Tan Ching Ong

A rule-based approach for robust clump splitting

This paper presents a robust rule-based approach for the splitting of binary clumps that are formed by objects of diverse shapes and sizes. First, the deepest boundary pixels, i.e., the concavity pixels in a clump, are detected using a fast and accurate scheme. Next, concavity-based rules are applied to generate the candidate split lines that join pairs of concavity pixels. A figure of merit is used to determine the best split line from the set of candidate lines. Experimental results show that the proposed approach is robust and accurate.

Nuclear Magnetic Resonance Structure and IgE Epitopes of Blo t 5, a Major Dust Mite Allergen

A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel α-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices α2 and α3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76–91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.

Structures of two major allergens, Bla g 4 and Per a 4, from cockroaches and their IgE binding epitopes.

Inhalant allergens from cockroaches are an important cause of asthma to millions of individuals worldwide. Here we report for the first time the structures of two major cockroach allergens, Bla g 4 and Per a 4, that adopt a typical lipocalin fold but with distinct structural features as compared with other known lipocalin allergens. Both Bla g 4 and Per a 4 contain two long-range disulfide bonds linking the N and C termini to a β-barrel. The C-terminal helix of Bla g 4 is bent and greatly extended toward the N terminus. Bla g 4 is found to be a monomer, whereas Per a 4 exists as a dimer in solution with a novel dimeric interface involving residues from loops at the top and bottom of the β-barrel. Putative ligand binding sites of both allergens are determined by docking of the juvenile hormone III inside the β-barrel and found to interact with the ligand using non-conserved residues. Bla g 4 and Per a 4 are found to be cross-reactive in sera IgE binding, at least in the Singaporean Chinese population tested. A major IgE binding epitope unique to Per a 4 is found on the loops at the bottom of the β-barrel that may aid the development of hypoallergens for immunotherapy.

NMR structure and IgE epitopes of Blo t 21, a major dust mite allergen from Blomia tropicalis

Background: Blo t 21 is a paralogue of the group 5 allergen with comparable allergenicity. Results: Blo t 21 has an additional conformational IgE epitope and only a low to moderate cross-reactivity with Blo t 5. Conclusion: Blo t 21 represents a novel group of major allergen in the dust mite Blomia tropicalis. Significance: The structure and IgE epitopes of Blo t 21 allow design of hypoallergen for immunotherapy. Blo t 21 is a paralogue of the group 5 allergen, Blo t 5, a major allergen from the dust mite Blomia tropicalis. Blo t 21 has moderate sequence identity (40.7%) to Blo t 5 and low to moderate cross-reactivity to Blo t 5. In B. tropicalis, the most prevalent and allergenic allergens are in the order of Blo t 21, Blo t 5, and Blo t 7. Here, we determined the NMR solution structure of Blo t 21, which represents the first structure of the group 21 dust mite allergen. The structure of Blo t 21 closely resembles the structures of Blo t 5 and Der p 5, comprising three anti-parallel α-helices arranged in a helical bundle. Using site-directed mutagenesis and specific IgE binding ELISA, Blo t 21 was found to contain both conserved and unique charged IgE epitope residues at the L2 loop region and on helix α3. Cross-inhibition assays confirmed that Blo t 21 has a low to moderate cross-reactivity with Blo t 5 and Der p 5 and represents a novel group of major allergen in B. tropicalis. In addition to group 5 allergens, Blo t 21 has also a low to moderate cross-reactivity with group 21 allergens from Dermatophagoides mites, confirming that B. tropicalis is a major and distinct source of dust mite allergens.

Crystal Structure of Der f 7, a Dust Mite Allergen from Dermatophagoides farinae.

Background Der f 7 is the group 7 allergen from the dust mite Dermatophagoides farinae, homologous to the major allergen Der p 7 from D. pteronyssinus. Monoclonal antibody that bind to residues Leu48 and Phe50 was found to inhibit IgE binding to residue Asp159, which is important for the cross-reactivity between Der f 7 and Der p 7. Methodology/Principal Findings Here, we report the crystal structure of Der f 7 that shows an elongated and curved molecule consisting of two anti-parallel β-sheets – one 4-stranded and the other 5-stranded – that wrap around a long C-terminal helix. The overall fold of Der f 7 is similar to Der p 7 but key difference was found in the β1–β2 loop region. In Der f 7, Leu48 and Phe50 are in close proximity to Asp159, explaining why monoclonal antibody binding to Leu48 and Phe50 can inhibit IgE binding to Asp159. Both Der f 7 and Der p 7 bind weakly to polymyxin B via a similar binding site that is formed by the N-terminal helix, the 4-stranded β-sheet and the C-terminal helix. The thermal stability of Der f 7 is significantly lower than that of Der p 7, and the stabilities of both allergens are highly depend on pH. Conclusion/Significance Der f 7 is homologous to Der p 7 in terms of the amino acid sequence and overall 3D structure but with significant differences in the region proximal to the IgE epitope and in thermal stability. The crystal structure of Der f 7 provides a basis for studying the function and allergenicity of this group of allergens.

Determinants of indoor allergens in tropical child care centers

Limited data are available about indoor allergen determinants in child care centers (CCCs) especially in the tropics. This information is important epidemiologically and clinically considering many children attend CCCs. The purpose of this study was to determine the allergen concentrations in CCCs and their associations with CCC characteristics and indoor air quality (IAQ). A panel of indoor allergens including Der p 1, Blo t 5, Fel d 1, Can f 1, Mus m 1, Bla g 1 and Asp f 1 were evaluated from dusts vacuumed from classroom floors of CCCs. Allergen levels were assayed with antibody‐based bioplex array or enzyme‐linked immunosorbent assays. Indoor temperature, relative humidity and air exchange rates were measured and CCC characteristics inspected. Allergen levels were linearly regressed with CCC characteristics and IAQ. The dominant allergens found in classroom floors were Der p 1, Blo t 5 and Fel d 1. Lower indoor temperatures were associated with higher Der p 1 while lower ventilation rates were associated with higher Blo t 5 concentrations. Prevalence of cat owners was found to be a predictor for Fel d 1 concentrations. Full or partial carpeting is associated with higher dust mite allergen levels. These findings provide information for future indoor allergen exposure assessment studies in CCCs and can be used for intervention with regard to allergen avoidance.

Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex suspension array.

We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing results showed complete concordance in genotype assignments. The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping. It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.

Segmentation of microscope cell images via adaptive eigenfilters

This paper presents the use of a PCA based approach to segment cells from RGB light microscope images. The proposed segmentation is accurate and robust under uneven illumination, lighting variation, and noise. Principal component analysis (PCA) is first applied to the RGB color bands of the image. The image corresponding to the principal component has significantly better contrast over the original image. A set of eigenfilters is then obtained by applying PCA to local neighborhoods of this image. A pair of filters from this set, corresponding to the second and third largest eigenvalues, resembles ramp edge filters with orientations that adapt to the image. These edge filters are used to obtain the edgemap of the image. We define a criterion that enables accurate detection of valid edges of cells while suppressing noise.

Fern spore and pollen airspora profile of Singapore

Monitoring atmospheric fern spore and pollen loads in Singapore was initiated in June 1990. Aside from the more numerous fungal spores, fern spores and pollen grains made up 6.2–8.6% and 4.4–5.4% of the total airspora sampled, respectively. The most frequently encountered fern spores, in descending order, were those of Nephrolepisauriculata, Dicranopterislinearis, Stenochlaenapalustris, Aspleniumnidus, Pteridiumaquilinum, and Dicranopteriscurranii. For pollen grains, the most frequently encountered, in descending order, were Elaeisguineensis, Casuarinaequisetifolia, Acaciaauriculiformis, Kyllingiapolyphylla, Podocarpus, and Poaceae pollen grains. Seasonal patterns for individual fern spore or pollen types were discernible despite the relatively uniform tropical climate in Singapore. The fern spore and pollen calendar for the period 1991–1995 was compared to that of 2005–2006 as a follow-up study to keep abreast with the rapidly changing landscape of Singapore. Diurnal patterns showing a late morning to afternoon peak period were seen in fern spores, while the peak period was in the morning for pollen types studied with the exception to oil palm pollen. Additionally, association between fern spore and pollen counts and local meteorological conditions were also analyzed and found to be highly correlated. This study has thus identified the fern spores and pollen airspora components, and determined the calendars, as well as diurnal profiles of the Singapore airspora and provides invaluable information for allergy studies by highlighting the trigger sources present in the environment.

Collembola are unlikely to cause human dermatitis

Abstract There have been several unconfirmed case reports of dermatitis caused by Collembola (springtails). We recently investigated two nurses with dermatitis suspected to be caused by Drepanura Schött (Collembola: Entomobryidae). IgE antibodies to Collembola proteins were not detected in sera from the nurses and skin tests with the Collembola extract and crushed whole Collembola were negative in both the nurses and volunteers. This study suggests that the springtail Drepanura may not cause human dermatitis and that other organisms and organic matter that are also found in the moist environment inhabited by Collembola might instead be responsible.