parasite.

Animals↗
PubMed2004

Metronidazole induces programmed cell death in the protozoan parasite Blastocystis hominis.

Previous studies by the authors have shown that the protozoan parasite Blastocystis hominis succumbed to a cytotoxic monoclonal antibody with a number of cellular and biochemical features characteristic of apoptosis in higher eukaryotes. The present study reports that apoptosis-like features are also observed in growing cultures of axenic B. hominis upon exposure to metronidazole, a drug commonly used for the treatment of blastocystosis. Upon treatment with the drug, B. hominis cells displayed key morphological and biochemical features of programmed cell death (PCD), viz. nuclear condensation and nicked DNA in nucleus, reduced cytoplasmic volume, externalization of phosphatidylserine and maintenance of plasma membrane integrity with increasing permeability. This present study also supports the authors' previously postulated novel function for the B. hominis central vacuole in PCD; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space. The implications and possible roles of PCD in B. hominis are discussed.

Animals↗
PubMed2002

Recent advances in Blastocystis hominis research: hot spots in terra incognita.

Despite being discovered more than 80 years ago, progress in Blastocystis research has been gradual and challenging, due to the small number of laboratories currently working on this protozoan parasite. To date, the morphology of Blastocystis hominis has been extensively studied by light and electron microscopy but all other aspects of its biology remain little explored areas. However, the availability of numerous and varied molecular tools and their application to the study of Blastocystis has brought us closer to understanding its biology. The purpose of this review is to describe and discuss recent advances in B. hominis research, with particular focus on new, and sometimes controversial, information that has shed light on its genetic heterogeneity, taxonomic links, mode of transmission, in vitro culture and pathogenesis. We also discuss recent observations that B. hominis has the capacity to undergo programmed cell death; a phenomenon similarly reported for many other unicellular organisms. There are still many gaps in our knowledge of this parasite. Although there is a growing body of evidence suggesting that B. hominis can be pathogenic under specific conditions, there are also other studies that indicated otherwise. Indeed, more studies are warranted before this controversial issue can be resolved. There is an urgent need for the identification and/or development of an animal model so that questions on its pathogenesis can be better answered. Another area that requires attention is the development of methods for the transfection of foreign/altered genes into B. hominis in order to facilitate genetic experiments.

Animals↗
PubMedDate not supplied

Trypanosoma brucei MRE11 is non-essential but influences growth, homologous recombination and DNA double-strand break repair.

MRE11 is a conserved multi-functional protein that is important for maintaining genomic integrity in yeast and mammalian cells. By database searching, we identified a full-length candidate MRE11 on Trypanosoma brucei chromosome II. We subsequently cloned and sequenced the corresponding gene from the Lister 427 strain. MRE11 is a single copy gene that encodes an 83 kDa protein of 763 amino acids. GFP-MRE11 and Ty1-MRE11 fusion proteins localized to the nucleus of bloodstream and procyclic T. brucei. Interestingly, Ty1-MRE11 associated, to some extent, with telomeres of procyclic but not bloodstream forms. This association appears cell-cycle dependent, with the highest co-localization in G1 cells. We were able to generate an MRE11 null mutant in bloodstream forms, indicating that it is non-essential. However, the null mutant was impaired in homologous recombination, as evidenced by the reduced integration efficiency of transfected DNA. A conditional null mutant, containing a tetracycline-inducible ectopic Ty1-MRE11, exhibited reduced growth and plating efficiency and increased sensitivity to DNA double-strand breaks, induced by methyl methanesulphonate or ionizing radiation, in the absence of tetracycline.

Amino Acid Sequence↗