• Molecules and Cells
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• v.43 no.11
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• pp.909-920
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• 2020

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7635-7638
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• 2013

Background: Sperm-associated antigen 9 (SPAG9) has been recently proposed as a novel biomarker for early diagnosis of several human tumors, including ovarian, cervical and breast cancers. Its clinical value remains to be clarified for endometrial cancer (EC). In this study, we investigated the utility of serum SPAG9 levels in diagnosis of EC and its association with important clinicopathological parameters. Materials and Methods: This cross-sectional study was performed at a tertiary women's referral center in Ankara, Turkey. Preoperative serum samples were collected from patients surgically treated for endometrial cancer between June 2012-April 2013. Similar aged women with a biopsy proven benign endometrium were used as controls. Serum SPAG9 levels were measured with an enzyme-linked immunosorbent assay (ELISA) method and assessed for links with clinicopathological factors. Receiver operating characteristic (ROC) curve analysis was performed to assess power of SPAG9 levels for EC prediction. P values less than 0.05 were considered statistically significant. Results: A total of 63 women with EC and 27 with benign endometrium were included in the study. Mean age in the EC group was $58.7{\pm}1.1$. Median SPAG9 levels in the EC and control groups were 18.3 (range, 12.7-53.8) and 14.1 (range, 4.3-65.3), respectively (p<0.001). A cut-off value of 17 ng/ml for SPAG9 predicted presence of malignant endometrium with 74% sensitivity and 83% specificity [Area under curve (AUC)=0.82, p<0.001]. SPAG9 levels did not demonstrate any significant association with histological type, FIGO stage, tumor grade, size, myometrial invasion, lymphovascular space invasion, cervical involvement, adnexal involvement, peritoneal cytology or lymph node status (all p>0.05). Conclusions: Testing for SPAG9 may be useful for early detection of EC in asymptomatic high-risk women. Its role in post-treatment follow-up and early detection of recurrence should be assessed in future trials.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.4
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• pp.1657-1663
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• 2015

Background: Early diagnosis of hepatocellular carcinoma (HCC) is the most important step in successful treatment. However, it is usually rare due to the lack of a highly sensitive specific biomarker so that the HCC is usually fatal within few months after diagnosis. The aim of this work was to study the role of plasma nuclear factor kappa B (NF-${\kappa}B$) and serum peroxiredoxin 3 (PRDX3) as diagnostic biomarkers for early detection of HCC in a high-risk population. Materials and Methods: Plasma nuclear factor kappa B level (NF-${\kappa}B$) and serum peroxiredoxin 3 (PRDX3) levels were measured using enzyme linked immunosorbent assay (ELISA), in addition to alpha-fetoprotein (AFP) in 72 cirrhotic patients, 64 patients with HCC and 29 healthy controls. Results: NF-${\kappa}B$ and PRDX3 were significantly elevated in the HCC group in relation to the others. Higher area under curve (AUC) of 0.854 (for PRDX3) and 0.825 (for NF-${\kappa}B$) with sensitivity of 86.3% and 84.4% and specificity of 75.8% and 75.4% respectively, were found compared to AUC of alpha-fetoprotein (AFP) (0.65) with sensitivity of 72.4% and specificity of 64.3%. Conclusions: NF-${\kappa}B$ and PRDX3 may serve as early and sensitive biomarkers for early detection of HCC facilitating improved management. The role of nuclear factor kappa B (NF-${\kappa}B$) as a target for treatment of liver fibrosis and HCC must be widely evaluated.

• Korean Journal of Environmental Biology
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• v.26 no.4
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• pp.330-342
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• 2008

A comprehensive quality survey for PCDDs/PCDFs and coplanar PCBs as well as heavy metals (Cu, Zn, Cd and Pb) in sediments has been investigated in August 2006, Korea. Monitoring was undertaken at five streams representing different surrounding environments throughout Juwang and Gapyeong streams (reference sites), Jungrang stream (dense population site), Ansan stream (mixed small population and industrial site), and Siheung stream (heavy industrial site). The levels of heavy metal in samples were found to be significantly higher in sediment from Siheung stream compared to those of other stream sites. The heavy metal concentrations (dry weight basis) in sediment from Siheung stream were as follows; Cd (3.7 ${\mu}g$/g), Pb (1,295 ${\mu}g$/g), Cu (713.4 ${\mu}g$/g) and Zn (358.1 ${\mu}g$/g). Among 12 coplanar PCBs and 17 PCDDs/PCDFs selected as target compounds in this study, PCB (IUPAC no. 118) and OCDD were the most abundant congeners found in all sediment samples, followed by 1,2,3,4,6,7,8-HpCDD, OCDF and 1,2,3,4,6,7,8HpCDF as well as PCB (IUPAC no. 105). These results were shown to be in the same trend as the sediment samples of other countries. The levels of PCDDs/PCDFs/coplanar PCBs in sediment samples were expressed as concentrations and WHO- TEQ values. The PCDDs/PCDFs/coplanar PCBs concentrations and their WHO-TEQ values in sediment from Siheung stream were remarkably high. The levels detected were 788.16 pg/g and 36.080 pg WHO-TEQ/g dry weight for PCDDs/ PCDFs and 314 pg/g and 0.4189 pg WHO-TEQ/g dry weight for coplanar PCBs, respectively, beyond the safety level of sediment value 20 pg WHO-TEQ/g. Sediment samples of the five streams were also monitored by sensitive biomarkers using insect immune responses: hemocyte-spreading behavior and immune-associated enzyme activities of phospholipase A$_2$ (PLA$_2$) and phenoloxidase. Organic extracts of Siheung and Jungrang sediments significantly interfered with the hemocytespreading behavior, whereas those of Ansan, Gapyeong, and Juwang did not. These organic extracts did not inhibit the PLA$_2$ and phenoloxidase activities. However, phenoloxidase was highly susceptible to exposure to aqueous extracts in all site sediments. In comparison, PLA$_2$ activities of the hemocytes were significantly inhibited only by aqueous extracts of Siheung, Jungrang, and Gapyeong sediments, but not by those of Ansan and Juwang. Despite some disparity between bioand chemical monitoring results, the biomarkers can be recommended as a device warning the contamination of biohazard environmental chemicals because of a fast and inexpensive detection method.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4469-4475
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• 2012

Background: The potential use of hypomethylation of Long INterspersed Element 1 (LINE-1) and Alu elements (Alu) as a biomarker has been comprehensively assessed in several cancers, including head and neck squamous cell carcinoma (HNSCC). Failure to detect occult metastatic head and neck tumors on radical neck lymph node dissection can affect the therapeutic measures taken. Objective: The aim of this study was to investigate the LINE-1 and Alu methylation status and determine whether it can be applied for detection of occult metastatic tumors in HNSCC cases. Methods: We used the Combine Bisulfite Restriction Analysis (COBRA) technique to analyse LINE-1 and Alu methylation status. In addition to the methylation level, LINE-1 and Alu loci were classified based on the methylation statuses of two CpG dinucleotides in each allele as follows: hypermethylation ($^mC^mC$), hypomethylation ($^uC^uC$), and 2 forms of partial methylation ($^mC^uC$ and $^uC^mC$). Sixty-one lymph nodes were divided into 3 groups: 1) non-metastatic head and neck cancer (NM), 2) histologically negative for tumor cells of cases with metastatic head and neck cancer (LN), and 3) histologically positive for tumor cells (LP). Results: Alu methylation change was not significant. However, LINE-1 methylation of both LN and LP was altered, as demonstrated by the lower LINE-1 methylation levels (p<0.001), higher percentage of $^mC^uC$ (p<0.01), lower percentage of $^uC^mC$ (p<0.001) and higher percentage of $^uC^uC$ (p<0.001). Using receiver operating characteristic (ROC) curve analysis, $%^uC^mC$ and $%^mC^uC$ values revealed a high level of AUC at 0.806 and 0.716, respectively, in distinguishing LN from NM. Conclusion: The LINE-1 methylation changes in LN have the same pattern as that in LP. This epigenomic change may be due to the presence of occult metastatic tumor in LN cases.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3931-3936
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• 2013

Background: Iran is a country with very high incidences of stomach cancer, especially in Northern parts. Here we assessed prognostic value of serum screening biomarkers among people >50 years old for early detection of precancerous lesions in a hot spot for gastric carcinoma in Guilan Province, North Iran. Methods: A cross-sectional population-based survey was conducted on 1,390 residents of Lashtenasha city with the mean age (SD) of 61.8 (9.02) years old (50.8% females) to assess the association of gastrin and the pepsinogen (PG) I/II ratio with premalignant gastric lesions. Blood samples were taken for CBC, blood group, and serologic exams (PGI, PGII, and gastrin 17) from each subject. Expert gastroenterologists performed upper GI endoscopy and ROC curves were generated to determine appropriate cutoff points. Results: Mean values of PGI, PGII, PGI/PGII and gastrin were significantly different between patients with and without atrophy or metaplasia (P<0.05). To diagnose atrophy and intestinal metaplasia, a significantly higher AUC was observed for the PGI/PGII ratio (70 and 72%, respectively) compared to the PGI (56, 55%), PGII (63, 64%) and gastrin (59, 61%) (all p<0.001). Conclusions: Biomarker tests such as the PGI/II ratio can be used in the screening and diagnosis of subjects at high gastric cancer risk in our region.

• Analytical Science and Technology
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• v.25 no.6
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• pp.460-466
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• 2012

An environmentally friendly, so-called green, high performance liquid chromatography method was developed and validated for the determination of trans, trans-muconic acid (t,t-MA) in human urine as a biomarker of benzene exposure. After urinary t,t-MA was extracted and enriched using solid-phase extraction, a MF-Ph1 SG80 ($150mm{\times}2.0mm$ I.D., 5 ${\mu}m$) column with a mobile phase of 10 mM $KH_2PO_4$ containing 0.1% $H_3PO_4$ was used for isocratic separation of t,t-MA with UV detection at 259 nm. The calibration curve was constructed in the range of 0.1-5.0 mg/L with good linearity ($r^2$=0.9992). The intra-day and inter-day precision (as RSD) were 0.9-8.5% and 3.1-4.5%, respectively. The average recovery ranged from 97.5% to 101.7%. The green sample preparation and separation with no organic solvents were successfully achieved. The validated method would be suitable for the routine biological monitoring of benzene exposure in the occupational settings.

• Journal of Periodontal and Implant Science
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• v.46 no.5
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• pp.320-328
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• 2016

Purpose: Human saliva, as a vital part of the immune defense system, contains a number of distinct proteins and peptides. Recently human common salivary protein 1 (CSP1) has been identified as an abundant salivary protein and may play a role in promoting the binding of cariogenic bacteria to salivary pellicles. However, nothing else is known regarding the role of CSP1 in periodontology. The aim of this study was to quantify and compare CSP1 levels between healthy subjects and periodontal patients. Methods: This controlled clinical study was conducted in periodontally healthy individuals and patients with chronic periodontitis Chonbuk National University Hospital, with Institutional Review Board approval. Whole saliva samples were collected from 36 healthy subjects and 33 chronic periodontitis patients and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immune blotting were conducted to ensure that anti-CSP1 monoclonal antibody (mAb) binds to CSP1 in human saliva. A sandwich enzyme-linked immunosorbent assay (ELISA) system was house-fabricated using mAb-hCSP1#14 and mAb-hCSP1#4 as a capture and a detector mAb, respectively. The CSP1 concentrations in saliva from 36 healthy subjects and 33 periodontal patients were quantified using the CSP1 sandwich ELISA system, and the results were analyzed using the Student's t-test. Results: Immunoblot analysis using mAb-hCSP1 as a probe confirmed that CSP1 in human saliva existed as a single band with a molecular weight of approximately 27-kDa. The quantification of CSP1 concentrations by CSP1 ELISA showed that the median values (25th to 75th percentiles) of periodontal patients and healthy subjects were 9,474 ng/mL (range, 8,434.10,139 ng/mL) and 8,598 ng/mL (range, 7,421.9,877 ng/mL), respectively. The Student's t-test indicated the presence of a statistically significant difference between the 2 groups (P=0.024). Conclusions: The presence of a significant difference in CSP1 levels between healthy subjects and periodontal patients suggests that CSP1 may be a potential biomarker for the detection or screening of periodontitis patients.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.2301-2305
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• 2016

Background: The early detection of anthracycline- induced cardiotoxicity is very important since it might be useful in prevention of cardiac decompensation. This study was designed with the intent of assessing the usefulness of cardiac troponin T (cTnT) and NT- Pro BNP estimation in early prediction of anthracycline induced cardiotoxicity. Materials and Methods: In this prospective study histologically proven breast cancer patients who were scheduled to receive anthracycline containing combination chemotherapy as a part of multimodality treatment were enrolled. Baseline cardiac evaluation was performed by echocardiography (ECHO) and biomarkers like cardiac troponin T (cTnT) and N terminal- pro brain natriuretic peptide (NT- Pro BNP). All patients underwent cTnT and NT- Pro BNP estimation within 24 hours of each cycle of chemotherapy and were followed up after 6 months of initiation of chemotherapy. Any changes in follow up ECHO were compared to ECHO at baseline and cTnT and NT- Pro BNP levels after each cycle of anthracycline-based chemotherapy. Results: Initial data were obtained for 33 patients. Mean change in left ventricular diastolic diameter (LVDD) within 6 months was $0.154{\pm}0.433cms$ (p value=0.049). Seven out of 33 patients had an increase in biomarker cTnT levels (p value=0.5). A significant change in baseline and follow up LVDD was observed in patients with raised cTnT levels (p value=0.026) whereas no change was seen in ejection fraction (EF) and left atrial diameters (LAD) within 6 months of chemotherapy. NT- Pro BNP levels increased in significant number of patients (p value ${\leq}0.0001$) but no statistically significant change was observed in the ECHO parameters within 6 months. Conclusions: Functional monitoring is a poorly effective method in early estimation of anthracycline induced cardiac dysfunction. Estimation of biomarkers after chemotherapy may allow stratification of patients in various risk groups, thereby opening window for interventional strategies in order to prevent permanent damage to the myocardium.

• Journal of Veterinary Clinics
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• v.23 no.2
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• pp.144-148
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• 2006

Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis. Tartrate resistant acid phosphatase (TRAP) has been used for several years as a marker enzyme of bone-resorbing osteoclasts. This study investigated the activity of TRAP in media of apoptotic cell death-induced canine chondrocyte. We exposed canine chondrocyte to capsaicin and the results showed that capsaicin induced cell death in a dose dependent manner. And we measured TRAP activity in media of chondrocyte death induced by capsaicin treatment and the results capsaicin significantly increased the activity of TRAP in media for dose dependent. We also investigated whether the combination treatment with capsaicin and TRAIL enhance apoptotic cell death in canine chondrocyte. We exposed canine chondrocyte to capsaicin for 24 hrs at the indicated dose, and then treated with recombinant TRAIL protein for 24 hrs. TRAIL alone did not induce cell death after 24 hours, but the combined treatment of both induced more cell death compared with capsaicin alone in a dose dependent manner. Also, the combination treatment with capsaicin and TRAIL increased the activity of TRAP in culture media. These results suggest that TRAP can flow out into extracellular after chondrocyte damage, and TRAP may be a successful biomarker for detection of joint disease such as osteoarthritis.