• Journal of Microbiology and Biotechnology
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• v.33 no.11
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• pp.1506-1512
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• 2023

Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r² = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

• Korean Journal of Environment and Ecology
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• v.25 no.6
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• pp.853-860
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• 2011

We measured blood plasma parameters(cortisol, glucose, albumin) and glucocorticoid receptor(GCR) gene expression level of the Japanese eel(Anguilla japonica) exposed to an explosion noise for an hour in order to evaluate the effects of noise stress and to explore the possibility of these parameters as biomarkers on noise stress for one of this valuable aquaculture species. Plasma cortisol and glucose reached high levels with significant differences compared to the control group, whereas albumin showed a low value after 1 h of exposure. In addition, tissue distribution of GCR gene expression was studied by real-time RT-PCR of ten organs(brain, eye, gill, gonad, heart, intestine, kidney, liver, muscle and skin). Liver showed the highest level of expression in the control followed by gill, muscle and intestine. A time-course study revealed induction in liver, gill, muscle and intestine after 30 min or 1 h of noise exposure.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.29-34
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• 2010

Follicular cystic ovary (FCO) is one of the major causes of reproductive failure in cattle. Genetic alterations affect the function of diverse cells and/or tissues, which could be present in cystic ovaries. A microarray analysis was performed to screen differential gene expressions in follicular cystic follicles of cattle. In this study, we hypothesized that follicular cysts may be induced by changes in ion- and transporter-related gene expression. Microarray data showed that fibrinogen-gamma (FGG) and low density lipoprotein receptor-related protein 8 (LRP8) were up-regulated, while choline transporter-like protein 4 (SLC44A4), very long-chain acyl-CoA synthetase homolog 2 (SLC27A5), annexin 8 (ANXA8), and aquaporin 4 were down-regulated in follicular cystic follicles. A semi-quantitative RT-PCR was carried out to validate DEGs altered in follicular cystic follicles. Of six DEGs, three DEGs (FGG, SLC44A4, and aquaporin 4) showed a positive correlation between microarray and semi-quantitative PCR data. We focused on FGG, among three DEGs, which was highly up-regulated in follicular cystic follicles. The FGG mRNA was upregulated by 8.4-fold and by 1.7-fold in the bovine follicular cystic follicles as judged by microarray and RT-PCR analysis, respectively. However, there was no significant changes in the expression level of FGG protein in both follicular cystic follicles and granulosa cells isolated from follicular cystic follicles by Western blot analysis. Although this study does not reveal a positive correlation between the mRNA and protein level, FGG appears to be an important biomarker in the discrimination of follicular cyst from normal ovary.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.1
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• pp.299-304
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• 2014

Cholangiocarcinoma (CCA), a malignancy of biliary duct with a very poor prognosis, is the leading cause of cancer death in countries of the Mekong subregion. Liver fluke infection is the main etiological factor, but genetic variation has been recognized as also important in conferring susceptibility to CCA risk. Nuclear factor (erythroid derived 2)-like 2 (NRF2) is a key transcription factor in detoxification and antioxidant defense. Emerging evidence has demonstrated that genetic polymorphisms in the NRF2 gene may be associated with cancer development. The objectives of this study were to investigate the association of NRF2 genetic polymorphism with CCA risk and to evaluate the influence of the NRF2 genotype on survival time of affected patients. Single nucleotide polymorphisms (SNPs) of the NRF2 gene, including rs6726395: A/G, rs2886161: C/T, rs1806649: C/T, and rs10183914: C/T, were analyzed using TaqMan$^{(R)}$ SNP genotyping assays. Among 158 healthy northeastern Thai subjects, the allele frequencies were 41, 62, 94, and 92%, respectively. The correlation of NRF2 SNPs and CCA risk was analyzed in the 158 healthy subjects and 198 CCA patients, using unconditional logistic regression. The results showed that whereas the NRF2 SNPs were not associated with CCA risk (p>0.05), Kaplan-Meier analysis of 88 intrahepatic CCA patients showed median survival time with rs6726395 genotypes of GG and AA/AG to be $344{\pm}138$ (95%CI: 73-615) days and $172{\pm}37$ (95%CI: 100-244) days, respectively, (p<0.006). On multivariate Cox proportional hazard analysis, the GG genotype of rs6726395 was found to be associated with longer survival with a hazard ratio of 0.54 (95%CI: 0.31-0.94). In addition, non-papillary adenocarcinoma was associated with poor survival with a hazard ratio of 2.09 (95%CI: 1.16-3.75). The results suggest that the NRF2 rs6726395 polymorphism can be a potential prognostic biomarker for CCA patients.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.14 no.8
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• pp.4086-4092
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• 2013

Mycosporine-like amino acids (MAAs) are UV-absorbing substances, and diverse marine organisms have the evolved the capacity to diminished the direct and indirect damaging effects of environmental ultraviolet radiation by synthesis and accumulation of MAAs. In this study, we manufactured a radiofrequency (RF) generation device and applied to microalgal culture. $0.35{\pm}0.05$ mHz of RF was supplied to culture vessel for Chlamydomonas sp. and samples were harvested at the designated time intervals (1, 0.5, 1 and 2 hr). MAAs biosynthetic genes, dehydroquinate synthase homolog (DHQS-like) and nonribosomal peptide synthetase homolog (NRPS-like), were cloned from Chlamydomonas sp. and their gene expressions under the RF exposure were analyzed using qRT-PCR. DHQS-like and NRPS-like gene expressions of Chlamydomonas sp. exposed to RF were increased 1.46 and 1.19 fold at 1 hr, respectively. These results means that DHQS-like and NRPS-like genes can be good biomarker candidates for diagnosis of MAAs biosynthesis in the Chlamydomonas sp.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4281-4287
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• 2014

Background: Methylation of tumor suppressor genes has been investigated in all kinds of cancer. Tumor specific epigenetic alterations can be used as a molecular markers of malignancy, which can lead to better diagnosis, prognosis and therapy. Therefore, the aim of this study was to evaluate the association between gene hypermethylation and expression of fragile histidine triad (FHIT), glutathione S-transferase P1 (GSTP1) and p16 genes and various clinicopathologic characteristics in primary non-small cell lung carcinomas (NSCLC). Materials and Methods: The study included 28 primary non-small cell lung carcinomas, where an additional 28 tissue samples taken from apparently normal safety margin surrounding the tumors served as controls. Methylation-specific polymerase chain reaction (MSP) was performed to analyze the methylation status of FHIT, GSTP1 and p16 while their mRNA expression levels were measured using a real-time PCR assay with SYBR Green I. Results: The methylation frequencies of the genes tested in NSCLC specimens were 53.6% for FHIT, 25% for GSTP1, and 0% for p16, and the risk of FHIT hypermethylation increased among patients with NSCLC by 2.88, while the risk of GSTP1 hypermethylation increased by 2.33. Hypermethylation of FHIT gene showed a highly significant correlation with pathologic stage (p<0.01) and a significant correlation with smoking habit and FHIT mRNA expression level (p<0.05). In contrast, no correlation was observed between the methylation of GSTP1 or p16 and smoking habit or any other parameter investigated (p>0.05). Conclusions: Results of the present study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC. FHIT methylation may play a role in lung cancer later metastatic stages while GSTP1 methylation may rather play a role in the early pathogenesis.

• KSBB Journal
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• v.24 no.1
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• pp.80-88
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• 2009

Human prostatic acid phosphatase (PAP), with comprehensive homology to glandular kallikrein, are representative serum biomarkers of prostate cancer. Dendritic cell (DC), which is the potent antigen-presenting cells(APC) in the immune system, can induce strong T cell responses against viruses, microbial pathogens, and tumors. Therefore, the immunization using DC loaded with tumor-associated antigens is a powerful method for inducing anti-tumor immunity. The CTP (Cytoplasmic Transduction Peptide) technology developed by Creagene which can transport attached bio-polymers like nucleic acids or proteins into the cell with high permeation efficiency. As the active forms of PAP can mediate apoptotic processing, we used multimer forms of PAP as an inactive form for antigen pulsing of DCs. In this study, multimeric forms of CTP-rhPAP was obtained according to the advanced purification process and subsequently confirmed by gel filtration chromatography, western blot and Dynamic Light Scattering. Therefore, CTP-conjugated PA multimers transduced into the cytoplasm were efficiently presented on the cell surface without any harm effect on cells via MHC class I molecules and result in induction of a large number of effector cell.

• Toxicological Research
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• v.21 no.4
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• pp.339-345
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• 2005

Aflatoxins are produced by Aspergillus flavus, parasiticus that grows in improperly stored cereals. Aflatoxin $B_1\;(AFB_1)$ is a potent hepatocarcinogen in a variety of experimental animals including human beings. In spite of a high attention to the hepatocarcinogenecity of aflatoxins, the relative toxicity of other types $(AFB_2,\;AFG_1\;and\;AFG_2)$ of the toxins is not fully clarified. Sprague-Dawley male rats were orally administered with $AFB_1,\;AFB_2,\;AFG_1\;and\;AFG_2$ at the dose of 250, 1250, and $2500\;{\mu}g/kg$ body weight. Animals were then killed at 12, 24 or 48 hrs following aflatoxin adminstration. Subsequently the relative weight of liver was measured and histopathological examination on the liver was performed. Level of 8-OxodG and expression of ras gene in the liver was determined. The relative liver weights at high doses of $AFB_1\;and\;AFG_1$ was significantly low. The treatment of $AFB_1$ at the high dose of $2500\;{\mu}g/kg$ showed vacuolar degeneration and centrilobular hepatic necrosis with inflammatory cells. The pathological changes by $AFB_2\;AFG_1,\;and\;AFG_2$ were not clearly found. The formation of 8-OxodG by $AFB_1$ increased in a dose-dependent manner up to 24 hrs after a single treatment of $AFB_1$ thereafter decreased to the level of the control. The treatments of $AFB_2\;AFG_1,\;and\;AFG_2$ showed an inconsistent pattern in the formation of 8-OxodG in the liver of rats with increasing time. The expression of ras oncogene in the liver by $AFB_1$ at the dose of $1250\;{\mu}g/kg$ was increased twice compared to the control. The treatments of $AFB_2\;AFG_1,\;and\;AFG_2$ at all doses decreased the expression of ras in the liver. These results in the present study indicate that $AFB_1$ among aflatoxins with low comparable levels is the most toxic as determined by early biomarkers such as 8-OxodG formation and ras expression. However, the levels of 8-OxodG and ras as biomarkers were not useful to predict the relative hepatocarcinogenicity of aflatoxins to $AFB_1$ in the present model. Further studies are required to look for other biomarkers to predict carcinogenic potency of aflatoxins.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.2185-2193
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• 2016

Background: The pathogenesis of sporadic colorectal cancer (CRC) is influenced by the patient genetic background and environmental factors. Based on prior understanding, these are classified in two major pathways of genetic instability. Microsatellite instability (MSI) and CPG island methylator phenotype (CIMP) are categorized as features of the hypermethylated prototype, and chromosomal instability (CIN) is known to be indicative of the non-hypermethylated category. Secreted frizzled related protein 2 (SFRP2), APC1A in WNT signaling pathway and the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT), are frequently hypermethylated in colorectal cancer. Detection of methylated DNA as a biomarker by easy and inexpensive methods might improve the quality of life of patients with CRC via early detection of cancer or a precancerous condition. Aim: To evaluate the rate of SFRP2 and MGMT hypermethylation in both polyp tissue and serum of patients in south Iran as compared with matched control normal population corresponding samples. Materials and Methods: Methylation-specific PCR was used to detect hypermethylation in DNA extracted from 48 polypoid tissue samples and 25 healthy individuals. Results: Of total polyp samples, 89.5% had at least one promoter gene hypermethylation. The most frequent methylated locus was SFRP2 followed by MGMT-B (81.2 and 66.6 percent respectively). Serologic detection of hypermethylation was 95% sensitive as compared with polyp tissue. No hypermethylation was detected in normal tissue and serum and its detection in patients with polyps, especially of serrated type, was specific. Conclusions: Serologic investigation for detection of MGMT-B, SFRP2 hypermethylation could facilitate prioritization of high risk patients for colonoscopic polyp detection and excision.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.3
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• pp.1051-1056
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• 2015

Aberrant expression of genes in de novo lipogenesis (DNL) pathway were associated with various cancers, including hepatocellular carcinoma (HCC). Single nucleotide polymorphisms (SNPs) of DNL genes have been reported to be associated with prognosis of some malignancies. However, the effects of SNPs in DNL genes on overall survival of HCC patients receiving transarterial chemoembolization (TACE) treatment are still unknown. In present study, nine SNPs in three genes (ACLY, ACACA and FASN) in DNL pathway were genotyped using the Sequenom iPLEX genotyping system in a hospital-based cohort with 419 HCC patients treated with TACE, and their associations with HCC overall survival were evaluated by Cox proportional hazard regression analysis under three genetic models (additive, dominant and recessive). Although we did not find any significant results in total analysis (all p>0.05), our stratified data showed that SNP rs9912300 in ACLY gene was significantly associated with overall survival of HCC patients with lower AFP level and SNP rs11871275 in ACACA gene was significantly associated with overall survival of HCC patients with higher AFP level. We further identified the significant interactions between AFP level and SNP rs9912300 or rs11871275 in the joint analysis. Conclusively, our data suggest that genetic variations in genes of DNL pathway may be a potential biomarker for predicting clinical outcome of HCC patients treated with TACE.