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Publication Open Access Effect of amino acid polymorphisms of house dust mite der p 2 variants on allergic sensitization(2016) Sasipa Tanyaratsrisakul; Orathai Jirapongsananuruk; Bhakkawarat Kulwanich; Hales, Belinda J.; Thomas, Wayne R.; Surapon Piboonpocanun; Mahidol University. Institute of Molecular Biosciences; Mahidol University. Faculty of Medicine Siriraj Hospital. Department of Pediatricsvariants are found were compared by the affinity (IC₅₀) of IgE cross-reactivity to different variants and by direct IgE binding. Mice were immunized with the variants Der p 2.0101 and Der p 2.0110, and their IgG binding to Der p 2.0103, 2.0104, and 2... revealed that Der p 2.0104 was best for detecting IgE in both regions, followed by Der p 2.0101 with binding to other variants showing larger differences. Mouse anti-Der p 2.0101 antibodies had a high affinity of cross-reactivity but bound poorly to otherPublication Open Access Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis.(2014-03-20) Houghton, Raymond L.; Reed, Dana E.; Hubbard, Mark A.; Dillon, Michael J.; Chen, Hongjing; Currie, Bart J.; Mayo, Mark; Sarovich, Derek S.; Theobald, Vanessa; Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Gumphol Wongsuvan; Narisara Chantratita; นริศรา จันทราทิตย์; Peacock, Sharon J.; Hoffmaster, Alex R; Duval, Brea; Brett, Paul J.; Burtnick, Mary N.; AuCoin, David P.; AuCoin, David P.; Mahidol University. Faculty of Tropical Medicine. Department of Tropical Hygiene, Mahidol-Oxford Tropical Medicine Research Unit.; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology, and Mahidol-Oxford Tropical Medicine Research Unit.Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.Publication Open Access Enzymatic analysis of recombinant Japanese encephalitis virus NS2B(H)-NS3pro protease with fluorogenic model peptide substrates(2012-05) Muhammad Junaid; Chakard Chalayut; Anna Sehgelmeble Torrejon; Chanan Angsuthanasombat; Iryna Shutava; Maris Lapins; Jarl E. S. Wikberg; Gerd Katzenmeier; Mahidol University. Institute of Molecular Biosciences. Laboratory of Molecular and Cellular Microbiology(SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblottingPublication Open Access Third-stage Gnathostoma spinigerum larva excretory secretory antigens modulate function of Fc gamma receptor I-mediated monocytes in peripheral blood mononuclear cell culture(2016) Surachet Benjathummarak; Ratchanok Kumsiri; Supaporn Nuamtanong; Thareerat Kalambaheti; Jitra Waikagul; Nareerat Viseshakul; Yaowapa Maneerat; Mahidol University. Faculty of Tropical Medicine. Department of Tropical Pathology. Monocytes are innate immune cells that act as phagocytic and antigenpresenting cells and also play roles against helminthic infections via a complex interplay between other immune cells. Fc gamma receptor I (FcγRI) is a high-affinity receptorPublication Open Access Sensitivity and specificity of PS/AA-modified nanoparticles used in malaria detection(2012-11) Raweewan Thiramanas; Kulachart Jangpatarapongsa; Udom Asawapirom; Pramuan Tangboriboonrat; Duangporn Polpanich; Center for Innovation Development and Technology Transferand affinity binding via streptavidin (SA) and biotin interaction, were employed. The optimum ratio of Ab to NPs used in each immobilization procedure and the latex agglutination test based on the reaction between Ab conjugated NPs and malaria patient plasmaPublication Open Access Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ(2016) Jitlada Vasuvat; Atcha Montree; Sangduen Moonsom; Ubolsree Leartsakulpanich; Songsak Petmitr; Federico Focher; Wright, George E.; Porntip Chavalitshewinkoon‑Petmitr; Mahidol University. Faculty of Tropical Medicine. Department of ProtozoologyBackground: Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase δ is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme. Methods: The coding sequences of DNA polymerase δ catalytic subunit (PfPolδ-cat), DNA polymerase δ small subunit (PfPolδS) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC–MS/MS. PfPolδ-cat was biochemically characterized. The roles of PfPolδS and PfPCNA in PfPolδ-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPolδ-cat activity and on in vitro parasite growth using SYBR Green I assay. Results: The purified recombinant protein PfPolδ-cat, PfPolδS and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPolδ-cat and PfPolδS had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPolδ-cat possessed both DNA polymerase and 3′–5′ exonuclease activities. It used both Mg2+ and Mn2+ as cofactors and was inhibited by high KCl salt (>200 mM). PfPolδS stimulated PfPolδ-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPolδ-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 μM) and 7-acetoxypentyl-( 3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 μM). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 μM). Conclusions: Recombinant PfPolδ-cat, PfPolδS and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPolδ-cat. The high sensitivity of PfPolδ to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.Publication Open Access Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target(2014) Thidarat Suksangpleng; Ubolsree Leartsakulpanich; Saengduen Moonsom; Saranya Siribal; Usa Boonyuen; Wright, George E; Porntip Chavalitshewinkoon-Petmitr; Mahidol University. Faculty of Tropical Medicine. Department of Protozoology,Background: Based on resistance of currently used anti-malarials, a new anti-malarial drug target against Plasmodium falciparum is urgently needed. Damaged DNA cannot be transcribed without prior DNA repair; therefore, uracil-DNA glycosylase, playing an important role in base excision repair, may act as a candidate for a new anti-malarial drug target. Methods: Initially, the native PfUDG from parasite crude extract was partially purified using two columns, and the glycosylase activity was monitored. The existence of malarial UDG activity prompted the recombinant expression of PfUDG for further characterization. The PfUDG from chloroquine and pyrimethamine resistant P. falciparum strain K1 was amplified, cloned into the expression vector, and expressed in Escherichia coli. The recombinant PfUDG was analysed by SDS-PAGE and identified by LC-MS/MS. The three dimensional structure was modelled. Biochemical properties were characterized. Inhibitory effects of 12 uracil-derivatives on PfUDG activity were investigated. Inhibition of parasite growth was determined in vitro using SYBR Green I and compared with results from human cytotoxicity tests. Results: The native PfUDG was partially purified with a specific activity of 1,811.7 units/mg (113.2 fold purification). After cloning of 966-bp PCR product, the 40-kDa hexa-histidine tagged PfUDG was expressed and identified. The amino acid sequence of PfUDG showed only 24.8% similarity compared with the human enzyme. The biochemical characteristics of PfUDGs were quite similar. They were inhibited by uracil glycosylase inhibitor protein as found in other organisms. Interestingly, recombinant PfUDG was inhibited by two uracil-derived compounds; 1-methoxyethyl-6-(p-n-octylanilino) uracil (IC50 of 16.75 μM) and 6-(phenylhydrazino)uracil (IC50 of 77.5 μM). Both compounds also inhibited parasite growth with IC50s of 15.6 and 12.8 μM, respectively. Moreover, 1-methoxyethyl-6-(p-n-octylanilino)uracil was not toxic to HepG2 cells, with IC50 of > 160 μM while 6-(phenylhydrazino)uracil exhibited cytoxicity, with IC50 of 27.5 μM. Conclusions: The recombinant PfUDG was expressed, characterized and compared to partially purified native PfUDG. Their characteristics were not significantly different. PfUDG differs from human enzyme in its size and predicted amino acid sequence. Two uracil derivatives inhibited PfUDG and parasite growth; however, only one non-cytotoxic compound was found. Therefore, this selective compound can act as a lead compound for anti-malarial development in the future.Publication Open Access Interleukin-1beta interferes with signal transduction induced by neurotrophin-3 in cortical neurons.(2012) Rungtip Soiampornkula; Tongb, Liqi; Wipawan Thangnipona; Balazsb, Robert; Cotmanb, Carl W.; Tongb, LiqiIt was previously observed that IL-1beta interferes with BDNF-induced TrkB-mediated signal transduction and protection of cortical neurons from apoptosis evoked by deprivation from trophic support [Tong L., Balazs R., Soiampornkul R., Thangnipon W., Cotman C.W., 2007. Interleukin-1beta impairs brain derived neurotrophic factor-induced signal transduction. Neurobiol. Aging]. Here we investigated whether the effect of the cytokine on neurotrophin signaling is more general. The influence of IL-1beta on NT-3 signaling was therefore studied under conditions when NT-3 primarily activated the TrkC receptor. The cytokine reduced NT-3-induced activation of MAPK/ERK and Akt, but did not interfere with Trk receptor autophosphorylation. IL-1beta reduced tyrosine phosphorylation of the docking proteins, IRS-1 and Shc, which convey receptor activation to the downstream protein kinase cascades. These are the steps that are also inhibited by IL-1beta in BDNF-induced signal transduction. The functional consequences of the effect of IL-1beta on NT-3 signaling were severe, as NT-3 protection of the trophic support-deprived cortical neurons was abrogated. In view of the role in the maintenance and plasticity of neurons of ERK, Akt and CREB, which are activated by neurotrophins, elevated IL-1beta levels in the brain in Alzheimer's disease and other neurodegenerative diseases might contribute to the decline in cognitive functions before the pathological signs of the disease develop.Publication Open Access The crystal structure of JNK from Drosophila melanogaster reveals an evolutionarily conserved topology with that of mammalian JNK proteins(2015) Sarin Chimnaronk; Jatuporn Sitthiroongruang; Kanokporn Srisucharitpanit; Monrudee Srisaisup; Albert J. Ketterman; Panadda Boonserm; Mahidol University. Institute of Molecular BiosciencesBackground: The c-Jun N-terminal kinases (JNKs), members of the mitogen-activated protein kinase (MAPK) family, engage in diverse cellular responses to signals produced under normal development and stress conditions. In Drosophila, only one JNK member is present, whereas ten isoforms from three JNK genes (JNK1, 2, and 3) are present in mammalian cells. To date, several mammalian JNK structures have been determined, however, there has been no report of any insect JNK structure. Results: We report the first structure of JNK from Drosophila melanogaster (DJNK). The crystal structure of the unphosphorylated form of DJNK complexed with adenylyl imidodiphosphate (AMP-PNP) has been solved at 1.79 Å resolution. The fold and topology of DJNK are similar to those of mammalian JNK isoforms, demonstrating their evolutionarily conserved structures and functions. Structural comparisons of DJNK and the closely related mammalian JNKs also allow identification of putative catalytic residues, substrate-binding sites and conformational alterations upon docking interaction with Drosophila scaffold proteins. Conclusions: The DJNK structure reveals common features with those of the mammalian JNK isoforms, thereby allowing the mapping of putative catalytic and substrate binding sites. Additionally, structural changes upon peptide binding could be predicted based on the comparison with the closely-related JNK3 structure in complex with pepJIP1. This is the first structure of insect JNK reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of insect JNK.Publication Open Access Classification of P-glycoprotein-interacting compounds using machine-learning methods(2015-07) Watshara Shoombuatong; Apilak Worachartcheewan; Veda Prachayasittikul; Chanin Nantasenamat; Virapong Prachayasittikul; Mahidol University. Faculty of Medical Technology. Center of Data Mining and Biomedical InformaticsP-glycoprotein (Pgp) is a drug transporter that plays important roles in multidrug resistance and drug pharmacokinetics. The inhibition of Pgp has become a notable strategy for combating multidrug-resistant cancers and improving therapeutic outcomes. However, the polyspecific nature of Pgp, together with inconsistent results in experimental assays, renders the determination of endpoints for Pgp-interacting compounds a great challenge. In this study, the classification of a large set of 2,477 Pgp-interacting compounds (i.e., 1341 inhibitors, 913 non-inhibitors, 197 substrates and 26 non-substrates) was performed using several machine learning methods (i.e., decision tree induction, artificial neural network modelling and support vector machine) as a function of their physicochemical properties. The models provided good predictive performance, producing MCC values in the range of 0.739-1 for internal cross-validation and 0.665-1 for external validation. The study provided simple and interpretable models for important properties that influence the activity of Pgp-interacting compounds, which are potentially beneficial for screening and rational design of Pgp inhibitors that are of clinical importance.