Masaru Nikaido

In Vivo and In Vitro Studies Suggest a Possible Involvement of HPV Infection in the Early Stage of Breast Carcinogenesis via APOBEC3B Induction

High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction.

Development of multisample detection system using a tag insertion primer and an electrochemical DNA chip.

Abstract We have developed a novel multisample detection system by employing a technology combining a tag insertion primer and an electrochemical DNA chip. In the first application, Helicobacter species-infected mouse samples were detected. The primers that insert a different tag sequence in each sample were prepared, and loop-mediated isothermal amplification (LAMP) reaction was carried out. Then amplification products in which a part of the sequence was different in each sample could be obtained. The target sample in which these amplification products were mixed was injected into a cassette that included the DNA chip with immobilized probes. After the cassette was set in the DNA detection system, Genelyzer, the processes of hybridization, washing, and detection were performed by the system automatically. The positive and negative concordance rates of the existing nested polymerase chain reaction (PCR) method and this method were 100% (40/40 samples) and 97.3% (117/120 samples), respectively. This is a simple high-throughput method. Moreover, the cost per sample can be drastically lowered. Therefore, it is expected to contribute to the diagnosis of infectious agents in humans and animals.

High-risk human papillomavirus genotype detection by electrochemical DNA chip method.

High-risk human papillomavirus (HPV) genotypes are the major cause of cervical cancer. Hence, HPV genotype detection is a helpful preventive measure to combat cervical cancer. Recently, several HPV detection methods have been developed, each with different sensitivities and specificities. The objective of this study was to compare HPV high risk genotype detection by an electrochemical DNA chip system, a line probe assay (INNO- LiPA) and sequencing of the L1, E1 regions. A total of 361 cervical smears with different cytological findings were subjected to polymerase chain reaction-sequencing and electrochemical DNA chip assessment. Multiple infections were found in 21.9% (79/361) of the specimens, most prevalently in 20-29-year olds while the highest prevalence of HPV infection was found in the 30-39-year age group. The most prevalent genotype was HPV 16 at 28.2% (138/489) followed by HPV 52 at 9.6% (47/489), with the other types occurring at less than 9.0%. The electrochemical DNA chip results were compared with INNO-LiPA and sequencing (E1 and L1 regions) based on random selection of 273 specimens. The results obtained by the three methods were in agreement except for three cases. Direct sequencing detected only one predominant genotype including low risk HPV genotypes. INNO-LiPA identified multiple infections with various specific genotypes including some unclassified-risk genotypes. The electrochemical DNA chip was highly accurate, suitable for detection of single and multiple infections, allowed rapid detection, was less time-consuming and was easier to perform when compared with the other methods. It is concluded that for clinical and epidemiological studies, all genotyping methods are perfectly suitable and provide comparable results.

Long-life high-reliability Ir-coated dispenser cathode

A long-life high-reliability Ir-coated dispenser cathode has been developed. To study the life characteristics of Ir-coated dispenser cathode, which is provided with a surface alloy layer of εII, extended life tests were conducted over 10,000h. From this life data, this cathode was estimated to have a 200,000h life at 920°Cb with 0.8A/cm2, and to have a 100,000h life at 980°Cb with 2.5A/cm2before emission decreases by 4%. Further, the experiment equation for surface Ir concentration of the cathodes was deduced from the results of surface analysis. Using this equation, the surface alloy layer was evaluated as extremely stable without substantial degradation during cathode life. Thus, the Ir-coated dispenser cathode has been proved to be exceedingly durable and highly reliable.

Prevalence of Helicobacter in Laboratory Micein Thailand

Prevalence of Helicobacter is mostly unknown in laboratory animals in Thailand. The 221 mice feces/cecum from 8 universities, 2 pharmaceutical companies and 3 research institutions in Thailand were surveyed for the prevalence and distribution of Helicobacter species by using the Electrochemical DNA chip. Helicobacter were detected 23/46 samples in Specific Pathogen Free (SPF) and 168/175 in conventional condition. Prevalence of Helicobacter were 98%, 96%, 92% and 78% in South (n=40), Northeast (n=40), North (n=25) and Central area (n=116), respectively. Only Central area holds SPF facility resulting in Helicobacter prevalence that seems to be lower than other areas. Three species of Helicobacter were detected in feces/cecum samples by sequence analysis: H. rodentium (67.0%, 148 samples), Helicobacter sp. MIT 01-6451 (15.4%, 34 samples), and unidentified Helicobacter species (14.1%, 9 samples). The results suggested that H. rodentium is the most common species of Helicobacter in laboratory mice in Thailand.

Mobile Automatic Detection System for Bacillus anthracis using Electrochemical DNA Chip

A novel detection system for biological agents using Loop-Mediated Isothermal Amplification (LAMP) and an electrochemical DNA chip, Bio BulwarkTM, has been developed. Genomic DNAs/RNAs roughly extracted from biological agents were amplified by the LAMP method using target-specific primers and strand-displace polymerase. The amplified DNAs were processed to hybridization with the electrochemical DNA chip. Anodic current derived from hoechst 33258, an electrochemically active intercalator, was relative to the amount of the target DNA hybridized with target-specific probes on the DNA chip. All reactions and measurements were conducted using disposable cassettes, which included the reagents for amplification and the DNA chip, and a mobile automatic detection system. The preliminary study showed that accurate detection of B. anthracis was carried out within 70 minutes. This system would prevent or minimize damage in the event of a criminal attack involving a biological threat.

Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.

Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip

Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.