ASEAN Studies for Sustainable Development

In the present study, the magnetic fraction of fly ash was separated from the bulk material using a conventional magnet, yielding approximately 50% w/w. As the magnetic fraction of fly ash contains a high content of aluminum oxide, which is recognized as a phosphate adsorbent, and ferromagnetic iron oxides, it is potentially useful as a magnetic solid-phase extraction adsorbent of phosphoproteins. The results showed that the magnetic fraction of fly ash displayed a high enrichment factor of approximately 130-fold as a magnetic solid-phase extraction adsorbent. The phosphoprotein bound-magnetic fraction was efficiently isolated from the sample solution using a conventional magnet. The adsorbent was further applied to the isolation of phosphoproteins from biological samples, i.e., ovalbumin from egg white and caseins from milk. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enrichment of phosphoproteins revealed that the eluted fraction contained more purified phosphoproteins comparing to the initial diluted samples. In addition, the adsorbent may be reused with acceptable adsorption capacity at least three times. The magnetic solid-phase extraction using the magnetic fraction of fly ash as an adsorbent is effective, simple and reusable. Therefore, the magnetic fraction of fly ash may be an alternative low-cost magnetic adsorbent for the sequestration of phosphoproteins from complex samples.

PURPOSE. To investigate the role of mitochondrial DNA (mt DNA) background on the expression of Leber hereditary optic neuropathy (LHON) in Southeast Asian carriers of the G11778A mutation. METHODS. Complete mtDNA sequences were analyzed from 53 unrelated Southeast Asian G11778A LHON pedigrees in Thailand and 105 normal Thai controls, and mtDNA haplogroups were determined. Clinical phenotypes were tested for association with mtDNA haplogroup, with adjustment for potential confounders such as sex and age at onset. RESULTS. mtDNA subhaplogroup B was significantly associated with LHON. Follow-up analysis narrowed the association down to subhaplogroup B5a1 (P ϭ 0.008). Survival analyses with Cox's proportional hazards modeling on 469 samples (91 affected and 378 unaffected), adjusted for sex and heteroplasmy, revealed that haplogroup B5a1 tended to increase the risk of visual loss, but the trend was not statistically significant. Conversely, haplogroup F, the second most common haplogroup in the control population, was the least frequent haplogroup in LHON. This negative association was narrowed down to subhaplogroup F1 (P ϭ 0.00043), suggesting that haplogroup F1 confers a protective effect. The distributions of sex, age at onset and heteroplasmy were not significantly different among haplogroups. CONCLUSIONS. The specific mtDNA background B5a1 was significantly associated with Southeast Asian G11778A LHON and appeared to modify the risk of visual loss. (Invest Ophthalmol

Located only a short distance off the southernmost shore of the Greater Indian subcontinent, the island of Sri Lanka has long been inhabited by various ethnic populations. Mainly comprising the Vedda, Sinhalese (Up-and Low-country) and Tamil (Sri Lankan and Indian); their history of settlements on the island and the biological relationships among them have remained obscure. It has been hypothesized that the Vedda was probably the earliest inhabitants of the area, followed by Sinhalese and Tamil from the Indian mainland. This study, in which 271 individuals, representing the Sri Lankan ethnic populations mentioned, were typed for their mitochondrial DNA (mtDNA) hypervariable segment 1 (HVS-1) and part of hypervariable segment 2 (HVS-2), provides implications for their settlement history on the island. From the phylogenetic, principal coordinate and analysis of molecular variance results, the Vedda occupied a position separated from all other ethnic people of the island, who formed relatively close affiliations among themselves, suggesting a separate origin of the former. The haplotypes and analysis of molecular variance revealed that Vedda people's mitochondrial sequences are more related to the Sinhalese and Sri Lankan Tamils' than the Indian Tamils' sequences. MtDNA haplogroup analysis revealed that several West Eurasian haplogroups as well as Indian-specific mtDNA clades were found amongst the Sri Lankan populations. Through a comparison with the mtDNA HVS-1 and part of HVS-2 of Indian database, both Tamils and Sinhalese clusters were affiliated with Indian subcontinent populations than Vedda people who are believed to be the native population of the island of Sri Lanka.

Leber's Hereditary Optic Neuropathy (LHON) is one of the commonest mitochondrial diseases. It causes total blindness, and predominantly affects young males. For the disease to develop, it is necessary for an individual to carry one of the primary mtDNA mutations 11778G.A, 14484T.C or 3460G.A. However these mutations are not sufficient to cause disease, and they do not explain the characteristic features of LHON such as the higher prevalence in males, incomplete penetrance, and relatively later age of onset. In order to explore the roles of nuclear encoded mitochondrial proteins in development of LHON, we applied a proteomic approach to samples from affected and unaffected individuals from 3 pedigrees and from 5 unrelated controls. Two-dimensional electrophoresis followed by MS/MS analysis in the mitochondrial lysate identified 17 proteins which were differentially expressed between LHON cases and unrelated controls, and 24 proteins which were differentially expressed between unaffected relatives and unrelated controls. The proteomic data were successfully validated by western blot analysis of 3 selected proteins. All of the proteins identified in the study were mitochondrial proteins and most of them were down regulated in 11778G.A mutant fibroblasts. These proteins included: subunits of OXPHOS enzyme complexes, proteins involved in intermediary metabolic processes, nucleoid related proteins, chaperones, cristae remodelling proteins and an anti-oxidant enzyme. The protein profiles of both the affected and unaffected 11778G. A carriers shared many features which differed from those of unrelated control group, revealing similar proteomic responses to 11778G.A mutation in both affected and unaffected individuals. Differentially expressed proteins revealed two broad groups: a cluster of bioenergetic pathway proteins and a cluster involved in protein quality control system. Defects in these systems are likely to impede the function of retinal ganglion cells, and may lead to the development of LHON in synergy with the primary mtDNA mutation.

Leber Hereditary Optic Neuropathy (LHON) is one of the most common mitochondrial diseases that can cause complete blindness, particularly in young men. The majority of LHON cases are caused by one of the three prevalent mitochondrial DNA (mtDNA) mutations-G3460A/ND1, G11778A/ND4, and T14484C/ND6. However these mutations cannot explain the several remarkable features of LHON, therefore, the nuclear factors is suggested to be involved in the phenotypic expression of LHON. Our Previous findings demonstrated 29 nuclear-encoded mitochondrial proteins with altered expression pattern between affected and unaffected groups among Thai LHON patients. In the present study, heat shock protein 60 (Hsp60) a vital mitochondrial chaperone was investigated and found to be significantly down regulated in 11778G>A LHON affected fibroblast compared to the fibroblasts from either control individuals or from unaffected relatives. Therefore, the reduced expression level of Hsp60 in the fibroblast of affected LHON groups may suggest the defect in the protein turnover of the mitochondria, which might contribute to the pathogenesis of LHON.

Metal oxide affinity chromatography (MOAC) is one of the most commonly used techniques for selective isolation phosphoproteins and phosphopeptides. This technique is capable of capturing the phosphorylated biomolecules through the affinity of the phosphoryl group for metal oxides/hydroxides. Fly-ash (FA), a by-product of coal-combustion power plants, is primarily composed of oxides of silicon and metals, among which iron and titanium. A number of studies have demonstrated the potential of these metal oxides for phosphoprotein and phosphopeptide enrichment. FA is annually produced over hundred million tons worldwide and generally considered as hazardous waste. It is thus of great importance to enhance its utilization. Here we present the first demonstration of the utility of FA as a low-cost MOAC material for the enrichment of phosphoproteins. With an FA-microcolumn, phosphoproteins can be successfully sequestered from other proteins. FA-microcolumns are shown to be simple, cheap and...

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai-Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.

Intercellular communication is a crucial process for the multicellular community in both prokaryotes and eukaryotes. Indole has been recognized as a new member of the signal molecules which enables the regulated control of various bacterial phenotypes. To elucidate the interspecies relationship among enteric microorganisms via indole signaling, Klebsiella pneumoniae (KP) culture was treated with indole solution and examined for the pathogenicity by using various phenotypic tests. Both synthetic and naturally-produced indole preparations had no deteriorating effect on growth and autoaggregative capacity of KP. The results showed that biofilm formation of carbapenem-susceptible K. pneumoniae (KP-S) was clearly induced by indole exposure (& 2-10 folds), whereas no significant difference was observed in the resistant counterpart. In addition, the tolerance to b-lactam antibiotics of KP was altered upon exposure to indole and/ or derivatives assessed by Kirby-Bauer disk diffusion test. Taken together, our finding indicates the functional role of indole in changing or modulating pathogenic behaviors of other bacteria.

Plasmodium vivax is usually considered morbidity in endemic areas of Asia, Central and South America, and some part of Africa. In Thailand, previous studies indicated the genetic diversity of P. vivax in malaria-endemic regions such as the western part of Thailand bordering with Myanmar. The objective of the study is to investigate the genetic diversity of P. vivax circulating in Southern Thailand by using 3 antigenic markers and 8 microsatellite markers. Dried blood spots were collected from Chumphon, Phang Nga, Ranong and, Surat Thani provinces of Thailand. By PCR, 3 distinct sizes of PvMSP3α, 2 sizes of PvMSP3β and 2 sizes of PvMSP1 F2 were detected based on the length of PCR products, respectively. PCR/RFLP analyses of these antigen genes revealed high levels of genetic diversity. The genotyping of 8 microsatellite loci showed high genetic diversity as indicated by high alleles per locus and high expected heterozygosity (HE). The genotyping markers also showed multiple-clones of infection. Mixed genotypes were detected in 4.8% of PvMSP3α, 29.1% in PvMSP3β and 55.3% of microsatellite markers. These results showed that there was high genetic diversity of P. vivax isolated from Southern Thailand, indicating that the genetic diversity of P. vivax in this region was comparable to those observed other areas of Thailand.

Fly ash, a by-product generated from coal combustion, contains a high content of metal oxides which are recognized as efficient adsorptive materials for phosphate-containing molecules. This indicates the potential application of fly ash as a solid-phase extraction adsorbent to nucleic acids, the phosphate-containing biopolymers. However, little effort has been made to study and exploit fly ash properties for extracting nucleic acids. In this study, long doublestranded deoxyribonucleic acid (DNA) was used for examining the DNA adsorptivity of fly ash. On incubation with DNA, it required only two minutes for fly ash to adsorb DNA on the surface. This time was significantly shorter than other metal-oxide adsorbents such as Fe 3 O 4 @SiO 2 nanoparticles. In terms of desorption, DNA was rapidly eluted from the fly ash surface using phosphate buffer within a very short time of 5 minutes comparable to other metal oxide particles. Moreover, fly ash may be reused three times without significant loss of its adsorption capacity. Fly ash has been demonstrated to be a promising adsorbent in the DNA isolation process due to its low cost, reusability, and short-time extraction.

Artemisinin resistance (ART) has been confirmed in Greater Mekong Sub-region countries. Currently, C580Y mutation on Pfkelch13 gene is known as the molecular marker for the detection of ART. Rapid and accurate detection of ART in field study is essential to guide malaria containment and elimination interventions. A simple method for collection of malaria-infected blood is to spot the blood on filter paper and is fast and easy for transportation and storage in the field study. This study aims to evaluate LAMP-SNP assay for C580Y mutation detection by introducing an extra mismatched nucleotide at the 3’ end of the FIP primer. The LAMP-SNP assay was performed in a water bath held at a temperature of 56°C for 45 min. LAMP-SNP products were interpreted by both gel-electrophoresis and HNB-visualized changes in color. The method was then tested with 120 P. falciparum DNA from dried blood spot samples. In comparing the LAMP-SNP assay results with those from DNA sequencing of the clinical sa...

Erythrocytes deficient in glucose-6-phosphate dehydrogenase (G6PD) is more susceptible to oxidative damage from free radical derived compounds. The hemolysis triggered by oxidative agents such as primaquine (PQ) is used for the radical treatment of hypnozoites of P. vivax. Testing of G6PD screening before malaria treatment is not a common practice in Thailand, which poses patients at risk of hemolysis. This retrospective study aimed to investigate the prevalence of G6PD in malaria patients who live in Southern Thailand. Eight hundred eighty-one malaria patients were collected for 8-year from 2012 to 2019, including 785 (89.1%) of P. vivax, 61 (6.9%) of P. falciparum, 27 (3.1%) of P. knowlesi, and 8 (0.9%) of mixed infections. The DiaPlexC genotyping kit (Asian type) and PCR-RFLP were employed to determine the G6PD variants. The result showed that 5 different types of G6PD variants were identified in 26 cases (2.9%); 12/26 (46.2%) had Mahidol (487G>A) and 11/26 (42.3%) had Viangch...

Highly sensitive diagnostic tools are crucial for individual screening during an epidemic of leptospirosis. To aid in developing a diagnostic tool for the sensitive detection of pathogenic strains, a new approach targeting nucleic acid amplification that combines quantitative PCR (qPCR) and strand displacement isothermal amplification was evaluated. The effectiveness of the combined approach, a quantitative polymerase chain displacement reaction (qPCDR), was compared with a qPCR technique. The results showed that qPCDR presented higher sensitivity (at least tenfold) and shorter reaction time than the qPCR approach for pathogenic Leptospira spp. detection. Thus, the qPCDR-based technique developed in this study is a promising approach for pathogenic Leptospira spp. detection and the further development of a diagnostic kit.