Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Physiological and Genomic Features of a Novel Sulfur-Oxidizing Gammaproteobacterium Belonging to a Previously Uncultivated Symbiotic Lineage Isolated from a Hydrothermal Vent

Strain Hiromi 1, a sulfur-oxidizing gammaproteobacterium was isolated from a hydrothermal vent chimney in the Okinawa Trough and represents a novel genus that may include a phylogenetic group found as endosymbionts of deep-sea gastropods. The SSU rRNA gene sequence similarity between strain Hiromi 1 and the gastropod endosymbionts was approximately 97%. The strain was shown to grow both chemolithoautotrophically and chemolithoheterotrophically with an energy metabolism of sulfur oxidation and O₂ or nitrate reduction. Under chemolithoheterotrophic growth conditions, the strain utilized organic acids and proteinaceous compounds as the carbon and/or nitrogen sources but not the energy source. Various sugars did not support growth as a sole carbon source. The observation of chemolithoheterotrophy in this strain is in line with metagenomic analyses of endosymbionts suggesting the occurrence of chemolithoheterotrophy in gammaproteobacterial symbionts. Chemolithoheterotrophy and the presence of homologous genes for virulence- and quorum sensing-related functions suggest that the sulfur-oxidizing chomolithotrophic microbes seek animal bodies and microbial biofilm formation to obtain supplemental organic carbons in hydrothermal ecosystems.     

Studies on Flavor Components of Foodstuffs

Distribution of tetramethylpyrazine (T.M.P.) in Japanese fermented foodstuffs was investigated by more accurate analytical method.

Namely, the method was successful when trapping T.M.P. with picric acid after flash evaporation of the foodstuffs, followed by analysis with gas chromatography.

T.M.P. was detected in many Japanese fermented foodstuffs, especially in Miso, Soy sause and Natto, which suggests that alkylpyrazines may play an important role as flavor of those foodstuffs.     

Movement Patterns and Residency of the Critically Endangered Horseshoe Crab Tachypleus tridentatus in a Semi-Enclosed Bay Determined Using Acoustic Telemetry

The horseshoe crab Tachypleus tridentatus is critically endangered in Japan due to rapidly decreasing numbers resulting from the loss of tidal flats and sandy beaches, and the deterioration of coastal environments. We monitored the year-round migratory patterns and residency of this species in a coastal embayment at Tsuyazaki, Japan, using acoustic telemetry. Total 20 adult crabs (15 males and 5 females) were tagged with ultrasonic transmitters and tracked during two periods (2006–2008; n = 10 and 2007–2009; n = 10). Adult crabs were more active during periods of higher water temperatures and their activity peaked in July, during the spawning period. Water temperature appeared to be one of the key factors influencing the movement patterns for the species. Moreover, the crabs tended to be more active at night than in the day. The nocturnal activity pattern was clearly evident before and during the reproductive period (May–August). Tracking data also showed that one pair-bond was maintained for a maximum of 17 days after the pair-bonded female had spawned. Overall, 11 males (73% of 15 individuals) remained in the bay area over winter, whereas three females (60% of 5 individuals) overwintered outside of the bay. Telemetry data showed that over 60% (13 of 20) of tagged crabs overwintered within the bay where there are sandy beaches, mudflats, and scattered seagrass beds. This year-round residence by adult T. tridentatus in the bay area identifies it as a critical habitat for the management of this species, regardless of life-stage. Not only is it a comprehensive management strategy that effectively reflects this species’ habitat use patterns but also its implementation, such as the establishment of a protected area, would contribute to its conservation.     

Affinity Improvement of a Cancer-Targeted Antibody through Alanine-Induced Adjustment of Antigen-Antibody Interface

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Antifibrotic effect of methylated quercetin derivatives on TGFβ-induced hepatic stellate cells

Quercetin (QCT) and isorhamnetin (ISO), natural flavonoids, were both shown to possess antifibrotic activity in in vivo and in vitro models of hepatic fibrosis. Although ISO is a direct metabolite of QCT differing by a methyl group, it has been reported to be absorbed more adequately and eliminated slower than QCT after oral administration. Our aim of the study was to investigate biological effect of mono-methylated QCT derivatives against fibrosis using rat hepatic stellate cells (HSC-T6). All test derivatives were synthesized from QCT. HSC-T6 cells were induced by TGFβ and treated with derivatives followed by cell proliferation assay, immunofluorescence staining of αSMA, and gene expression analysis of fibrosis markers. All compounds showed a dose- and time-dependent antiproliferation effect. ISO, 3-O-methylquercetin (3MQ), and rhamnetin (RHA) reduced αSMA mRNA; 3MQ prevented the augmentation of collagen I mRNA; and compounds, except azaleatin and 3MQ, reduced Timp1 mRNA expression in TGFβ-induced HSCs. In conclusion, each compound had singular effect against different features of fibrosis depending on the position of methyl group although the further mechanism of action of compounds during fibrosis development remains to be investigated. These findings suggest that antifibrotic effect of quercetin can be enhanced by adding methyl group on functionally important position.     

Canine Mammary Tumours Are Affected by Frequent Copy Number Aberrations,including Amplification of MYC and Loss of PTEN

BackgroundCopy number aberrations frequently occur during the development of many cancers. Such events affect dosage of involved genes and may cause further genomic instability and progression of cancer. In this survey, canine SNP microarrays were used to study 117 canine mammary tumours from 69 dogs.ResultsWe found a high occurrence of copy number aberrations in canine mammary tumours, losses being more frequent than gains. Increased frequency of aberrations and loss of heterozygosity were positively correlated with increased malignancy in terms of histopathological diagnosis. One of the most highly recurrently amplified regions harbored the MYC gene. PTEN was located to a frequently lost region and also homozygously deleted in five tumours. Thus, deregulation of these genes due to copy number aberrations appears to be an important event in canine mammary tumour development. Other potential contributors to canine mammary tumour pathogenesis are COL9A3, INPP5A, CYP2E1 and RB1. The present study also shows that a more detailed analysis of chromosomal aberrations associated with histopathological parameters may aid in identifying specific genes associated with canine mammary tumour progression.ConclusionsThe high frequency of copy number aberrations is a prominent feature of canine mammary tumours as seen in other canine and human cancers. Our findings share several features with corresponding studies in human breast tumours and strengthen the dog as a suitable model organism for this disease.