No abstract

Dengue virus is responsible for 50-100 million new infections annually worldwide. The virus uses error-prone RNA polymerase during genome replication in a host, resulting in the formation of closely related viruses known as quasispecies. The availability of next-generation sequencing technology provides opportunities to analyze viral quasispecies. Before analysis, it is crucial to increase the amount of DNA because of the limited amounts of viral genomic material that can be isolated from a patient. However, using specific primers may overlook the occurrence of possible variations at primer binding sites. To address this problem, the performance of two sequence-independent amplification methods was compared for whole genome amplification (WGA): phi29 DNA polymerase-based WGA and whole transcriptome amplification (WTA). Both methods have the ability to provide complete coverage of the dengue genome from template amounts as low as 1 ng. However, WTA showed greater efficiency in terms of yield (WTA: ~10 μg; phi29-based WGA: ~500 ng) and lower amplification bias. In conclusion, the WTA amplification kit was shown to perform substantially better than phi29 DNA polymerase-based WGA in terms of both final concentration and amplification bias in amplifying small genomes, such as that of the dengue virus.

Oxidative stress can induce vascular endothelial dysfunction in diabetic and hyperlipidemia patients. Leucaena leucocephala (Lam.) de Wit has been reported of possessing antioxidant, antidiabetic and anticholiesterase activity; as well as the toxic substance called mimosine. Aims: To determine antioxidant effects of Leucaena leucocephala (Lam.) de Wit Leave extracts in oxidative stress induced vascular endothelial function. Methods and Material: Leave extracts were determined for antioxidant activity. Either or both of oxidized low density lipoprotein (oxLDL) and glucose were applied to induce oxidative stress condition in human umbilical vein cultured (HUVCs) to observe superoxide dismutase (SOD) activity, nitric oxide (NO) level and morphological changes. Results: Total polyphenol and flavonoid were 51.04 ± 0.91 mg GAE/g and 0.13 ± 0.01 mg catechin/g of dried weight (DW), respectively. Free radical reduction efficiency of crude extract observed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay showed IC 50 value of 329.6 µg of vitamin C equivalent/mg of extracts. Frap value was showed 428.54 ± 15.32 mM FeII equivalent/g of DW. The result observing in HUVCs showed that comparing to the control, SOD activity, NO and MDA level were maintained in 0.05 mg/mL of L. leucocephala treated group, but NO and MDA level were lowered when comparing with oxLDL and glucose-induced oxidative stress. No change was observed in 0.05 mg/mL of L. leucocephala treated group, comparing with control group. Conclusion: This study has been performed to exhibit the antioxidant activity of L. leucocephala in endothelium functions and has been found to have an appropriate concentration at 0.05 mg/mL in reducing oxidative stress condition in impaired fasting blood glucose patients. Nevertheless, the optimal level for toxic activity in inhibition of cancer angiogenesis should be further investigated.