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Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a

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Background: Polygonum odoratum is an indigenous vegetable that has been used as a favoring agent and also used as a Thai traditional medicine to treat flatulence. </P><P> Objective: To analyze active ingredients, total phenolic and total flavonoid contents, antiinflammatory and antioxidant activities from leaf and stem extracts of P. odoratum. </P><P> Methods: Leaves and stems were dried and extracted by using methanol, dichloromethane and water for obtaining Methanolic Leaf Extract (MLE), Methanolic Stem Extract (MTE), Dichloromethane Leaf Extract (DLE), Dichloromethane Stem Extract (DTE), Water Leaf Extract (WLE) and Water Stem Extract (WTE). The extracts were quantified for total phenolic and total flavonoid contents by spectrophotometry and active compounds were analyzed by using GC-MS. Antioxidant activity was determined by ABTS and DPPH radicals scavenging assays. Anti-inflammatory activity was tested by the inhibition of nitric oxide production in RAW 264.7 macrophage cells induced by lipopolysaccharide. </P><P> Results: The DLE exhibited the most potent anti-inflammatory effect by inhibiting nitric oxide production in a concentration-dependent manner (IC50 = 53.75&#177;0.72 &#181;g/mL). MLE exhibited strong antioxidant activity and contained the highest concentration of phenolic compounds (52.59&#177;0.58 mg gallic acid equivalent/g extract) and flavonoid (19.97+0.11 mg quercetin equivalent/g extract). E-15-Heptadecenal and 3, 7, 11, 15-tetramethyl-2- hexadecen-1-ol were found predominantly in the methanol extracts. </P><P> Conclusion: The leaf extract of P. odoratum showed potent anti-inflammatory and antioxidant activities, mediated by DLE and MLE, respectively.

Genetic hemochromatosis (HH) is a common inherited disorder in populations of European origin in which different types of genetic hemochromatosis (type 1–4) have been characterized. Most hemochromatosis-type 1 patients are homozygotes or compound heterozygotes for two HFE mutations C282Y and H63D. Studies of several non-HFE iron overload families led to identification of mutations in hemojuvelin and hepcidin (juvenile form-HFE2A and B), transferrin receptor 2 (HFE3) and ferroportin (HFE4) as a cause of different forms of hemochromatosis. In the Far East, inherited hemochromatosis has rarely been reported and may have been misdiagnosed due to the high prevalence of secondary iron loading from hemoglobin disorders. This report describes, for the first time, non-HFE iron overload in patients from Southeast Asia. The affected Thai family presented with a distinctive clinical phenotype including macrocytosis and elevated transferrin saturation (>95%), increased non-transferrin bound iron (NTBI) as well as raised serum ferritin and marked hepatic hemochromatosis. Our patients tolerated therapeutic phlebotomy well. DNAs from peripheral blood leukocytes were firstly analyzed for three common HFE mutations (C282Y, H63D and IVS5+1 G→A). Subsequently, we screened all coding sequences, promoters and exon/intron boundaries of the HFE, HAMP, TfR2, HJV and SLC40A1 genes using denaturing high performance liquid chromatography (DHPLC). The entire coding region and splice sites of these genes were amplified and directly sequenced. We identified a novel mutation (C326Y) in ferroportin (SLC40A1, IREG-1, MTP-1), a membrane iron transport protein due to a G→A substitution at nucleotide 1281 in exon 7. This mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis using Sfa NI. Six hundred Thai and two hundred Vietnamese chromosomes were analyzed for the C326Y mutation by RFLP analysis and it was not detected in any of the healthy controls studied. This result suggested that the G→A substitution is not a common polymorphism and is likely to be the causative mutation for the phenotype in this family. Previous reported mutations of ferroportin, including A77D and V162del, which lead to type IV hemochromatosis, were characterized by increased serum ferritin despite normal transferrin saturation, in contrast to our patients’ phenotype. These autosomal dominant mutants are postulated to lead to disease due to loss of iron exporting function. Preliminary in vivo assay using transient transfection of wild-type and ferroportin mutants in HeLa or 293T cells revealed, as expected, a loss of function and diminished surface membrane localisation in A77D and V162del mutants. Surprisingly, the C326Y mutant was indistinguishable from wt ferroportin in both iron status of the cell and protein localization suggesting different pathophysiology leading to iron overload in our patients.

Oxidative stress and inflammation are 2 major contributors to numerous life-threatening disorders, including vascular pathologies. Shallots (Allium ascalonicum) are a type of red onion which grows in Southeast Asia. Bulbs of this plant are used both as a food ingredient and in traditional medicine. This study attempted to investigate the possible ways that juice extracted from Thai shallot (A.ascalonicum cv. Chiangmai) bulbs could be used in the prevention of cardiovascular complications. The antioxidative and anti-inflammatory effects of shallot juice extract (SHE) on human vascular endothelial cells (EA.hy926) were investigated. Cell viability was evaluated by MTT assay, membrane lipid peroxidation by thiobarbituric acid reactive substances assay, intracellular reactive oxygen species (ROS) production by the fluorescent probe 6-carboxy-2'-7'-dichlorofluoresceine, and interleukin-6 (IL-6) released by ELISA. The shallot juice showed extremely low cytotoxicity against EA.hy926 cells, with IC50 of 41.9 and 27.3 mg/ml for 24 h- and 48 h-incubation, respectively. SHE reduced the iron-induced malondialdehyde production in a dose-dependent manner. The extract also demonstrated antioxidant activity as shown by a significant reduction of H2O2-induced ROS production at a low concentration (< 200 mg/ml). Furthermore, SHE significantly attenuated the level of IL-6 released during lipopolysaccharide stimulation (p < 0.05). It is of interest that the juice extracted from Thai shallot bulbs demonstrated both cellular antioxidant and anti-inflammatory properties in endothelial cell models, combined with a reduction in toxicity. Shallot extract could be considered as a nutraceutical for the prevention or management of vascular diseases as it is related to oxidative stress and inflammation.

Background The resistance of Plasmodium falciparum to artemisinin-based (ART) drugs, the front-line drug family used in artemisinin-based combination therapy (ACT) for treatment of malaria, is of great concern. Mutations in the kelch13 (k13) gene (for example, those resulting in the Cys580Tyr [C580Y] variant) were identified as genetic markers for ART-resistant parasites, which suggests they are associated with resistance mechanisms. However, not all resistant parasites contain a k13 mutation, and clearly greater understanding of resistance mechanisms is required. A genome-wide association study (GWAS) found single nucleotide polymorphisms associated with ART-resistance in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2), and crt (chloroquine resistance transporter), in addition to k13 gene mutations, suggesting that these alleles contribute to the resistance phenotype. The importance of the FD and ARPS10 variants in ART resistance was then studied since both proteins likely function in the apicoplast, which is a location distinct from that of K13. Methods The reported mutations were introduced, together with a mutation to produce the k13-C580Y variant into the ART-sensitive 3D7 parasite line and the effect on ART-susceptibility using the 0−3 h ring survival assay (RSA0−3 h) was investigated. Results and conclusion Introducing both fd-D193Y and arps10-V127M into a k13-C580Y-containing parasite, but not a wild-type k13 parasite, increased survival of the parasite in the RSA0−3 h. The results suggest epistasis of arps10 and k13, with arps10-V127M a modifier of ART susceptibility in different k13 allele backgrounds.

Cellular and mitochondrial damage can be caused by labile iron pool (LIP) and mediated by reactive oxygen species (ROS). Livers of the thalassemias have highly increased levels of LIP and ROS. Green tea extract (GTE) and epigallocatechin 3-gallatte (EGCG) can potentially protect liver inflammation, fibrosis and cancer due to their anti-oxidative and iron-chelating activities. We studied the effects of GTE and EGCG on intracellular LIP and ROS, and mitochondrial membrane potential (ΔΨ_m) in mouse hepatocyte and HepG2 cell cultures using specific fluorescent techniques. Treatment with GTE (12.5 - 25 mg/dl) and EGCG (25 - 50 μM) significantly lowered levels of ΔΨ_m in the mouse hepatocytes; however, combined treatment of 25 μM DFP with GTE and EGCG did not enhance the decrease of hepatic ΔΨ_m. The results showed that GTE and EGCG effectively removed the intracellular LIP and ROS, and relieved the mitochondria membrane collapse of the liver cells, suggesting a hepatoprotective effect of green tea extract and EGCG in the hepatocytes with iron overload. Their actions might be related to iron-chelating and free radical-scavenging capacities. Whether the effects can improve iron overload and oxidative stress in thalassemia patients remains to be seen upon further examination