Publications by authors named "Atchara Paemanee"

41 Publications

Caspase-3, a shrimp phosphorylated hemocytic protein is necessary to control YHV infection.

Fish Shellfish Immunol 2021 Apr 14;114:36-48. Epub 2021 Apr 14.

Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Phutthamonthon 4 Rd, Salaya, Nakhon Pathom, 73170, Thailand. Electronic address:

By using immunohistochemistry detection, yellow head virus (YHV) was found to replicate in granule-containing hemocytes including semi-granular hemocytes (SGC) and granular hemocytes (GC) during the early phase (24 h post injection) of YHV-infected shrimp. Higher signal of YHV infection was found in GC more than in SGC. Comparative phosphoproteomic profiles between YHV-infected and non-infected GC reveal a number of phosphoproteins with different expression levels. The phosphoprotein spot with later on identified as caspase-3 in YHV-infected GC is most interesting. Blocking caspase-3 function using a specific inhibitor (Ac-DEVD-CMK) demonstrated high replication of YHV and consequently, high shrimp mortality. The immunohistochemistry results confirmed the high viral load in shrimp that caspase-3 activity was blocked. Caspase-3 is regulated through a variety of posttranslational modifications, including phosphorylation. Analysis of phosphorylation sites of shrimp caspase-3 revealed phosphorylation sites at serine residue. Taken together, caspase-3 is a hemocytic protein isolated from shrimp granular hemocytes with a role in anti-YHV response and regulated through the phosphorylation process.
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http://dx.doi.org/10.1016/j.fsi.2021.04.007DOI Listing
April 2021

Black rice cultivar from Java Island of Indonesia revealed genomic, proteomic, and anthocyanin nutritional value.

Acta Biochim Pol 2021 Mar;68(1):55-63

1Research Center of Smart Molecule of Natural Genetics Resources, Brawijaya University, Indonesia; 2Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Indonesia.

Black rice is considered to be functional food containing anthocyanins as bioactive compounds. This study examined the genomic and proteomic patterns in local black rice from Java Island, Indonesia, with attention to the mechanism of anthocyanin synthesis. Three kinds of black rice from Java Island, including black rice from East Java (BREJ), black rice from Central Java (BRCJ), and black rice from West Java (BRWJ), were studied in comparison to white rice (WREJ) and red rice (RREJ). Genomic profiling was done by simple sequence repeat (SSR) analysis, and sequencing of red coleoptile (Rc) and glycosyltransferase (GT) genes, followed by in silico analysis. Total anthocyanin was investigated by ultra-high performance liquid chromatography- diode array detector (UHPLC-DAD). The proteomic profiles were determined by liquid-chromatography and mass spectrometry of tryptic peptides. The SSR profiles showed a specific band in each black rice variant. The Rc gene exon-2 sequences were similar in the three black rice cultivars. The GT gene sequence was identified as a new variant that correlates with the purple stem, leaf, bran, and whole grain morphology seen exclusively in the BRWJ cultivar. The anthocyanin composition in Java black rice is diverse. The highest cyanidin level was seen in BRWJ and the highest level of peonidin-3-O-glucoside in BREJ. Proteomic profiling of the black rice cultivars demonstrated that the expression of proteins that might be related to the levels of anthocyanin synthesis varied. These studies conclude that the genomic, proteomic and anthocyanins composition of Java black rice cultivars may be used the improvement of their functional nutrition values.
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http://dx.doi.org/10.18388/abp.2020_5386DOI Listing
March 2021

Andrographolide and Its 14-Aryloxy Analogues Inhibit Zika and Dengue Virus Infection.

Molecules 2020 Oct 30;25(21). Epub 2020 Oct 30.

School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 211816, China.

Andrographolide is a labdene diterpenoid with potential applications against a number of viruses, including the mosquito-transmitted dengue virus (DENV). In this study, we evaluated the anti-viral activity of three 14-aryloxy analogues (ZAD-1 to ZAD-3) of andrographolide against Zika virus (ZIKV) and DENV. Interestingly, one analogue, ZAD-1, showed better activity against both ZIKV and DENV than the parental andrographolide. A two-dimension (2D) proteomic analysis of human A549 cells treated with ZAD-1 compared to cells treated with andrographolide identified four differentially expressed proteins (heat shock 70 kDa protein 1 (HSPA1A), phosphoglycerate kinase 1 (PGK1), transketolase (TKT) and GTP-binding nuclear protein Ran (Ran)). Western blot analysis confirmed that ZAD-1 treatment downregulated expression of HSPA1A and upregulated expression of PGK1 as compared to andrographolide treatment. These results suggest that 14-aryloxy analogues of andrographolide have the potential for further development as anti-DENV and anti-ZIKV agents.
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http://dx.doi.org/10.3390/molecules25215037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662321PMC
October 2020

Proteomic analysis of monkey kidney LLC-MK2 cells infected with a Thai strain Zika virus.

Arch Virol 2019 Mar 5;164(3):725-737. Epub 2019 Jan 5.

Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonton Sai 4, Salaya, Nakorn Pathom, 73170, Thailand.

Zika virus (ZIKV) has been endemic in Southeast Asian countries for several years, but the presence of the virus has not been associated with significant outbreaks of infection unlike other countries around the world where the Asian lineage ZIKV was introduced recently. However, few studies have been undertaken using the endemic virus. The Thai isolate was shown to have a similar tissue tropism to an African isolate of ZIKV, albeit that the Thai isolate infected cells at a lower level as compared to the African isolate. To further understand the pathogenesis of the Thai isolate, a 2D-gel proteomic analysis was undertaken of ZIKV infected LLC-MK2 cells. Seven proteins (superoxide dismutase [Mn], peroxiredoxin 2, ATP synthase subunit alpha, annexin A5 and annexin A1, carnitine o-palmitoyltransferase 2 and cytoskeleton-associated protein 2) were identified as differentially regulated. Of four proteins selected for validation, three (superoxide dismutase [Mn], peroxiredoxin 2, ATP synthase subunit alpha, and annexin A1) were shown to be differentially regulated at both the transcriptional and translational levels. The proteins identified were primarily involved in energy production both directly, and indirectly through mediation of autophagy, as well as in the response to oxidative stress, possibly occurring as a consequence of increased energy production. This study provides further new information on the pathogenesis of ZIKV.
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http://dx.doi.org/10.1007/s00705-018-04137-1DOI Listing
March 2019

A proteomic analysis of the anti-dengue virus activity of andrographolide.

Biomed Pharmacother 2019 Jan 3;109:322-332. Epub 2018 Nov 3.

Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon Sai 4, Salaya, Nakorn Pathom 73170, Thailand. Electronic address:

Andrographolide is a major bioactive constituent of Andrographis paniculata that has been shown in vitro to have antiviral activity against a number of viruses, including the mosquito transmitted dengue virus (DENV). However, how andrographolide exerts an anti-DENV effect remains unclear. This study therefore sought to further understand the mechanism of action of andrographolide in inhibiting DENV infection of liver cells using a proteomic based approach. Both 1 dimension (D) and 2D proteome systems were used. Initial data was generated through andrographolide treatment of HepG2 cells without DENV infection (1D analysis), while subsequent data was generated through a combination of andrographolide treatment and DENV infection (2D analysis). A total of 17 (1D) and 18 (2D) proteins were identified as differentially regulated. The analyses identified proteins involved in chaperone activities, as well as energy production. In particular evidence suggested an important role for GRP78 and the unfolded protein response in mediating the anti-DENV activity of andrographolide, which might, in part, explain the broad antiviral activity of andrographolide.
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http://dx.doi.org/10.1016/j.biopha.2018.10.054DOI Listing
January 2019

Novel bioactive peptides demonstrating anti-dengue virus activity isolated from the Asian medicinal plant Acacia Catechu.

Chem Biol Drug Des 2019 02 10;93(2):100-109. Epub 2018 Oct 10.

Division of Molecular Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.

The therapeutic activities of food-derived bioactive proteins and peptides are attracting increased attention within the research community. Medicinal plants used in traditional medicines are an excellent source of bioactive proteins and peptides, especially those traditionally prepared by water extraction for use as tea or food supplement. In this study, novel bioactive peptides were isolated from enzymatic digests of 33 Thai medicinal plants. The inhibitory activity of each against dengue virus (DENV) infection was investigated. Of 33 plants, peptides from Acacia catechu extract demonstrated the most pronounced anti-DENV activity. Half maximal inhibitory concentration of 0.18 μg/ml effectively inhibited DENV foci formation. Treatment with 1.25 μg/ml crude peptide extract could reduce virus production less than 100-fold with no observable cell toxicity. Peptide sequences were determined by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Two bioactive peptides isolated from Acacia catechu inhibited DENV foci formation >90% at the concentration of 50 μM; therefore, they are recommended for further investigation as antiviral peptides against DENV infection.
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http://dx.doi.org/10.1111/cbdd.13400DOI Listing
February 2019

Interferon gamma induces cellular protein alteration and increases replication of porcine circovirus type 2 in PK-15 cells.

Arch Virol 2018 Nov 23;163(11):2947-2957. Epub 2018 Jul 23.

Center of Advanced Studies in Agriculture and Food, Kasetsart University, Bangkok, 10900, Thailand.

Porcine circovirus type 2 (PCV2) infections may lead to the development of subclinical signs or chronic systemic syndromes, collectively known as "porcine circovirus-associated disease" (PCVAD) in swine. Interferon gamma (IFN-γ) is known to enhance PCV2 replication in vitro, and immune mediators may act as pivotal factors in triggering PCV2 infection progression toward PCVAD. We determined the effects of IFN-γ on PCV2 replication in PK-15 cells. PCV2 was cultured in the presence or absence of exogenous swine IFN-γ (swIFNγ). Growth curve analysis in PK-15 cells revealed that PCV2 could replicate to a significantly higher titer in swIFNγ medium. To investigate the host cell response upon PVC2 infection, differential expression of proteins in PCV2-infected PK-15 cells with or without swIFNγ stimulation was analyzed by proteomics (LC-MS/MS) analysis. A large proportion of the differentially expressed proteins in swIFNγ-treated PCV2-infected cells were found to be involved in apoptosis, cellular stress responses, cell survival/proliferation pathways, and inflammatory responses. We further confirmed the expression of these differentially expressed proteins at the mRNA levels by qRT-PCR. PCV2 infection in PK-15 cells in the presence of IFN-γ resulted in upregulation of cellular proteins in responses to stress, cell survival, and cell proliferation (Hsp90, MAP3K7, RAS-GTPase, c-myc, and 14-3-3 epsilon) as well as in an increase in the levels of proteins (CASP9 and TRAF5) related to the apoptosis pathways. Thus, PCV2 exploits several cellular biological processes through IFN activation for enhancing viral replication. This is the first evidence of IFN-γ promoting PCV2 replication in vitro via a mechanism similar to that used by several human viruses.
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http://dx.doi.org/10.1007/s00705-018-3944-1DOI Listing
November 2018

Screening of melatonin, α-tocopherol, folic acid, acetyl-L-carnitine and resveratrol for anti-dengue 2 virus activity.

BMC Res Notes 2018 May 16;11(1):307. Epub 2018 May 16.

Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonthol Sai 4, Salaya, Nakorn Pathom, 73170, Thailand.

Objective: Infections with the mosquito transmitted dengue virus (DENV) are a significant public health burden in many parts of the world. Despite the introduction of a commercial vaccine in some parts of the world, the majority of the populations at risk of infection remain unprotected against this disease, and there is currently no treatment for DENV infection. Natural compounds offer the prospect of cheap and sustainable therapeutics to reduce the disease burden during infection, and thus potentially alleviate the risk of more severe disease. This study evaluated the potential anti-DENV 2 activity of five natural compounds namely melatonin, α-tocopherol, folic acid, acetyl-L-carnitine and resveratrol in two different cell lines.

Results: Screening of the compounds showed that one compound (acetyl-L-carnitine) showed no effect on DENV infection, three compounds (melatonin, α-tocopherol and folic acid) slightly increased levels of infection, while the 5th compound, resveratrol, showed some limited anti-DENV activity, with resveratrol reducing virus output with an EC of less than 25 μM. These results suggest that some commonly taken natural compounds may have beneficial effects on DENV infection, but that others may potentially add to the disease burden.
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http://dx.doi.org/10.1186/s13104-018-3417-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5956857PMC
May 2018

Simultaneous saccharification and viscosity reduction of cassava pulp using a multi-component starch- and cell-wall degrading enzyme for bioethanol production.

3 Biotech 2017 Oct 28;7(5):290. Epub 2017 Aug 28.

Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Klong Luang, 12120 Pathum Thani Thailand.

In this study, an efficient ethanol production process using simultaneous saccharification and viscosity reduction of raw cassava pulp with no prior high temperature pre-gelatinization/liquefaction step was developed using a crude starch- and cell wall-degrading enzyme preparation from BCC17849. Proteomic analysis revealed that the enzyme comprised a complex mixture of endo- and exo-acting amylases, cellulases, xylanases, and pectina ses belonging to various glycosyl hydrolase families. Enzymatic hydrolysis efficiency was dependent on the initial solid loading in the reaction. Reduction in mixture viscosity was observed with a rapid decrease in complex viscosity from 3785 to 0.45 Pa s with the enzyme dosage of 2.19 mg/g on a dried weight basis within the first 2 h, which resulted from partial destruction of the plant cell wall fiber and degradation of the released starch granules by the enzymes as shown by scanning electron microscopy. Saccharification of cassava pulp at an initial solid of 16% (w/v) in a bench-scale bioreactor resulted in 736.4 mg glucose/g, which is equivalent to 82.92% glucose yield based on the total starch and glucan in the substrate, after 96 h at 40 °C. Simultaneous saccharification and fermentation of cassava pulp by with the uncooked enzymatic process led to a final ethanol concentration of 6.98% w/v, equivalent to 96.7% theoretical yield based on the total starch and cellulose content. The results demonstrated potential of the enzyme for low-energy processing of cassava pulp in biofuel industry.
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http://dx.doi.org/10.1007/s13205-017-0924-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573685PMC
October 2017

Nevirapine induced mitochondrial dysfunction in HepG2 cells.

Sci Rep 2017 08 23;7(1):9194. Epub 2017 Aug 23.

Institute of Molecular Biosciences, Mahidol University, Bangkok, Thailand.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor frequently used in combination with other antiretroviral agents for highly active antiretroviral therapy (HAART) of patients infected with the human immunodeficiency virus type 1 (HIV-1). However NVP can cause serious, life-threatening complications. Hepatotoxicity is one of the most severe adverse effects, particularly in HIV patients with chronic hepatitis C virus co-infection as these patients can develop liver toxicity after a relatively short course of treatment. However, the mechanism of NVP-associated hepatotoxicity remains unclear. This study sought to investigate the effect of NVP on protein expression in liver cells using a proteomic approach. HepG2 cells were treated or not treated with NVP and proteins were subsequently resolved by two-dimensional gel electrophoresis. A total of 33 differentially regulated proteins were identified, of which nearly 40% (13/33) were mitochondrial proteins. While no obvious differences were observed between NVP treated and untreated cells after staining mitochondria with mitotracker, RT-PCR expression analysis of three mitochondrially encoded genes showed all were significantly up-regulated in NVP treated cells. Mitochondrial dysfunction was observed in response to treatment even with slightly sub-optimal therapeutic treatment concentrations of NVP. This study shows that NVP induces mitochondrial dysregulation in HepG2 cells.
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http://dx.doi.org/10.1038/s41598-017-09321-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569014PMC
August 2017

Comparative proteomic analysis of Chlamydomonas reinhardtii control and a salinity-tolerant strain revealed a differential protein expression pattern.

Planta 2017 Nov 7;246(5):843-856. Epub 2017 Jul 7.

Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Ngamwongwan Rd., Bangkok, 10900, Thailand.

Main Conclusion: Proteins involved in membrane transport and trafficking, stress and defense, iron uptake and metabolism, as well as proteolytic enzymes, were remarkably up-regulated in the salinity-tolerant strain of Chlamydomonas reinhardtii. Excessive concentration of NaCl in the environment can cause adverse effects on plants and microalgae. Successful adaptation of plants to long-term salinity stress requires complex cellular adjustments at different levels from molecular, biochemical and physiological processes. In this study, we developed a salinity-tolerant strain (ST) of the model unicellular green alga, Chlamydomonas reinhardtii, capable of growing in medium containing 300 mM NaCl. Comparative proteomic analyses were performed to assess differential protein expression pattern between the ST and the control progenitor cells. Proteins involved in membrane transport and trafficking, stress and defense, iron uptake and metabolism, as well as protein degradation, were remarkably up-regulated in the ST cells, suggesting the importance of these processes in acclimation mechanisms to salinity stress. Moreover, 2-DE-based proteomic also revealed putative salinity-specific post-translational modifications (PTMs) on several important housekeeping proteins. Discussions were made regarding the roles of these differentially expressed proteins and the putative PTMs in cellular adaptation to long-term salinity stress.
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http://dx.doi.org/10.1007/s00425-017-2734-4DOI Listing
November 2017

The significance of proline and glutamate on butanol chaotropic stress in 168.

Biotechnol Biofuels 2017 11;10:122. Epub 2017 May 11.

Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330 Thailand.

Background: Butanol is an intensively used industrial solvent and an attractive alternative biofuel, but the bioproduction suffers from its high toxicity. Among the native butanol producers and heterologous butanol-producing hosts, 168 exhibited relatively higher butanol tolerance. Nevertheless, organic solvent tolerance mechanisms in Bacilli and Gram-positive bacteria have relatively less information. Thus, this study aimed to elucidate butanol stress responses that may involve in unique tolerance of 168 to butanol and other alcohol biocommodities.

Results: Using comparative proteomics approach and molecular analysis of butanol-challenged 168, 108 butanol-responsive proteins were revealed, and classified into seven groups according to their biological functions. While parts of them may be similar to the proteins reportedly involved in solvent stress response in other Gram-positive bacteria, significant role of proline in the proline-glutamate-arginine metabolism was substantiated. Detection of intracellular proline and glutamate accumulation, as well as glutamate transient conversion during butanol exposure confirmed their necessity, especially proline, for cellular butanol tolerance. Disruption of the particular genes in proline biosynthesis pathways clarified the essential role of the anabolic ProB-ProA-ProI system over the osmoadaptive ProH-ProA-ProJ system for cellular protection in response to butanol exposure. Molecular modifications to increase gene dosage for proline biosynthesis as well as for glutamate acquisition enhanced butanol tolerance of 168 up to 1.8% (vol/vol) under the conditions tested.

Conclusion: This work revealed the important role of proline as an effective compatible solute that is required to protect cells against butanol chaotropic effect and to maintain cellular functions in 168 during butanol exposure. Nevertheless, the accumulation of intracellular proline against butanol stress required a metabolic conversion of glutamate through the specific biosynthetic ProB-ProA-ProI route. Thus, exogenous addition of glutamate, but not proline, enhanced butanol tolerance. These findings serve as a practical knowledge to enhance 168 butanol tolerance, and demonstrate means to engineer the bacterial host to promote higher butanol/alcohol tolerance of 168 for the production of butanol and other alcohol biocommodities.
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http://dx.doi.org/10.1186/s13068-017-0811-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425972PMC
May 2017

Glycoproteomics analysis of plasma proteins associated with Opisthorchis viverrini infection-induced cholangiocarcinoma in hamster model.

Asian Pac J Trop Med 2016 12 9;9(12):1165-1171. Epub 2016 Nov 9.

Graduate Program in Bioclinical Sciences, Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma, Chulabhorn International College of Medicine, Thailand; Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma, Chulabhorn International College of Medicine, Thailand; Excellence Center in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma, Thammasat University, Thailand. Electronic address:

Objective: To apply lectin affinity chromatography and glycoproteomics-based LC-MS/MS to preliminarily investigate the possible potential plasma biomarkers of Opisthorchis viverrini (OV)-associated CCA in OV/dimethylnitrosamine (DMN)-induced CCA hamster model.

Methods: Nine Syrian hamsters were divided into 3 groups as follows (n = 3 each): normal (healthy control group); OV group; and OV/DMN group (CCA group). Pooled plasma samples collected from animals in each group at the 6th month post-infection with OV metacercarae were subjected to glycoproteomics analysis. Glycoproteins in the pooled sample from each group were initially isolated by concanavalin A (ConA)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LC-MS/MS.

Results: Among the 24 ConA-binding glycoproteins isolated, two proteins, N-myc downstream regulated gene 1 (NDRG1) and fetuin-B (FETUB) were found up-regulated only in the samples from the OV and control groups, but not in the OV/DMN (CCA) groups. On the other hand, one protein, i.e., NSFL1 cofactor p47 isoform ×3 (NSFL1C) was found only in the samples from OV/DMN (CCA) and control groups, but not in the OV group. The remaining 21 proteins were upregulated in the samples from all groups.

Conclusions: NDRG1, FETUB and NSFL1C glycoproteins isolated by ConA-based affinity chromatography could be potential biomarkers for CCA. Plasma samples with negative for NDRG1 and FETUB proteins but positive for NSFL1C are likely to be OV-associated CCA. Nevertheless, this conclusion remains to be confirmed whether this battery test can discriminate OV-associated CCA from other risk factors.
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http://dx.doi.org/10.1016/j.apjtm.2016.09.013DOI Listing
December 2016

Salivary Gland Proteome during Adult Development and after Blood Feeding of Female Anopheles dissidens Mosquitoes (Diptera: Culicidae).

PLoS One 2016;11(9):e0163810. Epub 2016 Sep 26.

Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, United Kingdom.

Understanding changes in mosquito salivary proteins during the time that sporozoite maturation occurs and after blood feeding may give information regarding the roles of salivary proteins during the malarial transmission. Anopheles dissidens (formerly Anopheles barbirostris species A1) is a potential vector of Plasmodium vivax in Thailand. In this study, analyses of the proteomic profiles of female An. dissidens salivary glands during adult development and after blood feeding were carried out using two-dimensional gel electrophoresis coupled with nano-liquid chromatography-mass spectrometry. Results showed at least 17 major salivary gland proteins present from day one to day 21 post emergence at 8 different time points sampled. Although there was variation observed, the patterns of protein expression could be placed into one of four groups. Fifteen protein spots showed significant depletion after blood feeding with the percentages of the amount of depletion ranging from 8.5% to 68.11%. The overall results identified various proteins, including a putative mucin-like protein, an anti-platelet protein, a long form D7 salivary protein, a putative gVAG protein precursor, a D7-related 3.2 protein, gSG7 salivary proteins, and a gSG6 protein. These results allow better understanding of the changes of the salivary proteins during the adult mosquito development. They also provide candidate proteins to investigate any possible link or not between sporozoite maturation, or survival of skin stage sporozoites, and salivary proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163810PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036837PMC
September 2016

Nevirapine induces apoptosis in liver (HepG2) cells.

Asian Pac J Trop Med 2016 Jun 16;9(6):547-53. Epub 2016 Apr 16.

Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthol Sai 4, Salaya, Nakorn Pathom 73170, Thailand. Electronic address:

Objective: To generate insights into the mechanism of NVP induced hepatotoxicity.

Methods: Liver (HepG2) cells were cultured with various concentrations of NVP. This cell line was chosen because it has low expression of cytochrome P450, allowing evaluation of the effects of NVP rather than specific metabolites. Cytotoxicity was determined using a proliferation assay and cell numbers were monitored using trypan blue exclusion assay for long term culture experiments and apoptosis induction was determined by morphological and biochemical investigation.

Results: HepG2 cells treated with the highest concentration of NVP tested (819 μM) initially showed a rounded morphology and all cells had died by week three of exposure. Nuclear condensation and fragmentation, increased Annexin V/propidium iodide staining and caspase 9 activation all supported the induction of apoptosis in HepG2 cells in response to NVP treatment.

Conclusions: There is a clear induction of apoptosis in response to NVP which suggests that NVP has significant cytotoxicity, over and above any cytotoxicity of metabolites and may contribute directly to patient hepatotoxicity.
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http://dx.doi.org/10.1016/j.apjtm.2016.04.015DOI Listing
June 2016

Application of GelC-MS/MS to Proteomic Profiling of Chikungunya Virus Infection: Preparation of Peptides for Analysis.

Methods Mol Biol 2016 ;1426:179-93

Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamontol Sai 4, Salaya, Nakorn Pathom, Thailand.

Gel-enhanced liquid chromatography coupled with tandem mass spectrometry (GeLC-MS/MS) is a labor intensive, but relatively straightforward methodology that generates high proteome coverage which can be applied to the proteome analysis of a range of starting materials such as cells or patient specimens. Sample proteins are resolved electrophoretically in one dimension through a sodium dodecyl sulfate (SDS) polyacrylamide gel after which the lanes are sliced into sections. The sections are further diced and the gel cubes generated are subjected to in-gel tryptic digestion. The resultant peptides can then be analyzed by tandem mass spectroscopy to identify the proteins by database searching. The methodology can routinely detect several thousand proteins in one analysis. The protocol we describe here has been used with both cells in culture that have been infected with chikungunya virus and specimens from Chikungunya fever patients. This protocol details the process for generating peptides for subsequent mass spectroscopic and bioinformatic analysis.
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http://dx.doi.org/10.1007/978-1-4939-3618-2_16DOI Listing
December 2017

Mass spectrometric analysis of host cell proteins interacting with dengue virus nonstructural protein 1 in dengue virus-infected HepG2 cells.

Biochim Biophys Acta 2016 09 21;1864(9):1270-1280. Epub 2016 Apr 21.

Division of Dengue Hemorrhagic Fever Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10700, Thailand. Electronic address:

Dengue virus (DENV) infection is a leading cause of the mosquito-borne infectious diseases that affect humans worldwide. Virus-host interactions appear to play significant roles in DENV replication and the pathogenesis of DENV infection. Nonstructural protein 1 (NS1) of DENV is likely involved in these processes; however, its associations with host cell proteins in DENV infection remain unclear. In this study, we used a combination of techniques (immunoprecipitation, in-solution trypsin digestion, and LC-MS/MS) to identify the host cell proteins that interact with cell-associated NS1 in an in vitro model of DENV infection in the human hepatocyte HepG2 cell line. Thirty-six novel host cell proteins were identified as potential DENV NS1-interacting partners. A large number of these proteins had characteristic binding or catalytic activities, and were involved in cellular metabolism. Coimmunoprecipitation and colocalization assays confirmed the interactions of DENV NS1 and human NIMA-related kinase 2 (NEK2), thousand and one amino acid protein kinase 1 (TAO1), and component of oligomeric Golgi complex 1 (COG1) proteins in virus-infected cells. This study reports a novel set of DENV NS1-interacting host cell proteins in the HepG2 cell line and proposes possible roles for human NEK2, TAO1, and COG1 in DENV infection.
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http://dx.doi.org/10.1016/j.bbapap.2016.04.008DOI Listing
September 2016

Mitochondrial Changes in β0-Thalassemia/Hb E Disease.

PLoS One 2016 19;11(4):e0153831. Epub 2016 Apr 19.

Institute of Molecular Biosciences, Mahidol University, Salaya campus, 25/25 Phuttamontol Sai 4, Salaya, Nakorn Pathom, 73170, Thailand.

The compound β°-thalassemia/Hb E hemoglobinopathy is characterized by an unusually large range of presentation from essentially asymptomatic to a severe transfusion dependent state. While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation. Mitochondria are subcellular organelles that are pivotal in a number of cellular processes including oxidative phosphorylation and apoptosis. A mitochondrial protein enriched proteome was determined and validated from erythroblasts from normal controls and β°-thalassemia/Hb E patients of different severities. Mitochondria were evaluated through the use of mitotracker staining, analysis of relative mitochondrial genome number and evaluation of mitochondrial gene expression in addition to assay of overall cellular redox status through the use of alamarBlue assays. Fifty differentially regulated mitochondrial proteins were identified. Mitotracker staining revealed significant differences in staining between normal control erythroblasts and those from β°-thalassemia/Hb E patients. Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells. Significant differences were seen in redox status as evaluated by alamarBlue staining in newly isolated CD34+ cells. Mitochondria mediate oxidative phosphorylation and apoptosis, both of which are known to be dysregulated in differentiating erythrocytes from β°-thalassemia/Hb E patients. The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153831PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836671PMC
September 2016

Identification of Hsp90 as a species independent H5N1 avian influenza A virus PB2 interacting protein.

Comp Immunol Microbiol Infect Dis 2015 Dec 23;43:28-35. Epub 2015 Oct 23.

Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamontol Sai 4, Salaya 73170, Nakorn Pathom, Thailand. Electronic address:

The avian influenza polymerase protein PB2 subunit is an important mediator of cross species adaptation and adaptation to mammalian cells is strongly but not exclusively associated with an adaptive mutation of the codon at position 627 of the PB2 protein which alters the glutamate normally found at this position to a lysine. This study sought to identify host cell factors in both mammalian and avian cells that interacted in a species specific or species independent manner. Two PB2 fusion proteins differing only in codon 627 were generated and transfected into mammalian and avian cells and interacting proteins identified through co-immunoprecipitation. A number of proteins including Hsp90 were identified and further investigation showed that Hsp90 interacted with both isoforms of PB2 in both mammalian and avian cells. Hsp90 is thus identified as a species independent interacting protein, further confirming that this protein may be a suitable target for anti-influenza drug development.
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http://dx.doi.org/10.1016/j.cimid.2015.10.001DOI Listing
December 2015

Comparative Proteomics of Activated THP-1 Cells Infected with Mycobacterium tuberculosis Identifies Putative Clearance Biomarkers for Tuberculosis Treatment.

PLoS One 2015 27;10(7):e0134168. Epub 2015 Jul 27.

Department of Microbiology and Research and Diagnostic Center for Emerging Infectious Diseases (RCEID), Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.

Biomarkers for determining clearance of Mycobacterium tuberculosis (Mtb) infection during anti-tuberculosis therapy or following exposure could facilitate enhanced monitoring and treatment. We screened for biomarkers indicating clearance of Mtb infection in vitro. A comparative proteomic analysis was performed using GeLC MSI/MS. Intracellular and secreted proteomes from activated THP-1 cells infected with the Mtb H37Rv strain (MOI = 1) and treated with isoniazid and rifampicin for 1 day (infection stage) and 5 days (clearance stage) were analyzed. Host proteins associated with early infection (n = 82), clearance (n = 121), sustained in both conditions (n = 34) and suppressed by infection (n = 46) were elucidated. Of the potential clearance markers, SSFA2 and CAECAM18 showed the highest and lowest protein intensities, respectively. A western blot of CAECAM18 validated the LC MS/MS result. For three clearance markers (SSFA2, PARP14 and PSME4), in vivo clinical validation was concordantly reported in previous patient cohorts. A network analysis revealed that clearance markers were enriched amongst four protein interaction networks centered on: (i) CD44/CCND1, (ii) IFN-β1/NF-κB, (iii) TP53/TGF-β and (iv) IFN-γ/CCL2. After infection, proteins associated with proliferation, and recruitment of immune cells appeared to be enriched possibly reflecting recruitment of defense mechanisms. Counteracting proteins (CASP3 vs. Akt and NF-κB vs. TP53) associated with apoptosis regulation and its networks were enriched among the early and sustained infection biomarkers, indicating host-pathogen competition. The BRCA1/2 network was suppressed during infection, suggesting that cell proliferation suppression is a feature of Mtb survival. Our study provides insights into the mechanisms of host-Mtb interaction by comparing the stages of infection clearance. The identified clearance biomarkers may be useful in monitoring tuberculosis treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134168PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516286PMC
May 2016

Novel Serum Biomarkers to Differentiate Cholangiocarcinoma from Benign Biliary Tract Diseases Using a Proteomic Approach.

Dis Markers 2015 28;2015:105358. Epub 2015 Apr 28.

Department of Biochemistry, Faculty of Science, Mahidol University, 272 Rama VI Road, Phayathai, Rajdhevi, Bangkok 10400, Thailand.

Background And Aim: Cholangiocarcinoma (CCA) is the most frequent biliary malignancy, which poses high mortality rate due to lack of early detection. Hence, most CCA cases are present at the advanced to late stages with local or distant metastasis at the time of diagnosis. Currently available tumor markers including CA19-9 and CEA are inefficient and of limited usage due to low sensitivity and specificity. Here, we attempt to identify serum tumor markers for CCA that can effectively distinguish CCA from benign biliary tract diseases (BBTDs).

Methods: Serum samples from 19 CCA patients and 17 BBTDs were separated by SDS-PAGE followed with LC-MS/MS and were subjected to statistical analysis and cross-validation to identify proteins whose abundance was significantly elevated or suppressed in CCA samples compared to BBTDs.

Results: In addition to identifying several proteins previously known to be differentially expressed in CCA and BBTDs, we also discovered a number of molecules that were previously not associated with CCA. These included FAM19A5, MAGED4B, KIAA0321, RBAK, and UPF3B.

Conclusions: Novel serum biomarkers to distinguish CCA from BBTDs were identified using a proteomic approach. Further validation of these proteins has the potential to provide a biomarker for differentiating CCA from BBTDs.
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http://dx.doi.org/10.1155/2015/105358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427802PMC
February 2016

Comparative Proteomic Analysis of Human Cholangiocarcinoma Cell Lines: S100A2 as a Potential Candidate Protein Inducer of Invasion.

Dis Markers 2015 27;2015:629367. Epub 2015 Apr 27.

Department of Biochemistry, Faculty of Science, Mahidol University, 272 Rama VI Road, Bangkok 10400, Thailand.

Cholangiocarcinoma (CCA) is a bile duct cancer, commonly found in Asia including Thailand and especially in the northeastern region of Thailand. To identify the proteins involved in carcinogenesis and metastasis of CCA, protein expression profiles of high-invasive KKU-M213 and low-invasive KKU-100 cell lines were compared using a comparative GeLC-MS/MS proteomics analysis. A total of 651 differentially expressed proteins were detected of which 27 protein candidates were identified as having functions involved in cell motility. A total of 22 proteins were significantly upregulated in KKU-M213, whereas 5 proteins were downregulated in KKU-M213. S100A2, a calcium-binding protein in S100 protein family, is upregulated in KKU-M213. S100A2 is implicated in metastasis development in several cancers. The protein expression level of S100A2 was verified by Western blot analysis. Intriguingly, high-invasive KKU-M213 cells showed higher expression of S100A2 than KKU-100 cells, consistent with proteomic data, suggesting that S100A2 may be a key protein involved in the progression of CCA. However, the biological function of S100A2 in cholangiocarcinoma remains to be elucidated. S100A2 might be a potential biomarker as well as a novel therapeutic target in CCA metastasis.
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http://dx.doi.org/10.1155/2015/629367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426780PMC
February 2016

Plasmodium vivax inhibits erythroid cell growth through altered phosphorylation of the cytoskeletal protein ezrin.

Malar J 2015 Mar 31;14:138. Epub 2015 Mar 31.

Department of Pathobiology, Faculty of Science, Mahidol University, 272 Rama VI Rd., Ratchathewi District, 10400, Bangkok, Thailand.

Background: The underlying causes of severe malarial anaemia are multifactorial. In previously reports, Plasmodium vivax was found to be able to directly inhibited erythroid cell proliferation and differentiation. The molecular mechanisms underlying the suppression of erythropoiesis by P. vivax are remarkably complex and remain unclear. In this study, a phosphoproteomic approach was performed to dissect the molecular mechanism of phosphoprotein regulation, which is involved in the inhibitory effect of parasites on erythroid cell development.

Methods: This study describes the first comparative phosphoproteome analysis of growing erythroid cells (gECs), derived from human haematopoietic stem cells, exposed to lysates of infected erythrocytes (IE)/uninfected erythrocytes (UE) for 24, 48 and 72 h. This study utilized IMAC phosphoprotein isolation directly coupled with LC MS/MS analysis.

Results: Lysed IE significantly inhibited gEC growth at 48 and 72 h and cell division resulting in the accumulation of cells in G0 phase. The relative levels of forty four phosphoproteins were determined from gECs exposed to IE/UE for 24-72 h and compared with the media control using the label-free quantitation technique. Interestingly, the levels of three phosphoproteins: ezrin, alpha actinin-1, and Rho kinase were significantly (p < 0.05) altered. These proteins display interactions and are involved in the regulation of the cellular cytoskeleton. Particularly affected was ezrin (phosphorylated at Thr567), which is normally localized to gEC cell extension peripheral processes. Following exposure to IE, for 48-72 h, the ezrin signal intensity was weak or absent. This result suggests that phospho-ezrin is important for actin cytoskeleton regulation during erythroid cell growth and division.

Conclusions: These findings suggest that parasite proteins are able to inhibit erythroid cell growth by down-regulation of ezrin phosphorylation, leading to ineffective erythropoiesis ultimately resulting in severe malarial anaemia. A better understanding of the mechanisms of ineffective erythropoiesis may be beneficial in the development of therapeutic strategies to prevent severe malarial anaemia.
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http://dx.doi.org/10.1186/s12936-015-0648-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392472PMC
March 2015

Comparative plasma protein profiling of hemoglobin H disease.

Dis Markers 2014 15;2014:340214. Epub 2014 Jun 15.

Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Nakhon Pathom 73170, Thailand.

HbH and HbH-constant spring (HbH-CS) are the most common forms of α-thalassemia detected in the Thai population. The accumulation of excess β globin chains in these diseases results in increased red cell hemolysis, and patients with HbH-CS normally have a more severe clinical presentation than patients with HbH disease. This study aimed to detect alterations in the expression of plasma proteins of HbH and HbH-CS patients as compared to normal plasma. Platelet poor plasma was separated from HbH and HbH-CS and normal subjects and differential plasma proteins were detected using two-dimensional gel electrophoresis and identified using LC/MS/MS. A total of 14 differentially expressed proteins were detected of which 5 proteins were upregulated and 9 were downregulated. Most of the differentially expressed proteins are liver secreted proteins involved in hemolysis, oxidative stress response, and hemoglobin degradation. Seven proteins were found to be differentially expressed between HbH and HbH-CS. Levels of haptoglobin, a hemoglobin scavenging protein, were significantly increased in HbH patients as compared to HbH-CS patients. The identification of differentially expressed proteins may lead to a better understanding of the biological events underlying the clinical presentation of HbH and HbH-CS patients and can have application as hemolytic markers or severity predictors.
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http://dx.doi.org/10.1155/2014/340214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082903PMC
April 2015

Shotgun proteomics analysis of proliferating STRO-1-positive human dental pulp cell after exposure to nacreous water-soluble matrix.

Clin Oral Investig 2015 Mar 13;19(2):261-70. Epub 2014 Jun 13.

Department of Oral Medicine and Periodontology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand,

Introduction: For dental treatment, dentin regeneration is required after a tooth injury with dental pulp exposure. The effects of the water-soluble matrix (WSM) extracted from the nacreous layer of the bivalve Pinctada maxima on human dental pulp cells in vitro were challenging and useful for clinical application.

Material And Methods: The biological activity of the STRO-1-positive human dental pulp cells in response to WSM compared to Dulbecco's modified Eagle medium (DMEM) as a normal control was monitored. The cell survival rate was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Proteomic profiles among inducers and noninducers with time dependency were compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS).

Results: The human dental pulp cells cultured in nacreous WSM exhibited higher relative cell viability than those in DMEM with similar morphological appearance. Significant changes were found in the relative abundance of 44 proteins in cells after exposure to WSM for 2 weeks. They play a role in cell adhesion, cell proliferation, metabolic process, signal transduction, stress response, transcription, translation, and transport.

Conclusion: These results indicate that WSM of P. maxima has the ability to induce proliferation of human dental pulp cells.

Clinical Relevance: This finding initiated the study to evaluate the suitability of nacre as biomaterial for dentistry.
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http://dx.doi.org/10.1007/s00784-014-1256-8DOI Listing
March 2015

Comprehensive proteomic analysis of white blood cells from chikungunya fever patients of different severities.

J Transl Med 2014 Apr 11;12:96. Epub 2014 Apr 11.

Institute of Molecular Biosciences, Mahidol University, Salaya Campus 25/25 Phuttamonthol Sai 4, Nakorn Pathom 73170, Thailand.

Background: Chikungunya fever (CHIKF) is a recently re-emerged mosquito transmitted viral disease caused by the chikungunya virus (CHIKV), an Alphavirus belonging to the family Togaviridae. Infection of humans with CHIKV can result in CHIKF of variable severity, although the factors mediating disease severity remain poorly defined.

Methods: White blood cells were isolated from blood samples collected during the 2009-2010 CHIKF outbreak in Thailand. Clinical presentation and viral load data were used to classify samples into three groups, namely non chikungunya fever (non-CHIKF), mild CHIKF, and severe CHIKF. Five samples from each group were analyzed for protein expression by GeLC-MS/MS.

Results: CHIKV proteins (structural and non-structural) were found only in CHIKF samples. A total of 3505 human proteins were identified, with 68 proteins only present in non-CHIKF samples. A total of 240 proteins were found only in CHIKF samples, of which 65 and 46 were found only in mild and severe CHIKF samples respectively. Proteins with altered expression mapped predominantly to cellular signaling pathways (including toll-like receptor and PI3K-Akt signaling) although many other processes showed altered expression as a result of CHIKV infection. Expression of proteins consistent with the activation of the inflammasome was detected, and quantitation of (pro)-caspase 1 at the protein and RNA levels showed an association with disease severity.

Conclusions: This study confirms the infection of at least a component of white blood cells by CHIKV, and shows that CHIKV infection results in activation of the inflammasome in a manner that is associated with disease severity.
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http://dx.doi.org/10.1186/1479-5876-12-96DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022080PMC
April 2014

Identification of salivary gland proteins depleted after blood feeding in the malaria vector Anopheles campestris-like mosquitoes (Diptera: Culicidae).

PLoS One 2014 5;9(3):e90809. Epub 2014 Mar 5.

Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.

Malaria sporozoites must invade the salivary glands of mosquitoes for maturation before transmission to vertebrate hosts. The duration of the sporogonic cycle within the mosquitoes ranges from 10 to 21 days depending on the parasite species and temperature. During blood feeding salivary gland proteins are injected into the vertebrate host, along with malaria sporozoites in the case of an infected mosquito. To identify salivary gland proteins depleted after blood feeding of female Anopheles campestris-like, a potential malaria vector of Plasmodium vivax in Thailand, two-dimensional gel electrophoresis and nano-liquid chromatography-mass spectrometry techniques were used. Results showed that 19 major proteins were significantly depleted in three to four day-old mosquitoes fed on a first blood meal. For the mosquitoes fed the second blood meal on day 14 after the first blood meal, 14 major proteins were significantly decreased in amount. The significantly depleted proteins in both groups included apyrase, 5'-nucleotidase/apyrase, D7, D7-related 1, short form D7r1, gSG6, anti-platelet protein, serine/threonine-protein kinase rio3, putative sil1, cyclophilin A, hypothetical protein Phum_PHUM512530, AGAP007618-PA, and two non-significant hit proteins. To our knowledge, this study presents for the first time the salivary gland proteins that are involved in the second blood feeding on the day corresponding to the transmission period of the sporozoites to new mammalian hosts. This information serves as a basis for future work concerning the possible role of these proteins in the parasite transmission and the physiological processes that occur during the blood feeding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090809PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944739PMC
November 2014

Salivary gland proteome of the human malaria vector, Anopheles campestris-like (Diptera: Culicidae).

Parasitol Res 2013 Mar 22;112(3):1065-75. Epub 2012 Dec 22.

Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand.

Anopheles campestris-like is proven to be a high-potential vector of Plasmodium vivax in Thailand. In this study, A. campestris-like salivary gland proteins were determined and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis, and nano-liquid chromatography-mass spectrometry. The total amount of salivary gland proteins in the mosquitoes aged 3-5 days was approximately 0.1 ± 0.05 μg/male and 1.38 ± 0.01 μg/female. SDS-PAGE analysis revealed at least 12 major proteins found in the female salivary glands and each morphological region of the female glands contained different major proteins. Two-dimensional gel electrophoresis showed approximately 20 major and several minor protein spots displaying relative molecular masses from 10 to 72 kDa with electric points ranging from 3.9 to 10. At least 15 glycoproteins were detected in the female glands. Similar electrophoretic protein profiles were detected comparing the male and proximal-lateral lobes of the female glands, suggesting that these lobes are responsible for sugar feeding. Blood-feeding proteins, i.e., putative 5'-nucleotidase/apyrase, anti-platelet protein, long-form D7 salivary protein, D7-related 1 protein, and gSG6, were detected in the distal-lateral lobes (DL) and/or medial lobes (ML) of the female glands. The major spots related to housekeeping proteins from other arthropod species including Culex quinquefasciatus serine/threonine-protein kinase rio3 expressed in both male and female glands, Ixodes scapularis putative sil1 expressed in DL and ML, and I. scapularis putative cyclophilin A expressed in DL. These results provide information for further study on the salivary gland proteins of A. campestris-like that are involved in hematophagy and disease transmission.
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http://dx.doi.org/10.1007/s00436-012-3233-yDOI Listing
March 2013

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.).

Food Chem 2012 Oct 28;134(3):1533-41. Epub 2012 Mar 28.

Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Biochemical Research Unit for Feed Utilisation Assessment, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Center for Advanced Studies in Tropical Natural Resources (CASTNAR), National Research University-Kasetsart University (NRU-KU), Kasetsart University, Bangkok 10900, Thailand.

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification - by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose - was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0-11.0 and an optimal temperature of approximately 55-60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis-Menten constant (Km) and catalytic constant (Kcat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s(-1), respectively. The catalytic efficiency (Kcat/Km) was 238 s(-1) mM(-1).
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http://dx.doi.org/10.1016/j.foodchem.2012.03.074DOI Listing
October 2012

Identification of prohibitin as a Chikungunya virus receptor protein.

J Med Virol 2012 Nov;84(11):1757-70

Institute of Molecular Biosciences, Mahidol University, Nakorn Pathom, Thailand.

Chikungunya virus (CHIKV) has recently re-emerged causing millions of infections in countries around the Indian Ocean. While CHIKV has a broad host cell range and productively infects a number of different cell types, macrophages have been identified as a potential viral reservoir serving to increase the duration of symptoms. To date no CHIKV interacting protein has been characterized and this study sought to identify CHIKV binding proteins expressed on target cell membranes. Two-dimensional virus overlay identified prohibitin (PHB) as a microglial cell expressed CHIKV binding protein. Co-localization, co-immunoprecipitation as well as antibody and siRNA mediated infection inhibition studies all confirmed a role for PHB in mediating internalization of CHIKV into microglial cells. PHB is the first identified CHIKV receptor protein, and this study is evidence that PHB may play a role in the internalization of multiple viruses.
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http://dx.doi.org/10.1002/jmv.23403DOI Listing
November 2012