Distribution of LGR5 + Cells and Associated Implications during the Early Stage of Gastric Tumorigenesis

Lgr5 was identified as a promising gastrointestinal tract stem cell marker in mice. Lineage tracing indicates that Lgr5 ⁺ cells may not only be the cells responsible for the origin of tumors; they may also be the so-called cancer stem cells. In the present study, we investigated the presence of Lgr5 ⁺ cells and their biological significance in normal human gastric mucosa and gastric tumors. RNAscope, a newly developed RNA in situ hybridization technique, specifically labeled Lgr5 ⁺ cells at the basal glands of the gastric antrum. Notably, the number of Lgr5 ⁺ cells was remarkably increased in intestinal metaplasia. In total, 76% of gastric adenomas and 43% of early gastric carcinomas were positive for LGR5. Lgr5 ⁺ cells were found more frequently in low-grade tumors with active Wnt signaling and an intestinal gland type, suggesting that LGR5 is likely involved in the very early stages of Wnt-driven tumorigenesis in the stomach. Interestingly, similar to stem cells in normal tissues, Lgr5 ⁺ cells were often restricted to the base of the tumor glands, and such Lgr5 ⁺ restriction was associated with high levels of intestinal stem cell markers such as EPHB2, OLFM4, and ASCL2. Thus, our findings show that Lgr5 ⁺ cells are present at the base of the antral glands in the human stomach and that this cell population significantly expands in intestinal metaplasias. Furthermore, Lgr5 ⁺ cells are seen in a large number of gastric tumors ; their frequent basal arrangements and coexpression of ISC markers support the idea that Lgr5 ⁺ cells act as stem cells during the early stage of intestinal-type gastric tumorigenesis.     

Cultivation of a hybrid of free-living gametophytes between Undariopsis peterseniana and Undaria pinnatifida: morphological aspects and cultivation period

The kelp Undariopsis peterseniana is warm-water-tolerant, and consequently, there is currently considerable interest in developing commercial cultivation techniques for this species in Korea. U. peterseniana plants have been successfully transferred to the northern coast of Korea beyond their original habitat in Jeju Island (33°30′08.65″N, 126°55′39.02″E). In this study, we cultured a hybrid kelp consisting of a cross between free-living gametophytes of U. peterseniana and U. pinnatifida in an attempt to extend the culture period of Undaria which is an important species for both the abalone industry and for commercial seaweed mariculture for human food applications. Morphological characters and cultivation period were compared between the parent thalli and the hybrid. The cultivation experiment was conducted in Wando, on the southern coast of Korea (34°26′18.68″N, 127°05′43.88″E). The morphological characteristics of the hybrid thalli were intermediate between the two species having shallow pinnated blades and a reduced reproductive organ. Hybrid thalli showed faster growth rates, 1.5 times greater biomass, and a longer cultivation period than the parent thalli. The hybrid strain possessed characteristics that indicate it could be used as an alternative kelp source to supply the abalone feed industry.     

Detection of uncharged or feebly charged small molecules by field-effect transistor biosensors

This paper describes a new technique for the detection of uncharged or feebly charged small molecules (<400Da) using Si field-effect transistor (FET) biosensors that are signal-enhanced by gold nanoparticle (NP) charges under dry measurement conditions. NP charges are quickly induced by a chemical deposition (that is, Au deposition) and the indirect competitive immunogold assay, and strongly enhance the electrical signals of the FET biosensors. For the validation of signal enhancement of FET biosensors based on NP charges and detection of uncharged or feebly charged small molecules, mycotoxins (MTXs) of aflatoxin-B1 (AFB1), zearalenone (ZEN), and ochratoxin-A (OTA) were used as target molecules. According to our experimental results, the signal is 100 times more enhanced than the use of the existing solution FET biosensing techniques. Furthermore, this method enables the FET biosensor to quantitatively detect target molecules, regardless of the ionic strengths, isoelectric points (pI), or pHs of the measured sample solutions.     

Antifungal activity and mode of action of silver nano-particles on Candida albicans

In this study, the antifungal effects of silver nano-particles (nano-Ag) and their mode of action were investigated. Nano-Ag showed antifungal effects on fungi tested with low hemolytic effects against human erythrocytes. To elucidate the antifungal mode of action of nano-Ag, flow cytometry analysis, a glucose-release test, transmission electron microscopy (TEM) and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH), as a plasma membrane probe, were performed with Candida albicans. The results suggest nano-Ag may exert an antifungal activity by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity. The present study indicates nano-Ag has considerable antifungal activity, deserving further investigation for clinical applications. K.-J. Kim and W. S. Sung contributed equally to this work and should be considered co-first authors.     

Malachite green induces cardiovascular defects in developing zebrafish (Danio rerio) embryos by blocking VEGFR-2 signaling

Malachite green (MG) is a triphenyl methane dye used in various fields that demonstrates high toxicity to bacteria and mammalian cells. When bud stage zebrafish embryos were treated with MG at 125, 150, and 175 ppb for 14 h, the development of trunk including intersomitic vessels was inhibited in MG-treated flk-1-GFP transgenic embyos. MG clearly induced whole growth retardation. MG induced severe cell death in trunk intersomite region of zebrafish embryos and in human vascular endothelial cells in a dose-dependent manner. MG inhibited heart rates and cardiac looping. MG attenuated whole blood formation and inhibited vascular endothelial growth factor (VEGF)-induced receptor (R)-2 phosphorylation in vascular endothelial cells. In conclusion, MG significantly alters the cardiovascular development causing growth retardation in zebrafish through the blocking VEGFR-2 activation in early cardiovascular development. It suggests that MG may be an environmental toxic agent with the potential to induce embryonic cardiovascular defects in vertebrates.     

Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18^®:DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.     

Endoplasmic reticulum stress response is involved in Mycobacterium tuberculosis protein ESAT-6-mediated apoptosis

Mycobacterium tuberculosis (Mtb) infection leads to the induction of the apoptotic response, which is associated with bacilli killing. The early secreted mycobacterial antigen ESAT-6 of Mtb has been shown to induce apoptosis in human macrophages and epithelial cells. In the present study, we demonstrate that the stimulation of human epithelial A549 cells by ESAT-6 induces the endoplasmic reticulum (ER) stress response. We observed that ESAT-6 treatment increases intracellular Ca²⁺ concentration, which results in ROS accumulation, and therefore induces the onset of ER stress-induced apoptosis. Our results uncover a novel apoptotic mechanism of ESAT-6 through ER stress responses in pathologic conditions such as tuberculosis.     

Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains

An Agrobacterium vitis-specific DNA fragment (pAVS3) was generated from PCR polymorphic bands amplified by primer URP 2R. A. vitis specificity of this fragment was confirmed by Southern hybridization with genomic DNA from different Agrobacterium species. Sequence-characterized amplified region (SCAR) markers were developed for A. vitis specific detection, using 24-mer oligonucleotide primers designed from the flanking ends of the 670 bp insert in pAVS3. The SCAR primers amplified target sequences only from A. vitis strains and not from other Agrobacterium species or other bacterial genera. First round PCR detected bacterial cells between 5×10² and 1×10³ cfu/ml and the detection sensitivity was increased to as few as 2 cfu/ml by nested PCR. This PCR protocol can be used to confirm the potential presence of infectious A. vitis strains in soil and furthermore, can identify A. vitis strains from naturally infected crown galls.