Differentiation of Scomber japonicus from Scomber scombrus by using a single locked nucleic acid probe.

Mackerel is marketed at prices according to the species type, Scomber japonicus and Scomber scombrus. Distinguishing these two species with the naked eye is difficult, and their differentiation becomes more difficult after they are processed by cooking, thereby leading to counterfeiting issues. Thus, in this study, we developed a method to differentiate S. japonicus from S. scombrus by detecting polymorphisms in mitochondrial 16 s rRNA gene by using fluorescence melting curve analysis and locked nucleic acid probes. Our method could distinguish S. japonicus from S. scombrus in a single experiment by using a single probe. The probes developed matched exactly with S. japonicus and had a melting temperature of 64 °C. However, the probes were mismatched with S. scombrus, resulting in a lower melting temperature of 46 °C. The high specificity of the locked nucleic acid probes resulted in this large difference in the melting temperatures.

High specific genotyping method using short target probe and helper probe.

Differentiating 1-bp differences using real-time PCR often leads to false-positive results. Therefore, we developed a fluorescence melting curve analysis (FMCA) method with a short target probe and helper probe labeled with a fluorophore and quencher, respectively. This fluorophore and quencher were designed to be near each other when the probes were hybridized to template DNA. The target probe was designed with a shorter length to facilitate a dramatic shift in melting temperature (Tm) upon encountering mismatched hybridization. In FMCA, when the temperature approached the target probe Tm, the target probe would begin to denature from the template DNA, and at the target probe Tm, the fluorescence signal increased markedly. Here, we examined 1-bp differences using the developed method with mitochondrial DNA from Larimichthys polyactis and Larimichthys crocea. Application of this method permitted specific genotype identification for all cases with no cross-reactivity, even when both templates were added to the same tube.

Genotyping of velvet antlers for identification of country of origin using mitochondrial DNA and fluorescence melting curve analysis with locked nucleic acid probes.

Velvet antlers are used medicinally in Asia and possess various therapeutic effects. Prices are set according to the country of origin, which is unidentifiable to the naked eye, and therefore counterfeiting is prevalent. Additionally, antlers of the Canadian elk, which can generate chronic wasting disease, are prevalently smuggled and distributed in the market. Thus, a method for identifying the country of origin of velvet antlers was developed, using polymorphisms in mitochondrial DNA, fluorescence melting curve analysis and analysis of locked nucleic acids (LNA). This combined method is capable of identifying five genotypes of velvet antlers in a single experiment using two probes. It also has advantages in multiplexing, simplicity and efficiency in genotyping, when compared to real-time PCR or microarrays. The developed method can be used to improve identification rates in the velvet antler market and, by extension, research based on polymorphisms in DNA sequences.

Identification of time-dependent biomarkers and effects of exposure to volatile organic compounds using high-throughput analysis.

Volatile organic compounds (VOCs) can be easily taken up by humans, leading to various diseases, such as respiratory system and central nervous system disorders. Environmental risk assessment is generally conducted using traditional tests, which may be time-consuming and technically challenging. Therefore, analysis of the effects of VOCs, such as toluene, ethylbenzene, and xylene, may be improved by use of novel, high-throughput methods, such as microarray analysis. In this study, we examined the effects of VOCs exposure in humans on gene expression and methylation using microarray analysis. We recruited participants who had short-term exposure, long-term exposure, or no exposure. We then analyzed changes in gene expression in blood samples from these participants. A total of 866 genes were upregulated, while 366 genes were downregulated in the short-term exposure group. Similarly, in the long-term exposure group, a total of 852 and 480 genes were up- or downregulated, respectively. Hierarchical clustering analysis was used to divide the clustered genes into nine clusters to investigate the expression of variations in accordance with the exposure period. And the methylation microarray was performed at the same time to see whether this expression variation is related to the epigenetic study. Finally, we have 5 genes that were upregulated and 12 genes that were downregulated, gradually and respectively, so these genes are expected to function as biomarkers of the duration of exposure to VOCs. Further research is required to determine the time-dependent effects of VOCs on epigenetic regulation of gene expression. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1563-1570, 2016.

Association analysis of toluene exposure time with high-throughput mRNA expressions and methylation patterns using in vivo samples.

The emission of volatile organic compounds (VOCs) resulting from outdoor air pollution can contribute to major public health problems. However, there has been limited research on the health effects in humans from the inhalation of VOCs. Therefore, this study conducted an in vivo analysis of the effects of toluene, one of the most commonly used chemicals in many industries, on gene expression and methylation over time using the high-throughput technique of microarray analysis. We separated participants into three groups (control, short-term exposure, and long-term exposure) to investigate the influence of toluene exposure time on gene expression. We then comprehensively analyzed and investigated the correlation between variations in gene expression and the occurrence of methylation. Twenty-six genes were upregulated and hypomethylated, while 32 genes were downregulated and hypermethylated. The pathways of these genes were confirmed to be associated with cell survival and the immune system. Based on our findings, these genes can help predict the effects of time-dependent exposure to toluene on human health. Thus, observations from our data may have implications for the identification of biomarkers of toluene exposure.

Multiplex genotyping based on the melting temperature of a single locked nucleic acid probe.

Locked nucleic acid (LNA) is a modified RNA nucleotide that can be incorporated at specific positions to generate probes with the desired length, melting temperature (TM), and specificity. Here, we describe a method of multiplex genotyping based on dramatic shifts in the TM of a single dual-labeled LNA probe. Using this method, two varieties of the hairtail fish Trichiurus lepturus can be distinguished from each other, as well as from Trichiurus japonicus, based on a 1- to 2-bp difference in a fragment of mitochondrial cytochrome oxidase subunit 1. The shift in TM was 15 °C for a 1-bp mismatch and 27 °C for a 2-bp mismatch, indicating remarkable specificity. We anticipate that the method will be widely useful in applications such as species identification that require accurate, multiplex, and efficient detection of DNA polymorphisms.

Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences.

Previous fluorescence melting curve analysis (FMCA) used intercalating dyes, and this method has restricted application. Therefore, FMCA methods such as probe-based FMCA and molecular beacons were studied. However, the usual dual-labeled probes do not possess adequate fluorescence quenching ability and sufficient specificity, and molecular beacons with the necessary stem structures are hard to design. Therefore, we have developed a peptide nucleic acid (PNA)-based FMCA method. PNA oligonucleotide can have a much higher melting temperature (Tm) value than DNA. Therefore, short PNA probes can have adequate Tm values for FMCA, and short probes can have higher specificity and accuracy in FMCA. Moreover, dual-labeled PNA probes have self-quenching ability via single-strand base stacking, which makes PNA more favorable. In addition, this method can facilitate simultaneous identification of multiple DNA templates. In conventional real-time polymerase chain reaction (PCR), one fluorescence channel can identify only one DNA template. However, this method uses two fluorescence channels to detect three types of DNA. Experiments were performed with one to three different DNA sequences mixed in a single tube. This method can be used to identify multiple DNA sequences in a single tube with high specificity and high clarity.

Application of fluorescence melting curve analysis for dual DNA detection using single peptide nucleic acid probe.

Peptide nucleic acid (PNA) is an artificially synthesized polymer. PNA oligomers show greater specificity in binding to complementary DNAs. Using this PNA, fluorescence melting curve analysis (FMCA) for dual detection was established. Genomic DNA of Mycoplasma fermentans and Mycoplasma hyorhinis was used as a template DNA model. By using one PNA probe, M. fermentans and M. hyorhinis could be detected and distinguished simultaneously in a single tube. The developed PNA probe is a dual-labeled probe with fluorescence and quencher dye. The PNA probe perfectly matches the M. fermentans 16s rRNA gene, with a melting temperature of 72°C. On the other hand, the developed PNA probe resulted in a mismatch with the 16s rRNA gene of M. hyorhinis, with a melting temperature of 44-45°C. The melting temperature of M. hyorhinis was 27-28°C lower than that of M. fermentans. Due to PNA's high specificity, this larger melting temperature gap is easy to create. FMCA using PNA offers an alternative method for specific DNA detection.

Prognostic Value of Metabolic Tumor Volume Measured by (18)F-FDG PET/CT in Locally Advanced Head and Neck Squamous Cell Carcinomas Treated by Surgery.

PURPOSE: We assessed the prognostic value of metabolic tumor volume (MTV) measured using(18)F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) inpatients with locally advanced head and neck squamous cell carcinoma (HNSCC). METHODS: We retrospectively reviewed 56 patients (51 men, five women; mean age 56.0 ± 8.8years) who had locally advanced HNSCC and underwent FDG PET/CT for initial evaluation. All patients had surgical resection and radiotherapy with or without concurrent chemotherapy. The peak standardized uptake value (SUVpeak) and MTV of the target lesion, including primary HNSCC andmetastatic cervical lymph nodes, were measured from FDG PET/CT images. We compared SUVpeak, MTV, and clinicopathologic variables such as age, Eastern Cooperative Oncology Group (ECOG) performance status, pN stage, pT stage, TNM stage, histologic grade and treatment modality to disease-free survival (DFS) and overall survival (OS). RESULTS: On the initial FDG PET/CT scans, the median SUVpeak was 7.8 (range, 1.8-19.0) and MTV was17.0 cm(3) (range, 0.1-131.0 cm(3)). The estimated 2-year DFS and OS rates were 67.2% and 81.8%. The cutoff points of SUVpeak 6.2 and MTV 20.7 cm(3) were the best discriminative values for predicting clinical outcome. MTV and ECOG performance status were significantly related to DFS and OS on univariate and multivariate analyses (p < 0.05). CONCLUSION: The MTV obtained from initial FDG PET/CT scan is a significant prognostic factor for disease recurrence and mortality in locally advanced HNSCC treated with surgery and radiotherapy with or without chemotherapy.

Primary radiation therapy in patients with localized orbital marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT Lymphoma).

PURPOSE: To evaluate the outcomes of patients with localized orbital marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT lymphoma) who were treated with radiotherapy (RT). METHODS AND MATERIALS: We retrospectively reviewed the records of 46 patients who were treated with RT for pathologically confirmed localized stage IE marginal zone B-cell lymphoma of MALT. The radiation dose ranged from 21.6 to 45 Gy (median, 30.6 Gy) at 1.8-2.0 Gy per fraction. Median follow-up duration was 32.3 months (range, 3.1-113.6 months). RESULTS: Forty-three patients (93%) achieved complete remission (CR), and three patients (7%) achieved partial remission (PR). Five-year relapse-free survival, cause-specific survival, and overall survival were 93%, 100%, and 100%, respectively. Among the patients with CR, two had recurrence at three sites. One patient relapsed locally and was successfully salvaged with reirradiation. The other patient relapsed in a distant site and was successfully treated with six cycles of CHOP chemotherapy. Late complications were noted in four patients. Two patients developed cataracts at 26 and 37 months after completion of RT. The other two patients developed nasolacrimal duct obstructions at 4 and 11 months after completion of RT. CONCLUSION: Our study showed that a modest dose of RT is an excellent treatment modality with low complication and recurrence rates. We suggest that a dose of 30.6 Gy is tolerable and sufficient for treating orbital MALT lymphoma. Even following recurrence, successful salvage is possible with RT or chemotherapy.