Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target.

BACKGROUND: Based on resistance of currently used anti-malarials, a new anti-malarial drug target against Plasmodium falciparum is urgently needed. Damaged DNA cannot be transcribed without prior DNA repair; therefore, uracil-DNA glycosylase, playing an important role in base excision repair, may act as a candidate for a new anti-malarial drug target. METHODS: Initially, the native PfUDG from parasite crude extract was partially purified using two columns, and the glycosylase activity was monitored. The existence of malarial UDG activity prompted the recombinant expression of PfUDG for further characterization. The PfUDG from chloroquine and pyrimethamine resistant P. falciparum strain K1 was amplified, cloned into the expression vector, and expressed in Escherichia coli. The recombinant PfUDG was analysed by SDS-PAGE and identified by LC-MS/MS. The three dimensional structure was modelled. Biochemical properties were characterized. Inhibitory effects of 12 uracil-derivatives on PfUDG activity were investigated. Inhibition of parasite growth was determined in vitro using SYBR Green I and compared with results from human cytotoxicity tests. RESULTS: The native PfUDG was partially purified with a specific activity of 1,811.7 units/mg (113.2 fold purification). After cloning of 966-bp PCR product, the 40-kDa hexa-histidine tagged PfUDG was expressed and identified. The amino acid sequence of PfUDG showed only 24.8% similarity compared with the human enzyme. The biochemical characteristics of PfUDGs were quite similar. They were inhibited by uracil glycosylase inhibitor protein as found in other organisms. Interestingly, recombinant PfUDG was inhibited by two uracil-derived compounds; 1-methoxyethyl-6-(p-n-octylanilino)uracil (IC50 of 16.75 µM) and 6-(phenylhydrazino)uracil (IC50 of 77.5 µM). Both compounds also inhibited parasite growth with IC50s of 15.6 and 12.8 µM, respectively. Moreover, 1-methoxyethyl-6-(p-n-octylanilino)uracil was not toxic to HepG2 cells, with IC50 of > 160 µM while 6-(phenylhydrazino)uracil exhibited cytoxicity, with IC50 of 27.5 µM. CONCLUSIONS: The recombinant PfUDG was expressed, characterized and compared to partially purified native PfUDG. Their characteristics were not significantly different. PfUDG differs from human enzyme in its size and predicted amino acid sequence. Two uracil derivatives inhibited PfUDG and parasite growth; however, only one non-cytotoxic compound was found. Therefore, this selective compound can act as a lead compound for anti-malarial development in the future.

Applicability of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection in high exposure risk setting.

OBJECTIVES: During the third wave, the growing number of COVID-19 case clusters reported countrywide in Thailand demonstrated the rapidly evolving characteristics of SARS-CoV-2, the causative agent of the COVID-19 pandemic. The rapid spread of COVID-19 infections had been extensively reported in public areas and construction camps, as well as in congested communities with poor sanitation. High demand for SARS-CoV-2 genome testing and quick reporting by an hour for case identification and isolation characterizes the COVID-19 crisis in Thailand. This situation leads to an urgent need for alternative molecular tests which are reliable, rapid, and cost-effective. METHODS: In this study, we assessed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP), using real-time reverse transcription-polymerase chain reaction (RT-PCR) as a reference standard, for active case finding in suspected (mostly asymptomatic) cases living in high-risk areas of Bangkok. RESULTS: The diagnostic performance of the RT-LAMP compared with real-time RT-PCR in specimens from 549 Thais were computed in a real-world field study setting. Our study demonstrated that RT-LAMP achieved robust identification of SARS-CoV-2 infection, with a diagnostic sensitivity and specificity of 91.67% and 100%, respectively. CONCLUSION: RT-LAMP is a reliable assay for SARS-CoV-2 detection and is scalable for use in the emergency response to a nationwide pandemic, despite resource limitations. The RT-LAMP real-world data derived from this field study validate its potential use in laboratory practice. RT-LAMP is a good choice as a laboratory-based SARS-CoV-2 molecular test when real-time RT-PCR is not available.

Identification of human malaria parasites and detection of mixed infection in Thai patients by nested PCR.

The species-specific nested PCR previously described by Snounou and others, for detecting the four species of human malaria parasites, is evaluated in the current study testing 40 blood samples from malaria patients admitted during July-September, 2003, at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Thailand. Parasite DNA of each blood sample was extracted and purified by QIAamp. DNA mini kit. Nested PCR was performed using genus-specific primers for the first PCR cycle and species-specific primer for the second cycle. Thin and thick smears were also made, stained with Giemsa, and examined by expert microscopists. Only one of 40 samples (2.5%) was identified as Plasmodium malariae infection by both microscopy and nested PCR. Twenty blood samples (50%) were identified as Plasmodium falciparum infections by both methods. However, 19 blood samples (47.5%) were reported as Plasmodium vivax infections by microscopic methods, whereas nested PCR could detect a mixed infection of Plasmodium vivax and Plasmodium falciparum in one sample taken from a young girl with 8 ameboid trophozoites of P. vivax per 200 white blood cells. These results demonstrated that the nested PCR assay surpasses microscopy and also offers a clear advantage in the detection of mixed infections, which is important not only for successful medical treatment, but also for the study of malaria epidemiology.

Molecular characterization of Plasmodium falciparum putative polynucleotide kinase/phosphatase.

Polynucleotide kinase/phosphatase (PNKP) is a bifunctional enzyme that can phosphorylate the 5'-OH termini and dephosphorylate the 3'-phosphate termini of DNA. It is a DNA repair enzyme involved in the processing of strand break termini, which permits subsequent repair proteins to replace missing nucleotides and rejoin broken strands. Little is known about DNA repair in Plasmodium falciparum, including the roles of PNKP in repairing parasite DNA. We identified a P. falciparum gene encoding a protein with 24% homology to human PNKP and thus suggestive of a putative PNKP. In this study, the PNKP gene of P. falciparum strain K1 (PfPNKP) was successfully cloned and expressed in E. coli as a GST-PfPNKP recombinant protein. MALDI-TOF/TOF analysis of the protein confirmed the identity of PfPNKP. Assays for enzymatic activity were carried out with a variety of single- and double-stranded substrates. Although 3'-phosphatase activity was detected, PfPNKP was observed to dephosphorylate single-stranded substrates or double-stranded substrates with a short 3'-single-stranded overhang, but not double-stranded substrates that mimicked single-strand breaks. We hypothesize that unlike human PNKP, PfPNKP may not be involved in single-strand break repair, since alternative terminal processing mechanisms can substitute for PfPNKP, and that PfPNKP DNA repair actions may be confined to overhanging termini of double-strand breaks.