Publications by authors named "Khajornsak Tragoolpua"

25 Publications

  • Page 1 of 1

Activity of Propolis Nanoparticles against HSV-2: Promising Approach to Inhibiting Infection and Replication.

Molecules 2022 Apr 15;27(8). Epub 2022 Apr 15.

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.

Herpes simplex type 2 (HSV-2) infection causes a significant life-long disease. Long-term side effects of antiviral drugs can lead to the emergence of drug resistance. Thus, propolis, a natural product derived from beehives, has been proposed to prevent or treat HSV-2 infections. Unfortunately, therapeutic applications of propolis are still limited due its poor solubility. To overcome this, a nanoparticle-based drug delivery system was employed. An ethanolic extract of propolis (EEP) was encapsulated in nanoparticles composed of poly(lactic-co-glycolic acid) and chitosan using a modified oil-in-water single emulsion by using the solvent evaporation method. The produced nanoparticles (EEP-NPs) had a spherical shape with a size of ~450 nm and presented satisfactory physicochemical properties, including positively charged surface (38.05 ± 7.65 mV), high entrapment efficiency (79.89 ± 13.92%), and sustained release profile. Moreover, EEP-NPs were less cytotoxic on Vero cells and exhibited anti-HSV-2 activity. EEP-NPs had a direct effect on the inactivation of viral particles, and also disrupted the virion entry and release from the host cells. A significant decrease in the expression levels of the HSV-2 replication-related genes (, , and ) was also observed. Our study suggests that EEP-NPs provide a strong anti-HSV-2 activity and serve as a promising platform for the treatment of HSV-2 infections.
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http://dx.doi.org/10.3390/molecules27082560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9032435PMC
April 2022

Prevalences of SARS-CoV-2 RNA and anti-SARS-CoV-2 among at-risk populations in Chiang Mai and Lamphun provinces, Thailand, during November 2020-January 2021.

PLoS One 2022 2;17(2):e0263127. Epub 2022 Feb 2.

Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiangmai, Thailand.

Non-healthcare workers with a high potential for exposure to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) may contribute to the virus spreading. Data among asymptomatic and high exposure risk populations is still scarce, in particular Chiang Mai and Lamphun provinces, Thailand. We conducted a cross-sectional observational study aiming to assess the prevalence of SARS-CoV-2 RNA positivity, anti-SARS-CoV-2 IgM/IgG, and potential associated factors among asymptomatic/mild symptomatic individuals with a high exposure risk in Chiang Mai and Lamphun provinces, during the second wave of outbreak in Thailand (November 2020-January 2021). Socio-demographic data was collected through an on-line questionnaire prior to collection of nasopharyngeal/throat swab samples and blood samples tested for SARS-CoV-2 RNA (DaAn Gene, China) and anti-SARS-CoV-2 IgM/IgG antibodies (commercial lateral flow immunoassays), respectively. Univariable and multivariable logistic regression analysis were used to analyze associated factors. None of 1,651 participants were found positive for SARS-CoV-2 RNA (0%, 95% confidence intervals, CI: 0-0.2). Fourteen were positive for anti-SARS-CoV-2 IgM/IgG antibodies (0.9%, 95% CI: 0.5-1.4), including 7 positives for IgM and 7 positives for IgG (0.4%, 95% CI: 0.2-0.9). Being over 50 years old was independently associated with virus exposure (OR: 5.8, 95% CI: 1.0-32.1%, p = 0.045). Despite high exposure risk, no current infection was found, and a very high proportion was still susceptible to SARS-CoV-2 infection and would clearly benefit from vaccination. Continuing active surveillance, rolling out of vaccination and monitoring response to vaccine will help better control the COVID-19 spread.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0263127PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8809620PMC
February 2022

Fungicidal Activity of Recombinant Javanicin against Is Associated with Intracellular Target(s) Involved in Carbohydrate and Energy Metabolic Processes.

Molecules 2021 Nov 20;26(22). Epub 2021 Nov 20.

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.

The occurrence of , the human fungal pathogen that primarily infects immunocompromised individuals, has been progressing at an alarming rate. The increased incidence of infection of with antifungal drugs resistance has become a global concern. Potential antifungal agents with extremely low toxicity are urgently needed. Herein, the biological activities of recombinant javanicin (r-javanicin) against were evaluated. A time-killing assay was performed and both concentration- and time-dependent antifungal activity of r-javanicin were indicated. The inhibitory effect of the peptide was initially observed at 4 h post-treatment and ultimately eradicated within 36 to 48 h. Fungal outer surface alteration was characterized by the scanning electron microscope (SEM) whereas a negligible change with slight shrinkage of external morphology was observed in r-javanicin treated cells. Confocal laser scanning microscopic analysis implied that the target(s) of r-javanicin is conceivably resided in the cell thereby allowing the peptide to penetrate across the membrane and accumulate throughout the fungal body. Finally, cryptococcal cells coped with r-javanicin were preliminarily investigated using label-free mass spectrometry-based proteomics. Combined with microscopic and proteomics analysis, it was clearly elucidated the peptide localized in the intracellular compartment where carbohydrate metabolism and energy production associated with glycolysis pathway and mitochondrial respiration, respectively, were principally interfered. Overall, r-javanicin would be an alternative candidate for further development of antifungal agents.
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http://dx.doi.org/10.3390/molecules26227011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8618071PMC
November 2021

Potentiality of Melittin-Loaded Niosomal Vesicles Against Vancomycin-Intermediate and Staphylococcal Skin Infection.

Int J Nanomedicine 2021 16;16:7639-7661. Epub 2021 Nov 16.

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.

Background: is an important human pathogen, especially causing skin and soft tissue infections (SSTIs). Over the decades, the infections caused by antibiotic-resistant strains have often become life-threatening. Consequently, exploration and development of competent approaches to combat these serious circumstances are urgently required.

Methods: The antibacterial activity of melittin (Mel) on , methicillin-resistant (MRSA) and clinical isolates of vancomycin-intermediate (VISA) was investigated by minimum inhibitory concentration (MIC) and time-killing assays. The localization of Mel on the bacterial cell was visualized by confocal laser scanning microscopy and its effect on the membrane was indicated based on propidium iodide uptake. The non-ionic surfactant vesicle (NISV) or niosome nanocarrier was established for Mel loading (Mel-loaded NISV) by the thin-film hydration method. Physicochemical and in vitro biological properties of Mel-loaded NISVs were characterized. The cellular uptake of Mel-loaded NISVs was evaluated by holotomography analysis. In addition, an ex vivo study was conducted on a porcine ear skin model to assess the permeation ability of Mel-loaded NISVs and their potential to inhibit bacterial skin infection.

Results: The effective inhibitory activity of Mel on skin pathogens was demonstrated. Among the tested strains, VISA was most susceptible to Mel. Regarding to its function, Mel targeted the bacterial cell envelope and disrupted cell membrane integrity. Mel-loaded NISVs were successfully fabricated with a nano-size of 120-200 nm and entrapment efficiency of greater than 90%. Moreover, Mel-loaded NISVs were taken up and accumulated in the intracellular space. Meanwhile, Mel was released and distributed throughout the cytosol and nucleus. Mel-loaded NISVs efficiently inhibited the growth of bacteria, particularly MRSA and VISA. Importantly, they not only penetrated epidermal and dermal skin layers, but also reduced the bacterial growth in infected skin.

Conclusion: Mel-loaded NISVs have a great potential to exhibit antibacterial activity. Therapeutic application of Mel-loaded NISVs could be further developed as an alternative platform for the treatment of skin infection via dermal and transdermal delivery.
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http://dx.doi.org/10.2147/IJN.S325901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8606986PMC
November 2021

Targeted Propolis-Loaded Poly (Butyl) Cyanoacrylate Nanoparticles: An Alternative Drug Delivery Tool for the Treatment of Cryptococcal Meningitis.

Front Pharmacol 2021 20;12:723727. Epub 2021 Aug 20.

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.

In this study, we describe a nano-carrier system for propolis that is able to cross an model of the blood-brain barrier (BBB) and effectively reduce the virulence of in animal models. Antimicrobial properties of propolis have been widely studied. However, propolis applications are limited by its low water solubility and poor bioavailability. Therefore, we recently formulated novel poly (n-butyl cyanoacrylate) nanoparticles (PBCA-NP) containing propolis. PBCA-NP are biocompatible, biodegradable and have been shown to effectively cross the BBB using apolipoprotein E (ApoE) as a ligand. Prepared nanoparticles were characterized for particle size, zeta potential, propolis entrapment efficiency and release. Additionally, the PBCA-NP were functionalized with polysorbate 80, which then specifically adsorbs ApoE. Using an BBB model of human brain microvascular endothelial cells hCMEC/D3, it was shown that fluorescence labelled ApoE-functionalized PBCA-NP were internalized by the cells and translocated across the cell monolayer. Propolis-loaded PBCA-NP had , antifungal activity against , which causes meningitis. To utilize the invertebrate model, larvae were infected with and treated with propolis-loaded PBCA-NP. The larvae exhibited normal behavior in toxicity testing, and treatment with propolis-loaded PBCA-NP increased survival in the -infected larvae group. In addition, following cryptococcal infection and then 7 days of treatment, the tissue fungal burden of mice treated with propolis-loaded PBCA-NP was significantly lower than control groups. Therefore, our ApoE-functionalized propolis-loaded PBCA-NP can be deemed as a potential targeted nanoparticle in the therapeutic treatment of cerebral cryptococcosis.
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http://dx.doi.org/10.3389/fphar.2021.723727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417799PMC
August 2021

Direct Detection of from Cerebrospinal Fluid, Positive Hemoculture, and Simultaneous Differentiation of Serotypes 1, 1/2, 2, and 14 within Single Reaction.

Pathogens 2021 Aug 6;10(8). Epub 2021 Aug 6.

Division of Clinical Microbiology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.

is an emerging zoonotic bacterium causing septicemia and meningitis in humans. Due to rapid disease progression, high mortality rate, and many underdiagnosed cases by time-consuming routine identification methods, alternative diagnostic testing is essential. Among 29 broadly accepted serotypes, serotypes 2 and 14 are high prevalent; however, many PCR assays showed an inability to differentiate serotype 2 from 1/2, and 1 from 14. In this study, we developed and validated a new multiplex PCR assay that facilitates the identification of only the 29 true serotypes of and simultaneously differentiates serotypes 1, 1/2, 2, and 14 within a single reaction. Importantly, the multiplex PCR could detect directly from positive hemocultures and CSF. The results revealed high sensitivity, specificity, and 100% accuracy with almost perfect agreement (κ = 1.0) compared to culture and serotyping methods. Direct detection enables a decrease in overall diagnosis time, rapid and efficient treatment, reduced fatality rates, and proficient disease control. This multiplex PCR offers a rapid, easy, and cost-effective method that can be applied in a routine laboratory. Furthermore, it is promising for developing point-of-care testing (POCT) for detection in the future.
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http://dx.doi.org/10.3390/pathogens10080996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8401787PMC
August 2021

Genotypic Distribution and a Potential Diagnostic Assay of Multidrug-Resistant Tuberculosis in Northern Thailand.

Infect Drug Resist 2020 30;13:3375-3382. Epub 2020 Sep 30.

Office for Research and Development Affairs, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

Introduction: Knowledge of the prevalence and distribution of multidrug-resistant tuberculosis (MDR-TB) genotypes in northern Thailand is still limited. An accurate, rapid, and cost-effective diagnostic of MDR-TB is crucial to improve treatment and control of increased MDR-TB.

Materials And Methods: The molecular diagnostic assays named "RIF-RD" and "INH-RD" were designed to detect rifampicin (RIF) and isoniazid (INH) resistance based on real-time PCR and high-resolution melting curve analysis. Applying the ∆T cutoff values, the RIF-RD and INH-RD were evaluated against the standard drug susceptibility testing (DST) using 107 and 103 clinical (Mtb) isolates from northern Thailand. DNA sequence analysis of partial , and promoter of 73 Mtb isolates, which included 30 MDR-TB, was performed to elucidate the mutations involved with RIF and INH resistance.

Results: When compared with the phenotypic DST, RIF-RD targeting showed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 83.9, 98.6, 96.9, and 92.0%, respectively. The multiplex reaction of the INH-RD targeted both and promoter showed high sensitivity, specificity, PPV, and NPV of 97.1, 94.2, 89.2, and 98.5%, respectively. Six patterns of mutation, predominately at codons 531 (50%) and 526 (40%) along with a rare S522L (3.33%) and D516V (3.33%), were detected. A single pattern of mutation (S315T) (63.3%) and four patterns of promoter mutation, predominately -15 (C>T), were found. Approximately, 17% of MDR-TB strains possessed double mutations within the and promoter.

Conclusion: Up to 86.7% and 96.7% of MDR-TB could be accurately detected by RIF-RD and INH-RD, emphasizing its usefulness as a low unit price assay for rapid screening of MDR-TB, with confirmation of INH resistance in low and middle-income countries. The MDR-TB genotypes provided will be beneficial for TB control and the development of drug-resistant TB diagnostic technology in the future.
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http://dx.doi.org/10.2147/IDR.S263082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533241PMC
September 2020

Melittin from Venom as a Promising Therapeutic Agent for Skin Cancer Treatment.

Antibiotics (Basel) 2020 Aug 14;9(8). Epub 2020 Aug 14.

Division of Microbiology, Department of Biology, Faculty of Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.

Melittin, a major component found in bee venom, is produced by the species of the honey bee. In this study, the effect of melittin derived from (Mel-AF), which is a wild honey bee species that is indigenous to Thailand, was investigated against human malignant melanoma (A375) cells. In this study, Mel-AF exhibited considerable potential in the anti-proliferative action of A375 cells. Subsequently, the cellular mechanism of Mel-AF that induced cell death was investigated in terms of apoptosis. As a result, gene and protein expression levels, which indicated the activation of cytochrome-c release and caspase-9 expression, eventually triggered the release of the caspase-3 executioner upon Mel-AF. We then determined that apoptosis-mediated cell death was carried out through the intrinsic mitochondrial pathway. Moreover, advanced abilities, including cell motility and invasion, were significantly suppressed. Mel-AF manipulated the actin arrangement via the trapping of stress fibers that were found underneath the membrane, which resulted in the defective actin cytoskeleton organization. Consequently, the expression of EGFR, a binding protein to F-actin, was also found to be suppressed. This outcome strongly supports the effects of Mel-AF in the inhibition of progressive malignant activity through the disruption of actin cytoskeleton-EGFR interaction and the EGFR signaling system. Thus, the findings of our current study indicate the potential usefulness of Mel-AF in cancer treatments as an apoptosis inducer and a potential actin-targeting agent.
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http://dx.doi.org/10.3390/antibiotics9080517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460526PMC
August 2020

Unveiling the Properties of Thai Stingless Bee Propolis via Diminishing Cell Wall-Associated Cryptococcal Melanin and Enhancing the Fungicidal Activity of Macrophages.

Antibiotics (Basel) 2020 Jul 17;9(7). Epub 2020 Jul 17.

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50000, Thailand.

, a life-threatening human yeast has the ability to produce melanin, which is one of the common virulence factors contributing to cryptococcal pathogenesis. This virulence factor is closely associated with the cryptococcal cell wall, specifically chitin and chitosan polysaccharides, a complex structure that is essential for maintaining cellular structure and integrity. In this study, we aim to investigate the effects of two stingless bee (SLB) propolis from and against cell wall-associated melanin in and its immune response in RAW 264.7 macrophage. The ethanolic extract of SLB propolis (EEP) has strongly exhibited anti-cryptococcal activity. Moreover, EEP from both sources reduced chitin/chitosan and melanin production against in a dose-dependent manner. Likewise, the mRNA expression level of and genes involved in the cryptococcal melanization pathway was significantly decreased at 2 mg/mL in EEP treatment. Additionally, pretreatment with EEP prior to yeast infection dramatically reduced intracellular replication of in RAW 264.7 macrophages in a dose-dependent manner. This study might be a new insight to use a natural powerful source, not only acting to target cell wall-associated molecules, but also being capable to explore a novel strategy by which dysregulation of these molecules leads to promote immunomodulatory activity.
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http://dx.doi.org/10.3390/antibiotics9070420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400477PMC
July 2020

In Vitro Antifungal and Antivirulence Activities of Biologically Synthesized Ethanolic Extract of Propolis-Loaded PLGA Nanoparticles against .

Evid Based Complement Alternat Med 2019 30;2019:3715481. Epub 2019 Nov 30.

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.

Propolis is a natural substance and consists of bioactive compounds, which gives it antioxidant and antimicrobial properties. However, the use of propolis is limited by the low solubility in aqueous solutions. Thus, nanoparticles may be likely to accomplish enhanced delivery of poorly water-soluble phytomedicine. The aim of the present study was to fabricate and evaluate the biological activity of ethanolic extract of propolis-loaded poly(lactic-co-glycolic acid) nanoparticles (EEP-NPs). The EEP-NPs were prepared using the oil-in-water (o/w) single-emulsion solvent evaporation technique. The physicochemical properties of EEP-NPs were characterized and tested on their cytotoxicity, antifungal activity, and impact on key virulence factors that contribute to pathogenesis of EEP-NPs were successfully synthesized and demonstrated higher antifungal activity than EEP in free form. Moreover, EEP-NPs exhibited less cytotoxicity on Vero cells and suppressed the virulence factors of , including adhesion, hyphal germination, biofilm formation, and invasion. Importantly, EEP-NPs exhibited a statistical decrease in the expression of hyphal adhesion-related genes, and , of . The results of this study revealed that EEP-NPs mediates a potent anticandidal activity and key virulence factors by reducing the gene-encoding virulence-associated hyphal- adhesion proteins of and, thereby, disrupting the morphologic presence and attenuating their virulence.
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http://dx.doi.org/10.1155/2019/3715481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6907039PMC
November 2019

A potential of propolis on major virulence factors of Cryptococcus neoformans.

Microb Pathog 2018 Oct 21;123:296-303. Epub 2018 Jul 21.

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, 50200, Thailand; Infectious Disease Research Unit (IDRU), Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, 50200, Thailand; Center of Biomolecular Therapy and Diagnostic, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, 50200, Thailand. Electronic address:

The present study was conducted to investigate the effects of a natural product from honeybees, named propolis, against Cryptococcus neoformans and its effect in the expression of putative virulence factors, such as capsular polysaccharides, melanin production and urease enzyme. Ethanol extract propolis (EEP) was first tested for its anti-cryptococcal activity and explored its impact on virulence factors in both phenotypes and enzyme activities. Moreover, the cryptococcal virulence genes were investigated using real time RT-PCR. The MIC value of EEP, 1 mg ml, displayed potent inhibition of C. neoformans cell viability. Of note is the high efficacy of sub-MIC concentrations (ranging from 0.5 to 0.125 mg ml) in decreasing the production of capsule, melanin, as well as laccase and urease enzyme activities. Importantly, EEP exhibited statistically decrease in the expression of gene-encoded virulence factors. In conclusion, EEP mediates C. neoformans growth inhibition and virulence factors by reducing the gene-encoding virulence-associated proteins and, thereby, disrupting the morphologic presence and attenuating their virulence. This study introduced EEP as regards anti-cryptococcal virulence factors activities; therefore, EEP would provide alternative ways of controlling the pathogenicity.
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http://dx.doi.org/10.1016/j.micpath.2018.07.028DOI Listing
October 2018

Comparison of two indirect ELISA coating antigens for the detection of dairy cow antibodies against Pasteurella multocida.

J Microbiol Methods 2018 02 12;145:20-27. Epub 2017 Dec 12.

Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand; Excellent Center in Veterinary Bioscience, Chiang Mai University, Chiang Mai, 50100, Thailand. Electronic address:

The ELISA is recognized as an efficient diagnostic tool for antibody detection, but there is no standard ELISA assay for detection of antibodies against hemorrhagic septicemia (HS) in cattle. The present study reports on an indirect ELISA assay for antibody detection of HS in dairy cows, and evaluates the sensitivity (Se) and specificity (Sp) of the method using a Bayesian approach. An indirect ELISA was developed with two types of heat extract antigens, Pasteurella multocida strains P-1256 and M-1404, as coating antigens. A checkerboard titration was employed using dairy cow sera immunized with P. multocida bacterin and colostrum-deprived calf sera. The concentrations of heat extract antigen (160μg/mL), sample serum (1:100) and goat anti-bovine immunoglobulin G labeled with horseradish peroxidase (1:2000) were optimal for the assay. The cut-off values were 0.147 and 0.128 for P-1256 and M-1404 coating antigens, and there were no differences in the results of tests with positive and negative sera (p<0.05). The characteristics of three diagnostic tests were evaluated using a one-population Bayesian model, assuming conditional dependence between two types of coating antigen-based ELISAs and indirect hemagglutination assay (IHA). A total of 415 sera samples from dairy cows without HS vaccination and no history of disease were tested. The Se and Sp of the P-1256 and M-1404 ELISAs were higher than those of the IHA. The Se and Sp of the P-1256 ELISA were 90.3% and 90.1%, while the Se and Sp of the M-1404 ELISA were 92.1% and 71.9%. The median values of Se and Sp from the IHA were 36.0% and 58.2%.
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http://dx.doi.org/10.1016/j.mimet.2017.12.005DOI Listing
February 2018

Secretome profile analysis of multidrug-resistant, monodrug-resistant and drug-susceptible Mycobacterium tuberculosis.

Arch Microbiol 2018 Mar 8;200(2):299-309. Epub 2017 Nov 8.

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, 110 Intawaroroj Rd., Sripoom, Chiang Mai, 50200, Thailand.

The emergence of drug-resistant tuberculosis has generated great concern in the control of tuberculosis and HIV/TB patients have established severe complications that are difficult to treat. Although, the gold standard of drug-susceptibility testing is highly accurate and efficient, it is time-consuming. Diagnostic biomarkers are, therefore, necessary in discriminating between infection from drug-resistant and drug-susceptible strains. One strategy that aids to effectively control tuberculosis is understanding the function of secreting proteins that mycobacteria use to manipulate the host cellular defenses. In this study, culture filtrate proteins from Mycobacterium tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains were gathered and profiled by shotgun-proteomics technique. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 837, 892, 838 and 850 were found in M. tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains, respectively. These proteins have been implicated in various cellular processes, including biological adhesion, biological regulation, developmental process, immune system process localization, cellular process, cellular component organization or biogenesis, metabolic process, and response to stimulus. Analysis based on STITCH database predicted the interaction of DNA topoisomerase I, 3-oxoacyl-(acyl-carrier protein) reductase, ESAT-6-like protein, putative prophage phiRv2 integrase, and 3-phosphoshikimate 1-carboxyvinyltransferase with isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin, suggesting putative roles in controlling the anti-tuberculosis ability. However, several proteins with no interaction with all first-line anti-tuberculosis drugs might be used as markers for mycobacterial identification.
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http://dx.doi.org/10.1007/s00203-017-1448-0DOI Listing
March 2018

Development and standardization of an in-house indirect ELISA for detection of duck antibody to fowl cholera.

J Microbiol Methods 2017 11 24;142:10-14. Epub 2017 Aug 24.

Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand; Excellent Center in Veterinary Bioscience, Chiang Mai University, Chiang Mai, 50100, Thailand. Electronic address:

Serological tests, such as agglutination and indirect hemagglutination assay (IHA), have been used to identify antibodies against Pasteurella multocida in poultry sera, but none are highly sensitive. An enzyme-linked immunosorbent assays (ELISA) has been used with varying degrees of success in attempts to monitor seroconversion in vaccinated poultry, but are not suitable for diagnosis. Commercial ELISA kits are available for chickens and turkeys, but not for ducks. The present study reports development and standardization of an in-house indirect ELISA for detection of duck antibody to fowl cholera. The characteristics of ELISA and IHA were analyzed using a one population Bayesian model assuming conditional dependence between the two diagnostic tests. An in-house indirect ELISA was developed using a heat extract antigen of P. multocida strain X-73 as a coating antigen and horseradish peroxidase conjugated goat anti-duck IgG antibody (dIgG-HRP). The checkerboard titration method was done using sera from ducks immunized with P. multocida bacterin as positive sera and 1day old duckling sera as negative sera. The heat extract antigen at 1μg/ml, sample serum at a dilution of 1:100, and dIgG-HRP 1:2000 were optimal concentrations for the assay. The cut-off value was 0.200. Of the duck sera, 89.05% (244/274) were considered seropositive by ELISA. Estimates for sensitivity and specificity of ELISA were higher than prior values with medians of 94.7% [95% posterior probability interval (PPI)=89.6-98.2%] and 87.2% (PPI=68.2-98.3%). Estimates for sensitivity of IHA were lower than prior values (median=97.6, PPI=93.2-99.7%) while the specificity was close to the prior value (median=76.5, PPI=65.8-85.4%). This finding suggests that an in-house indirect ELISA can be used to detect duck antibody to fowl cholera.
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http://dx.doi.org/10.1016/j.mimet.2017.08.018DOI Listing
November 2017

Downregulation of Extracellular Matrix Metalloproteinase Inducer by scFv-M6-1B9 Intrabody Suppresses Cervical Cancer Invasion Through Inhibition of Urokinase-Type Plasminogen Activator.

Cancer Biother Radiopharm 2017 Feb 24;32(1):1-8. Epub 2017 Jan 24.

1 Division of Clinical Microscopy, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University , Chiang Mai, Thailand .

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.
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http://dx.doi.org/10.1089/cbr.2016.2126DOI Listing
February 2017

Propolis extracts from the northern region of Thailand suppress cancer cell growth through induction of apoptosis pathways.

Invest New Drugs 2016 12 21;34(6):707-722. Epub 2016 Sep 21.

Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand, 50200.

The continual increase in mortality rates and number of cancer cases is a matter of serious concern in developing countries. The incorporation of natural products into classical cancer treatment approaches is a promising direction. The mechanisms of A549 and HeLa cancer cell death induction by ethanolic extracts of propolis samples from Phayao, Chiang Mai, and Nan provinces in northern Thailand were investigated in this study. The propolis extract from Chiang Mai showed the highest antioxidant activity and the greatest total phenolic content. The propolis extract from Nan also exhibited the highest total flavonoid content. The proliferation of A549 and HeLa cells grown in the presence of the propolis extracts was suppressed in a dose- and time-dependent manner. Moreover, treatment of both cancer cells with the propolis extracts showed DNA fragmentation and significantly increased the number of the apoptotic cells. On A549 cells, the extrinsic and intrinsic pathways of caspase enzymes were activated by the propolis extracts from Phayao and Chiang Mai. In the case of the propolis extract from Nan, the mechanisms involved apoptosis on the A549 cells were caspase-independent pathway. The extrinsic pathway of the caspase enzyme was triggered by all of the propolis extracts on HeLa cells. Finally, oral administration of the propolis granule produced from the propolis extract from Nan resulted in extended survival of tumour-bearing mice. Therefore, propolis extracts from the northern region of Thailand demonstrated pharmacological properties, both antioxidant and anticancer activities. From these findings, it is evident that propolis extracts can be considered as a naturally obtained agent extremely useful in cancer treatment.
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http://dx.doi.org/10.1007/s10637-016-0392-1DOI Listing
December 2016

Novel targeting of the lepB gene using PCR with confronting two-pair primers for simultaneous detection of Mycobacterium tuberculosis complex and Mycobacterium bovis.

J Med Microbiol 2016 Jan;65(1):36-43

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand.

Tuberculosis (TB), caused by members of the Mycobacterium tuberculosis complex (MTC), is the leading cause of infectious disease-related mortality worldwide. The standard method for TB diagnosis usually requires long periods of mycobacteria cultivation, leading to delayed diagnosis, inefficient treatment and widespread occurrence of the disease. Therefore, a rapid method for the detection and differentiation of MTC from other mycobacteria is essential for disease diagnosis. Here, we describe the potential of using the type I signal peptidase (lepB) gene as a novel target for TB diagnosis, based on confronting two-pair primers PCR (PCRCTPP) that can detect MTC and simultaneously differentiate M. bovis. The limit of detection of the developed technique was equivalent to 12–120 bacilli. PCR-CTPP was highly specific to only MTC and M. bovis, and no cross-reaction was detected in 27 DNA of the non-tuberculous mycobacterial and bacterial strains tested. Thirty-nine blinded clinical isolates and 72 sputum samples were used to validate the PCR-CTPP in comparison with the standard mycobacterial culture method. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PCR-CTPP were equal to 95, 100, 100 and 95 %, respectively, when tested with clinical isolates. Furthermore, upon testing with the sputum samples, the sensitivity, specificity, PPV and NPV were observed to be 84, 76, 90 and 67 %, respectively. Hence, this highly sensitive novel technique, which is rapid, easy to conduct and cost-effective, is a potential method for TB diagnosis and epidemiological studies, especially in resource-limited countries with a high TB burden.
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http://dx.doi.org/10.1099/jmm.0.000188DOI Listing
January 2016

Recombinant Multivalent EMMPRIN Extracellular Domain Induces U937 Human Leukemia Cell Apoptosis by Downregulation of Monocarboxylate Transporter 1 and Activation of Procaspase-9.

Appl Biochem Biotechnol 2015 Jul 31;176(6):1781-90. Epub 2015 May 31.

Division of Clinical Microscopy, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, 110 Intawaroros Road, Sripoom, Muang, Chiang Mai, 50200, Thailand,

This study was carried out to understand the effect of the recombinant multivalent extracellular matrix metalloproteinase inducer (EMMPRIN) extracellular domain, designated as rmEMMPRINex, on the apoptotic cell death of human leukemia U937 cells. Expression of monocarboxylate transporter 1 (MCT1) and caspase-9 in U937 treated with rmEMMPRINex was investigated in this study. Levels of membrane MCT1 and intracellular procaspase-9 were decreased in rmEMMPRINex-treated cells in comparison to controls. However, the expression of activated caspase-9 was undetectable. rmEMMPRINex also induced DNA fragmentation and apoptosis in U937 cells. Taken together, we concluded that interaction of rmEMMPRINex with U937 cells leads to inhibition of MCT1 membrane expression, intracellular activation of procaspase-9, followed by DNA fragmentation and apoptosis. This may contribute to the conceptual development of novel cancer drugs in the future.
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http://dx.doi.org/10.1007/s12010-015-1677-0DOI Listing
July 2015

Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody.

J Cancer 2015 20;6(3):276-86. Epub 2015 Jan 20.

1. Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer.
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http://dx.doi.org/10.7150/jca.10879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317764PMC
February 2015

Antioxidant and anti-cancer cell proliferation activity of propolis extracts from two extraction methods.

Asian Pac J Cancer Prev 2013 ;14(11):6991-5

Biotechnology, The Graduate School, Chiang Mai University, Chiang Mai, Thailand E-mail :

Antioxidant activity, total phenolic, total flavonoid compounds and cytotoxicity to cancer cell lines of propolis extracts from two extraction methods were investigated in this study. Propolis was collected from Phayao province and extracted with 70% ethanol using maceration and sonication techniques. The antioxidant activity was evaluated by DPPH assay. Total phenolic and flavonoid compounds were also determined. Moreover, the cytotoxicity of propolis was evaluated using MTT assay. The percentage propolis yield after extraction using maceration (18.1%) was higher than using sonication (15.7%). Nevertheless, antioxidant and flavonoid compounds of the sonication propolis extract were significant greater than using maceration. Propolis extract from sonication showed antioxidant activity by 3.30 ± 0.15 mg gallic acid equivalents/g extract. Total phenolic compound was 18.3 ± 3.30 mg gallic acid equivalents/g extract and flavonoid compound was 20.49 ± 0.62 mg quercetin/g extract. Additionally, propolis extracts from two extraction methods demonstrated the inhibitory effect on proliferation of A549 and HeLa cancer cell lines at 24, 48 and 72 hours in a dose-dependent manner. These results are of interest for the selection of the most appropriate method for preparation of propolis extracts as potential antioxidant and anticancer agents.
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http://dx.doi.org/10.7314/apjcp.2013.14.11.6991DOI Listing
August 2014

In situ adenovirus vaccination engages T effector cells against cancer.

Vaccine 2009 Jun 16;27(31):4225-39. Epub 2009 Apr 16.

Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA.

The efficacy of cancer immunotherapy is limited because of central and peripheral immune tolerance towards tumor-antigens. We propose a novel approach based on the fact that the immune system has not evolved tolerance towards adenoviruses (Ads) and that Ads have not evolved efficient mechanisms for immune-escape. The host-response to intratumoral Ad-vector injection in mice that were immunologically tolerant to neu-positive syngeneic mammary-cancer (MMC) was investigated. Intratumoral injection with replication-deficient, transgene-devoid Ad induced immune responses at two different anatomical sites: the tumor-draining lymph nodes and the tumor microenvironment. The lymph nodes supported the generation of both neu- and Ad-specific T effector cells, while inside the tumor microenvironment only Ad-specific T cells expanded. Importantly, Ad-specific T cells were anti-tumor-reactive despite the presence of active regulatory T cell-mediated immune tolerance inside MMC tumors and anti-tumor efficacy of Ad was increased by pre-immunization against Ad despite the production of Ad-neutralizing antibodies.
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http://dx.doi.org/10.1016/j.vaccine.2009.03.074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727281PMC
June 2009

Potent inhibition of OKT3-induced T cell proliferation and suppression of CD147 cell surface expression in HeLa cells by scFv-M6-1B9.

Immunobiology 2008 ;214(6):410-21

Division ofClinicalMicroscopy,DepartmentofMedicalTechnology,FacultyofAssociatedMedicalSciences, Chiang MaiUniversity,ChiangMai50200,Thailand.

CD147, a multifunctional type I transmembrane glycoprotein, has been implicated in various physiological and pathological processes. It is involved in signal transduction pathways and also plays a crucial role in the invasive and metastatic activity of malignant tumor cells. Diminished expression of this molecule has been shown to be beneficial in suppression of tumor progression. In a previous study, we generated and characterized a recombinant antibody fragment, scFv, which reacted specifically to CD147. In the present study, we further investigated the biological properties, function and the effect of generated scFv on CD147 expression. The in vitro study showed that soluble scFv-M6-1B9 produced from E. coli HB2151 bound to CD147 surface molecule and inhibited OKT3-induced T cell proliferation. Furthermore, soluble lysate of scFv-M6-1B9 from 293A cells, transduced with a scFv-M6-1B9 expressing adenovirus vector, recognized both recombinant and native CD147. These results indicate that scFv-M6-1B9 binds with high efficiency and specificity. Importantly, scFv-M6-1B9 intrabody reduced the expression of CD147 on the cell surface of HeLa cells suggesting that scFv-M6-1B9 is biologically active. In conclusion, our present study demonstrated that scFv-M6-1B9 has a great potential to target both the intracellular and the extracellular CD147. The generated scFv-M6-1B9 may be an effective agent to clarify the cellular function of CD147 and may aid in efforts to develop a novel treatment in various human carcinomas.
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http://dx.doi.org/10.1016/j.imbio.2008.12.006DOI Listing
April 2017

Hemoglobin E detection using PCR with confronting two-pair primers.

J Med Assoc Thai 2008 Nov;91(11):1677-80

Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, 110 Indrawaroros Rd, Sripoom, Mueang, Chiang Mai 50200, Thailand.

Objectives: To develop and apply the polymerase chain reaction with confronting two-pair primers (PCR-CTPP) for detection and identification of hemoglobin E (Hb E).

Material And Method: Fifty unrelated northern Thais were included in the present study. DNA was extracted from peripheral blood mononuclear cells and targeted to amplify by PCR-CTPP. The amplified product was analyzed and compared with the reference hemoglobin electrophoresis and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.

Results: The results validated a completely concordant among these three methods consisting of 74%, 24%, and 2% identified as normal, heterozygous, and homozygous Hb E type, respectively.

Conclusion: Successful Hb E genotyping by PCR-CTPP was introduced. It allows for confirming and simultaneously detection with other thalassemia mutations.
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November 2008

Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells.

BMC Biotechnol 2008 Jan 29;8. Epub 2008 Jan 29.

Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, 50200, Thailand.

Background: Expression of intracellular antibodies (intrabodies) has become a broadly applicable technology for generation of phenotypic knockouts in vivo. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer.

Results: The recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9) reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA) using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system.

Conclusion: The results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.
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http://dx.doi.org/10.1186/1472-6750-8-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258298PMC
January 2008

Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells.

Vet Parasitol 2002 Nov;109(3-4):191-201

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

We investigated the terminal location of NcSRS2, a surface antigen of Neospora caninum that has potential use for diagnosis, and demonstrated its importance as a vaccine component against neosporosis, in an insect-baculovirus expression system. To examine the role of the hydrophobic C-terminal tail in NcSRS2, four types of recombinant baculoviruses were constructed. Immunoblotting and N-terminal amino acid analysis revealed cleavage of a 6 kDa of the N-terminal signal peptide in the mature NcSRS2 protein. The recombinant NcSRS2 (rNcSRS2) lacking 25, and 62 amino acids from the termination codon were detected in supernatants from recombinant virus-infected cells, but not in recombinants with truncated 147 amino acids from the termination codon, and intact NcSRS2 gene (401 amino acids). By flow cytometric and confocal laser scanning microscopic analyses, the truncation of the hydrophobic C-terminal tail in NcSRS2 was shown to result in the reduction of protein expression on the cell surface relative to intact rNcSRS2. Except for the recombinant lacking the 147 C-terminal residues, three other rNcSRS2 were detected in the supernatants after treatment with phosphatidylinositol-specific phospholipase C. Our results demonstrate that the N. caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol-anchor molecule in insect cells, and that the hydrophobic C-terminal domain is an essential component for GPI-membrane attachment. We have likewise shown the usefulness of the insect-recombinant baculovirus system in the expression of rNcSRS2.
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http://dx.doi.org/10.1016/s0304-4017(02)00256-xDOI Listing
November 2002
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