CpG-island methylation study of liver fluke-related cholangiocarcinoma

Background: Genetic changes have been widely reported in association with cholangiocarcinoma (CCA), while epigenetic changes are poorly characterised. We aimed to further evaluate CpG-island hypermethylation in CCA at candidate loci, which may have potential as diagnostic or prognostic biomarkers. Methods: We analysed methylation of 26 CpG-islands in 102 liver fluke related-CCA and 29 adjacent normal samples using methylation-specific PCR (MSP). Methylation of interest loci was confirmed using pyrosequencing and/or combined bisulfite restriction analysis, and protein expression by immunohistochemistry. Results: A number of CpG-islands (OPCML, SFRP1, HIC1, PTEN and DcR1) showed frequency of hypermethylation in >28% of CCA, but not adjacent normal tissues. The results showed that 91% of CCA were methylated in at least one CpG-island. The OPCML was the most frequently methylated locus (72.5%) and was more frequently methylated in less differentiated CCA. Patients with methylated DcR1 had significantly longer overall survival (Median; 41.7 vs 21.7 weeks, P=0.027). Low-protein expression was found in >70% of CCA with methylation of OPCML or DcR1. Conclusion: Aberrant hypermethylation of certain loci is a common event in liver fluke-related CCA and may potentially contribute to cholangiocarcinogenesis. The OPCML and DcR1 might serve as methylation biomarkers in CCA that can be readily examined by MSP

High uptake and inward diffusion of iron fortificant in ultrasonicated milled rice

Nutritional iron content in milled rice is generally lost as a result of milling due to removal of aleurone layer. Iron fortification in milled rice is recommended to regain the lost nutrients and address the malnutrition issues. This work investigates the uptake of iron and its diffusion into the kernel on ultrasonic-treated milled rice. Rice samples were subjected to ultrasonic waves resulting in the formation of microporous surfaces and the creation of fissures in the milled rice. Sonication followed by soaking in aqueous iron solution resulted in the uptake of 321 ± 13.43 mg of iron per kg of rice, a 28-fold increase compared to the endogenous iron content of milled rice with retention of 82.9% upon washing and cooking. Cross-section mapping (μ-XRF) of the concentration of the fortificant into the uncooked grains showed inward diffusion at different rates reaching into the kernel core. Results also show that sonication decreased the amount of water-soluble phosphorus in rice suggesting the removal of potentially anti-nutrient phytic acid. Textural analysis of ultrasonic-treated iron-fortified rice premix revealed favorable properties that can be advantageous for its consumer acceptability. © 2020 Elsevier Lt

A novel TFF2 splice variant (Delta EX2TFF2) correlates with longer overall survival time in cholangiocarcinoma

Trefoil factor 2 (TFF2) is a member of trefoil factor family found to be overexpressed in many cancers including cholangiocarcinoma (CCA). The majority of studies have focused on wild-type TFF2 (wtTFF2) expression, but information regarding alternative splicing variants of TFF2 mRNA has not been reported. In this study, we aimed to identify and quantify a novel TFF2 splice variant in cholangiocarcinoma (CCA). Seventy-eight tumors and 15 normal adjacent tissues were quantified for the expression of the TFF2 splice variant relative to wild-type (wt) TFF2 mRNA using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). The ratio of TFF2 splice variant against wtTFF2 was analyzed for associations with clinical parameters. We found a novel TFF2 splice variant, exon 2 skipping (∆EX2TFF2), resulting in a stop codon (TAG) at exon 1. The ∆EX2TFF2/wtTFF2 ratio in tumors was significantly higher than in normal tissue (P<0.01). Interestingly, high ∆EX2TFF2/wtTFF2 ratio was significantly associated with good prognosis compared with low ratio (P=0.017). In contrast, the presence of wtTFF2 protein was associated with poor survival of CCA patients (P=0.034). This is the first report of a trefoil factor splice variant and its potential application as a prognostic biomarker in CCA