Changes in antioxidant status and DNA repair capacity are corroborated with molecular alterations in malignant thyroid tissue of patients with papillary thyroid cancer.

Introduction: Papillary thyroid cancer (PTC) accounts for approximately 80% of all thyroid cancer cases. The mechanism of PTC tumourigenesis is not fully understood, but oxidative imbalance is thought to play a role. To gain further insight, this study evaluated antioxidant status, DNA repair capacity and genetic alterations in individuals diagnosed with benign thyroid lesion in one lobe (BTG) and PTC lesion in another. Methods: Individuals with coexisting BTG and PTC lesions in their thyroid lobes were included in this study. Reactive oxygen species (ROS) level, ABTS radical scavenging activity, ferric reducing antioxidant capacity, glutathione peroxidase and superoxide dismutase activities were measured in the thyroid tissue lysate. The expression of selected genes and proteins associated with oxidative stress defence and DNA repair were analysed through quantitative real-time PCR and Western blotting. Molecular alterations in genomic DNA were analysed through whole-exome sequencing and the potentially pathogenic driver genes filtered through Cancer-Related Analysis of Variants Toolkit (CRAVAT) analysis were subjected to pathway enrichment analysis using Metascape. Results: Significantly higher ROS level was detected in the PTC compared to the BTG lesions. The PTC lesions had significantly higher expression of GPX1, SOD2 and OGG1 but significantly lower expression of CAT and PRDX1 genes than the BTG lesions. Pathway enrichment analysis identified "regulation of MAPK cascade," "positive regulation of ERK1 and ERK2 cascade" and "negative regulation of reactive oxygen species metabolic process" to be significantly enriched in the PTC lesions only. Four pathogenic genetic variants were identified in the PTC lesions; BRAF V600E, MAP2K7-rs2145142862, BCR-rs372013175 and CD24 NM_001291737.1:p.Gln23fs while MAP3K9 and G6PD were among 11 genes that were mutated in both BTG and PTC lesions. Conclusion: Our findings provided further insight into the connection between oxidative stress, DNA damage, and genetic changes associated with BTG-to-PTC transformation. The increased oxidative DNA damage due to the heightened ROS levels could have heralded the BTG-to-PTC transformation, potentially through mutations in the genes involved in the MAPK signalling pathway and stress-activated MAPK/JNK cascade. Further in-vitro functional analyses and studies involving a larger sample size would need to be carried out to validate the findings from this pilot study.

Alternanthera sessilis: Uncovering the nutritional and medicinal values of an edible weed.

ETHNOPHARMACOLOGICAL RELEVANCE: Weeds are often considered undesirable as they interfere with the habitat of native plants, and therefore they are underestimated and underutilised. In fact, some edible weeds have beneficial nutritional and medicinal values. Alternanthera sessilis (L.) R. Br. ex DC., an edible medicinal weed is a species of the Amaranthaceae family that consists of two cultivars: green and red. Local communities in different regions have traditionally consumed the plants as food and medicine, with the green cultivar being applied to relieve pain, treat wound healing, dysentery, asthma and hypertension, while the red cultivar is applied to prevent cardiovascular and liver diseases in general. AIM OF THE STUDY: The present review intends to provide an in-depth discussion and scientific basis of A. sessilis green and red's health-promoting properties in relation to their ethnobotanical use, nutritional components and bioactive compounds. MATERIALS AND METHODS: The literature search was conducted using relevant keywords on scientific search engines such as the Web of Science, Google Scholar, Medline and Scopus. RESULTS: A. sessilis shows potent antioxidant activity as a result of its diverse phytochemical constituents, such as polyphenols, terpenes, alkaloid and carotenoids in addition to its nutritional components: vitamin C, E and unsaturated fatty acids, which contribute to its various bioactive properties: anti-microbial and anthelmintic, anti-diabetic, lipid lowering, anti-inflammatory and analgesic activities, anti-cancer and other biological activities. Toxicity evaluation revealed the absence of adverse effect of A. sesslis extracts. CONCLUSION: A. sessilis has a great potential to be used as complementary medicine and ingredients for pharmaceuticals, nutraceuticals and functional foods, instead of being regarded as a pest. Prospects for enhancing the development and commercialisation of this edible medicinal weed as a high value health-promoting product are suggested.

Comparative Analysis of In Vitro Enzyme Inhibitory Activities and Phytochemicals from Platycladus orientalis (L.) Franco via Solvent Partitioning Method.

The extraction of plant bioactive compounds from Platycladus orientalis (L.) Franco remains a great challenge due to the different chemical groups. This study aimed to compare the bioactive compounds with enzyme inhibitory effect from P. orientalis via solvent partitioning method. Dried leaf samples were macerated and fractionated with six solvents of different polarities. The phenolic, flavonoid, tannin, saponin, alkaloid and pharmacological activities including anti-inflammatory, anti-diabetic, antioxidant and anti-glycation potential were compared across the six plant fractions. Toxicity assessment was performed with an in vivo brine shrimp model. The varying levels of bioactive compounds in ethyl acetate (phenolics, flavonoids), hexane (saponins, tannins) and chloroform (alkaloids) fractions clearly demonstrated the significant impact of solvent polarity on the extraction of bioactive compounds. The reducing potential (r = 0.67), IC50 of α-amylase inhibition (r = -0.71), IC50 of advanced glycation end-product inhibition (r = -0.93) and dicarbonyl compound inhibition (r = 0.57) in the plant fractions were correlated (p<0.05) with the flavonoids. Besides, the alkaloid, saponin and tannin were associated with cyclooxygenase-1 inhibitory activity. Principal component analysis confirmed that solvent polarity (23.9%) and plant extraction yield (37.1%) collectively contributed to 61% of bioactivity variation in P. orientalis. Among the six plant fractions, ethyl acetate fraction exhibited relatively high anti-inflammatory, anti-diabetic, antioxidant and anti-glycation potential while the non-toxic methanolic and aqueous fractions displayed optimal hyaluronidase and lipoxygenase inhibitory activities, respectively. The current study has identified semi-polar ethyl acetate fraction of P. orientalis as a good alternative source of bioactive compounds for future pharmaceutical product development.

Application of Proteomics in Pancreatic Ductal Adenocarcinoma Biomarker Investigations: A Review.

Pancreatic ductal adenocarcinoma (PDAC), a highly aggressive malignancy with a poor prognosis is usually detected at the advanced stage of the disease. The only US Food and Drug Administration-approved biomarker that is available for PDAC, CA 19-9, is most useful in monitoring treatment response among PDAC patients rather than for early detection. Moreover, when CA 19-9 is solely used for diagnostic purposes, it has only a recorded sensitivity of 79% and specificity of 82% in symptomatic individuals. Therefore, there is an urgent need to identify reliable biomarkers for diagnosis (specifically for the early diagnosis), ascertain prognosis as well as to monitor treatment response and tumour recurrence of PDAC. In recent years, proteomic technologies are growing exponentially at an accelerated rate for a wide range of applications in cancer research. In this review, we discussed the current status of biomarker research for PDAC using various proteomic technologies. This review will explore the potential perspective for understanding and identifying the unique alterations in protein expressions that could prove beneficial in discovering new robust biomarkers to detect PDAC at an early stage, ascertain prognosis of patients with the disease in addition to monitoring treatment response and tumour recurrence of patients.

Whole-exome sequencing and bioinformatic analyses revealed differences in gene mutation profiles in papillary thyroid cancer patients with and without benign thyroid goitre background.

Background: Papillary thyroid cancer (PTC) is the most common thyroid malignancy. Concurrent presence of cytomorphological benign thyroid goitre (BTG) and PTC lesion is often detected. Aberrant protein profiles were previously reported in patients with and without BTG cytomorphological background. This study aimed to evaluate gene mutation profiles to further understand the molecular mechanism underlying BTG, PTC without BTG background and PTC with BTG background. Methods: Patients were grouped according to the histopathological examination results: (i) BTG patients (n = 9), (ii) PTC patients without BTG background (PTCa, n = 8), and (iii) PTC patients with BTG background (PTCb, n = 5). Whole-exome sequencing (WES) was performed on genomic DNA extracted from thyroid tissue specimens. Nonsynonymous and splice-site variants with MAF of ≤ 1% in the 1000 Genomes Project were subjected to principal component analysis (PCA). PTC-specific SNVs were filtered against OncoKB and COSMIC while novel SNVs were screened through dbSNP and COSMIC databases. Functional impacts of the SNVs were predicted using PolyPhen-2 and SIFT. Protein-protein interaction (PPI) enrichment of the tumour-related genes was analysed using Metascape and MCODE algorithm. Results: PCA plots showed distinctive SNV profiles among the three groups. OncoKB and COSMIC database screening identified 36 tumour-related genes including BRCA2 and FANCD2 in all groups. BRAF and 19 additional genes were found only in PTCa and PTCb. "Pathways in cancer", "DNA repair" and "Fanconi anaemia pathway" were among the top networks shared by all groups. However, signalling pathways related to tyrosine kinases were the most significantly enriched in PTCa while "Jak-STAT signalling pathway" and "Notch signalling pathway" were the only significantly enriched in PTCb. Ten SNVs were PTC-specific of which two were novel; DCTN1 c.2786C>G (p.Ala929Gly) and TRRAP c.8735G>C (p.Ser2912Thr). Four out of the ten SNVs were unique to PTCa. Conclusion: Distinctive gene mutation patterns detected in this study corroborated the previous protein profile findings. We hypothesised that the PTCa and PTCb subtypes differed in the underlying molecular mechanisms involving tyrosine kinase, Jak-STAT and Notch signalling pathways. The potential applications of the SNVs in differentiating the benign from the PTC subtypes requires further validation in a larger sample size.

Evaluation of Anti-proliferative Effects of Barringtonia racemosa and Gallic Acid on Caco-2 Cells.

Barringtonia racemosa leaf water extract (BLE) had been shown to have high gallic acid (GA) content and BLE has been postulated to have anti-proliferative effects towards colorectal cancer. This study aims to further investigate the mechanism underlying the anti-proliferative effect of BLE in Caco-2 cells and to determine if GA is responsible for the observed effects. Both BLE and GA inhibited Caco-2 cells in a dose-dependent manner. Cells exposed to IC50 concentration of BLE and GA showed reduced antioxidant activities. GA-treated Caco-2 cells experienced higher oxidative stress compared to cells treated with BLE. Both BLE and GA significantly up-regulated the expression of SLC2A1. BLE but not GA, significantly down-regulated the expression of ADH4. Meanwhile, GA but not BLE, significantly up-regulated AKRIB10 and GLO1 but significantly down-regulated HAGH. Alterations in gene expression were coupled with changes in extracellular glucose and pyruvate levels. While BLE decreased intracellular pyruvate, GA did the opposite. Both intracellular and extracellular D-lactate were not affected by either BLE or GA. GA showed more pronounced effects on apoptosis while BLE irreversibly reduced cell percentage in the G0/G1 phase. In conclusion, this study demonstrates the multiple-actions of BLE against Caco-2 cells, potentially involving various polyphenolic compounds, including GA.

Solid-liquid extraction of bioactive compounds with antioxidant potential from Alternanthera sesillis (red) and identification of the polyphenols using UHPLC-QqQ-MS/MS.

Alternanthera sessilis (red) (ASR) is an edible herbal plant with many beneficial health effects. This study aimed to investigate the antioxidant components and antioxidant activities of the edible leaves and stems of ASR extracted using solvent of varying polarities namely water, ethanol, ethyl acetate and hexane. ASR leaf extracts showed higher in both antioxidant components and activities than the stem extracts. Among the antioxidant components, the ethanol leaf extract showed higher phenolic (77.29 ±â€¯1.02 mg GAE/g extract) content while the ethyl acetate leaf extract was rich in flavonoids (157.44 ±â€¯10.19 mg RE/g extract), carotenoids (782.97 ±â€¯10.78 mg BE/g extract) and betalains (betanin: 67.08 ±â€¯0.49 mg/g extract; amaranthin: 93.94 ±â€¯0.68 mg/g extract and betaxanthin: 53.92 ±â€¯0.88 mg/g extract). Nevertheless, the ethanol leaf extract showed the highest DPPH radical scavenging activity and ABTS radical cation scavenging activity. It also exhibited highest ferric reducing activity among all the extracts. Four polyphenolic compounds from ASR leaf, namely ferulic acid, rutin, quercetin and apigenin, were identified and quantified using ultra high performance liquid chromatography. The existence of these compounds was further verified using tandem mass spectrometry. These current results indicate that ASR leaf particularly the ethanol extract has the potential to be exploited as a source of natural antioxidants.

Analyses of antioxidant status and nucleotide alterations in genes encoding antioxidant enzymes in patients with benign and malignant thyroid disorders.

BACKGROUND: Synthesis of thyroid hormones and regulation of their metabolism involve free radicals that may affect redox balance in the body. Thyroid disorders causing variations in the levels of thyroid hormones may alter cellular oxidative stress. The aim of this study was to measure the antioxidant activities and biomarkers of oxidative stress in serum and red blood cells (RBC) of patients with benign and malignant thyroid disorders and to investigate if changes in the antioxidant activities in these patients were linked to alterations in genes encoding the antioxidant enzymes. METHODS: Forty-one patients with thyroid disorders from University of Malaya Medical Centre were recruited. They were categorised into four groups: multinodular goitre (MNG) (n = 18), follicular thyroid adenoma (FTA) (n = 7), papillary thyroid cancer (PTC) (n = 10), and follicular thyroid cancer (FTC) (n = 6). Serum and RBC of patients were analysed for antioxidant activities, antioxidant enzymes, and biomarkers of oxidative stress. Alterations in genes encoding the antioxidant enzymes were analysed using whole exome sequencing and PCR-DNA sequencing. RESULTS: Patients with thyroid disorders had significantly higher serum superoxide dismutase (SOD) and catalase (CAT) activities compared to control, but had lower activities in RBC. There were no significant changes in serum glutathione peroxidase (GPx) activity. Meanwhile, GPx activity in RBC was reduced in PTC and FTC, compared to control and the respective benign groups. Antioxidant activities in serum were decreased in the thyroid disorder groups when compared to the control group. The levels of malondialdehyde (MDA) were elevated in the serum of FTA group when compared to controls, while in the RBC, only the MNG and PTC groups showed higher MDA equivalents than control. Serum reactive oxygen species (ROS) levels in PTC group of both serum and RBC were significantly higher than control group. Whole exome sequencing has resulted in identification of 49 single nucleotide polymorphisms (SNPs) in MNG and PTC patients and their genotypic and allelic frequencies were calculated. Analyses of the relationship between serum enzyme activities and the total SNPs identified in both groups revealed no correlation. DISCUSSION: Different forms of thyroid disorders influence the levels of antioxidant status in the serum and RBC of these patients, implying varying capability of preventing oxidative stress. A more comprehensive study with a larger target population should be done in order to further evaluate the relationships between antioxidant enzymes gene polymorphisms and thyroid disorders, as well as strengthening the minor evidences provided in literatures.

Tissue and serum samples of patients with papillary thyroid cancer with and without benign background demonstrate different altered expression of proteins.

BACKGROUND: Papillary thyroid cancer (PTC) is mainly diagnosed using fine-needle aspiration biopsy. This most common form of well-differentiated thyroid cancer occurs with or without a background of benign thyroid goiter (BTG). METHODS: In the present study, a gel-based proteomics analysis was performed to analyse the expression of proteins in tissue and serum samples of PTC patients with (PTCb; n = 6) and without a history of BTG (PTCa; n = 8) relative to patients with BTG (n = 20). This was followed by confirmation of the levels of proteins which showed significant altered abundances of more than two-fold difference (p < 0.01) in the tissue and serum samples of the same subjects using ELISA. RESULTS: The data of our study showed that PTCa and PTCb distinguish themselves from BTG in the types of tissue and serum proteins of altered abundance. While higher levels of alpha-1 antitrypsin (A1AT) and heat shock 70 kDa protein were associated with PTCa, lower levels of A1AT, protein disulfide isomerase and ubiquitin-conjugating enzyme E2 N seemed apparent in the PTCb. In case of the serum proteins, higher abundances of A1AT and alpha 1-beta glycoprotein were detected in PTCa, while PTCb was associated with enhanced apolipoprotein A-IV and alpha 2-HS glycoprotein (AHSG). The different altered expression of tissue and serum A1AT as well as serum AHSG between PTCa and PTCb patients were also validated by ELISA. DISCUSSION: The distinctive altered abundances of the tissue and serum proteins form preliminary indications that PTCa and PTCb are two distinct cancers of the thyroid that are etiologically and mechanistically different although it is currently not possible to rule out that they may also be due other reasons such as the different stages of the malignant disease. These proteins stand to have a potential use as tissue or serum biomarkers to discriminate the three different thyroid neoplasms although this requires further validation in clinically representative populations.

Antioxidant-rich leaf extract of Barringtonia racemosa significantly alters the in vitro expression of genes encoding enzymes that are involved in methylglyoxal degradation III.

BACKGROUND: Barringtonia racemosa is a medicinal plant belonging to the Lecythidaceae family. The water extract of B. racemosa leaf (BLE) has been shown to be rich in polyphenols. Despite the diverse medicinal properties of B. racemosa, information on its major biological effects and the underlying molecular mechanisms are still lacking. METHODS: In this study, the effect of the antioxidant-rich BLE on gene expression in HepG2 cells was investigated using microarray analysis in order to shed more light on the molecular mechanism associated with the medicinal properties of the plant. RESULTS: Microarray analysis showed that a total of 138 genes were significantly altered in response to BLE treatment (p < 0.05) with a fold change difference of at least 1.5. SERPINE1 was the most significantly up-regulated gene at 2.8-fold while HAMP was the most significantly down-regulated gene at 6.5-fold. Ingenuity Pathways Analysis (IPA) revealed that "Cancer, cell death and survival, cellular movement" was the top network affected by the BLE with a score of 44. The top five canonical pathways associated with BLE were Methylglyoxal Degradation III followed by VDR/RXR activation, TR/RXR activation, PXR/RXR activation and gluconeogenesis. The expression of genes that encode for enzymes involved in methylglyoxal degradation (ADH4, AKR1B10 and AKR1C2) and glycolytic process (ENO3, ALDOC and SLC2A1) was significantly regulated. Owing to the Warburg effect, aerobic glycolysis in cancer cells may increase the level of methylglyoxal, a cytotoxic compound. CONCLUSIONS: BLE has the potential to be developed into a novel chemopreventive agent provided that the cytotoxic effects related to methylglyoxal accumulation are minimized in normal cells that rely on aerobic glycolysis for energy supply.

Protective effects of the extracts of Barringtonia racemosa shoots against oxidative damage in HepG2 cells.

Barringtonia racemosa is a tropical plant with medicinal values. In this study, the ability of the water extracts of the leaf (BLE) and stem (BSE) from the shoots to protect HepG2 cells against oxidative damage was studied. Five major polyphenolic compounds consisting of gallic acid, ellagic acid, protocatechuic acid, quercetin and kaempferol were identified using HPLC-DAD and ESI-MS. Cell viability assay revealed that BLE and BSE were non-cytotoxic (cell viabilities >80%) at concentration less than 250 µg/ml and 500 µg/ml, respectively. BLE and BSE improved cellular antioxidant status measured by FRAP assay and protected HepG2 cells against H2O2-induced cytotoxicity. The extracts also inhibited lipid peroxidation in HepG2 cells as well as the production of reactive oxygen species. BLE and BSE could also suppress the activities of superoxide dismutase and catalase during oxidative stress. The shoots of B. racemosa can be an alternative bioactive ingredient in the prevention of oxidative damage.

Polyphenols from the extract and fraction of T. indica seeds protected HepG2 cells against oxidative stress.

BACKGROUND: Tamarindus indica L. (T. indica) or locally known as "asam jawa" belongs to the family Leguminosae. T. indica seeds as by-products from the fruits were previously reported to contain high polyphenolic content. However, identification of their bioactive polyphenols using recent technologies is less well researched but nonetheless important. Hence, it was the aim of this study to provide further information on the polyphenolic content and antioxidant activities as well as to identify and quantify its bioactive polyphenols. METHODS: T. indica seeds were extracted with methanol and were then fractionated with different compositions of hexane, ethyl acetate and methanol. Polyphenolic contents were measured using Folin-Ciocalteu assay while antioxidant activities were measured using DPPH radical scavenging and ferric reducing (FRAP) activities. The cytotoxic activities of the crude extract and the active fraction were evaluated in HepG2 cells using MTT assay. The cells were then pre-treated with the IC20 concentrations and induced with H2O2 before measuring their cellular antioxidant activities including FRAP, DPPH, lipid peroxidation, ROS generation and antioxidant enzymes, SOD, GPx and CAT. Analyses of polyphenols in the crude extract and its active fraction were done using UHPLC and NMR. RESULTS: Amongst the 7 isolated fractions, fraction F3 showed the highest polyphenolic content and antioxidant activities. When HepG2 cells were treated with fraction F3 or the crude extract, the former demonstrated higher antioxidant activities. F3 also showed stronger inhibition of lipid peroxidation and ROS generation, and enhanced activities of SOD, GPx and CAT of HepG2 cells following H2O2-induced oxidative damage. UHPLC analyses revealed the presence of catechin, procyanidin B2, caffeic acid, ferulic acid, chloramphenicol, myricetin, morin, quercetin, apigenin and kaempferol, in the crude seed extract of T. indica. UHPLC and NMR analyses identified the presence of caffeic acid in fraction F3. Our studies were the first to report caffeic acid as the active polyphenol isolated from T. indica seeds which likely contributed to the potent antioxidant defense system of HepG2 cells. CONCLUSION: Results from this study indicate that caffeic acid together with other polyphenols in T. indica seeds can enhance the antioxidant activities of treated HepG2 cells which can provide protection against oxidative damage.

Investigation into the effects of antioxidant-rich extract of Tamarindus indica leaf on antioxidant enzyme activities, oxidative stress and gene expression profiles in HepG2 cells.

The leaf extract of Tamarindus indica L. (T. indica) had been reported to possess high phenolic content and showed high antioxidant activities. In this study, the effects of the antioxidant-rich leaf extract of the T. indica on lipid peroxidation, antioxidant enzyme activities, H2O2-induced ROS production and gene expression patterns were investigated in liver HepG2 cells. Lipid peroxidation and ROS production were inhibited and the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase was enhanced when the cells were treated with the antioxidant-rich leaf extract. cDNA microarray analysis revealed that 207 genes were significantly regulated by at least 1.5-fold (p < 0.05) in cells treated with the antioxidant-rich leaf extract. The expression of KNG1, SERPINC1, SERPIND1, SERPINE1, FGG, FGA, MVK, DHCR24, CYP24A1, ALDH6A1, EPHX1 and LEAP2 were amongst the highly regulated. When the significantly regulated genes were analyzed using Ingenuity Pathway Analysis software, "Lipid Metabolism, Small Molecule Biochemistry, Hematological Disease" was the top biological network affected by the leaf extract, with a score of 36. The top predicted canonical pathway affected by the leaf extract was the coagulation system (P < 2.80 × 10(-6)) followed by the superpathway of cholesterol biosynthesis (P < 2.17 × 10(-4)), intrinsic prothrombin pathway (P < 2.92 × 10(-4)), Immune Protection/Antimicrobial Response (P < 2.28 × 10(-3)) and xenobiotic metabolism signaling (P < 2.41 × 10(-3)). The antioxidant-rich leaf extract of T. indica also altered the expression of proteins that are involved in the Coagulation System and the Intrinsic Prothrombin Activation Pathway (KNG1, SERPINE1, FGG), Superpathway of Cholesterol Biosynthesis (MVK), Immune protection/antimicrobial response (IFNGR1, LEAP2, ANXA3 and MX1) and Xenobiotic Metabolism Signaling (ALDH6A1, ADH6). In conclusion, the antioxidant-rich leaf extract of T. indica inhibited lipid peroxidation and ROS production, enhanced antioxidant enzyme activities and significantly regulated the expression of genes and proteins involved with consequential impact on the coagulation system, cholesterol biosynthesis, xenobiotic metabolism signaling and antimicrobial response.

Functional analyses of C.2268dup in thyroid peroxidase gene associated with goitrous congenital hypothyroidism.

The c.2268dup mutation in thyroid peroxidase (TPO) gene was reported to be a founder mutation in Taiwanese patients with dyshormonogenetic congenital hypothyroidism (CH). The functional impact of the mutation is not well documented. In this study, homozygous c.2268dup mutation was detected in two Malaysian-Chinese sisters with goitrous CH. Normal and alternatively spliced TPO mRNA transcripts were present in thyroid tissues of the two sisters. The abnormal transcript contained 34 nucleotides originating from intron 12. The c.2268dup is predicted to generate a premature termination codon (PTC) at position 757 (p.Glu757X). Instead of restoring the normal reading frame, the alternatively spliced transcript has led to another stop codon at position 740 (p.Asp739ValfsX740). The two PTCs are located at 116 and 201 nucleotides upstream of the exons 13/14 junction fulfilling the requirement for a nonsense-mediated mRNA decay (NMD). Quantitative RT-PCR revealed an abundance of unidentified transcripts believed to be associated with the NMD. TPO enzyme activity was not detected in both patients, even though a faint TPO band of about 80 kD was present. In conclusion, the c.2268dup mutation leads to the formation of normal and alternatively spliced TPO mRNA transcripts with a consequential loss of TPO enzymatic activity in Malaysian-Chinese patients with goitrous CH.

Variable clinical phenotypes in a family with homozygous c.1159G>A mutation in the thyroid peroxidase gene.

BACKGROUND: Defects in the thyroid peroxidase (TPO) gene have been associated with goitrous congenital hypothyroidism (CH). CASE REPORT: In this study, we report 3 siblings possessing a homozygous mutation, c.1159G>A, but exhibiting different clinical phenotypes in a Malaysian-Malay family. The index patient was diagnosed with CH during a routine neonatal screening but the other 2 siblings appeared to be asymptomatic until the ages of 19 and 12.5, respectively, when they started to develop goiter. RESULTS AND CONCLUSION: The mutation was predicted to interrupt the correct splicing of pre-mRNA and also lead to structural alterations in the functional sites of the mutant TPO. The current results suggest the association of goiter development with a homozygous c.1159G>A mutation, but the CH in the index patient could be triggered by other genetic and epigenetic factors distinct from the c.1159G>A mutation.

Polyphenols in Barringtonia racemosa and their protection against oxidation of LDL, serum and haemoglobin.

The polyphenolic profiles and antioxidant activities of the water extracts of Barringtonia racemosa shoots (leaves and stems) were explored. Two methods, freeze drying and air drying, for preparation of the shoots, were also compared. Freeze drying was better as air drying caused 5-41% reduction of polyphenols. Three phenolic acids and three flavonoids were identified, using UHPLC. The descending order of polyphenols in the leaves and stems was gallic acid>ellagic acid>quercetin>protocatechuic acid>rutin>kaempferol. In vitro antioxidant analyses were performed using biological samples. In the LDL oxidation assay, B. racemosa leaf extract (IC50=73.0µg/ml) was better than stem extract (IC50=226µg/ml) at inhibiting the formation of TBARS and lipid hydroperoxides. Similar trends were observed for serum and haemoglobin oxidation. B. racemosa leaf extract was better than its stem extract in delaying the time required to oxidise haemoglobin to methaemoglobin. The high polyphenolic content of B. racemosa shoots could have contributed towards their antioxidative effects.

In vivo biochemical and gene expression analyses of the antioxidant activities and hypocholesterolaemic properties of Tamarindus indica fruit pulp extract.

BACKGROUND: Tamarindus indica (T. indica) is a medicinal plant with many biological activities including anti-diabetic, hypolipidaemic and anti-bacterial activities. A recent study demonstrated the hypolipidaemic effect of T. indica fruit pulp in hamsters. However, the biochemical and molecular mechanisms responsible for these effects have not been fully elucidated. Hence, the aims of this study were to evaluate the antioxidant activities and potential hypocholesterolaemic properties of T. indica, using in vitro and in vivo approaches. METHODOLOGY/PRINCIPAL FINDINGS: The in vitro study demonstrated that T. indica fruit pulp had significant amount of phenolic (244.9 ± 10.1 mg GAE/extract) and flavonoid (93.9 ± 2.6 mg RE/g extract) content and possessed antioxidant activities. In the in vivo study, hamsters fed with high-cholesterol diet for ten weeks showed elevated serum triglyceride, total cholesterol, HDL-C and LDL-C levels. Administration of T. indica fruit pulp to hypercholesterolaemic hamsters significantly lowered serum triglyceride, total cholesterol and LDL-C levels but had no effect on the HDL-C level. The lipid-lowering effect was accompanied with significant increase in the expression of Apo A1, Abcg5 and LDL receptor genes and significant decrease in the expression of HMG-CoA reductase and Mtp genes. Administration of T. indica fruit pulp to hypercholesterolaemic hamsters also protected against oxidative damage by increasing hepatic antioxidant enzymes, antioxidant activities and preventing hepatic lipid peroxidation. CONCLUSION/SIGNIFICANCE: It is postulated that tamarind fruit pulp exerts its hypocholesterolaemic effect by increasing cholesterol efflux, enhancing LDL-C uptake and clearance, suppressing triglyceride accumulation and inhibiting cholesterol biosynthesis. T. indica fruit pulp has potential antioxidative effects and is potentially protective against diet-induced hypercholesterolaemia.

A Novel, Homozygous c.1502T>G (p.Val501Gly) Mutation in the Thyroid peroxidase Gene in Malaysian Sisters with Congenital Hypothyroidism and Multinodular Goiter.

Congenital hypothyroidism (CH) with multinodular goiter (MNG) is uncommonly seen in children. However, CH associated with goiter is often caused by defective Thyroid peroxidase (TPO) gene. In this study, we screened for mutation(s) in the TPO gene in two siblings with CH and MNG and their healthy family members. The two sisters, born to consanguineous parents, were diagnosed with CH during infancy and received treatment since then. They developed MNG during childhood despite adequate L-thyroxine replacement and negative thyroid antibody screening. PCR-amplification of all exons using flanking primers followed by DNA sequencing revealed that the two sisters were homozygous for a novel c.1502T>G mutation. The mutation is predicted to substitute valine for glycine at a highly conserved amino acid residue 501 (p.Val501Gly). Other healthy family members were either heterozygotes or mutation-free. The mutation was not detected in 50 healthy unrelated individuals. In silico analyses using PolyPhen-2 and SIFT predicted that the p.Val501Gly mutation is functionally "damaging." Tertiary modeling showed structural alterations in the active site of the mutant TPO. In conclusion, a novel mutation, p.Val501Gly, in the TPO gene was detected expanding the mutation spectrum of TPO associated with CH and MNG.

Effects of Tamarindus indica fruit pulp extract on abundance of HepG2 cell lysate proteins and their possible consequential impact on metabolism and inflammation.

The fruit pulp extract of Tamarindus indica has been reported for its antioxidant and hypolipidemic properties. In this study, the methanol extract of T. indica fruit pulp was investigated for its effects on the abundance of HepG2 cell lysate proteins. Cell lysate was extracted from HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp. Approximately 2500 spots were resolved using two-dimensional gel electrophoresis and the abundance of 20 cellular proteins was found to be significantly reduced. Among the proteins of reduced abundance, fourteen, including six proteins involved in metabolism (including ethanolamine phosphate cytidylyltransferase), four mitochondrial proteins (including prohibitin and respiratory chain proteins), and four proteins involved in translation and splicing, were positively identified by mass spectrometry and database search. The identified HepG2 altered abundance proteins, when taken together and analyzed by Ingenuity Pathways Analysis (IPA) software, are suggestive of the effects of T. indica fruit pulp extract on metabolism and inflammation, which are modulated by LXR/RXR. In conclusion, the methanol fruit pulp extract of T. indica was shown to cause reduced abundance of HepG2 mitochondrial, metabolic, and regulatory proteins involved in oxidative phosphorylation, protein synthesis, and cellular metabolism.