ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis

Mutations in mitochondrial DNA (mtDNA) occur at high frequency in human tumors, but whether these mutations alter tumor cell behavior has been unclear. We used cytoplasmic hybrid (cybrid) technology to replace the endogenous mtDNA in a mouse tumor cell line that was poorly metastatic with mtDNA from a cell line that was highly metastatic, and vice versa. Using assays of metastasis in mice, we found that the recipient tumor cells acquired the metastatic potential of the transferred mtDNA. The mtDNA conferring high metastatic potential contained G13997A and 13885insC mutations in the gene encoding NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase subunit 6 (ND6). These mutations produced a deficiency in respiratory complex I activity and were associated with overproduction of reactive oxygen species (ROS). Pretreatment of the highly metastatic tumor cells with ROS scavengers suppressed their metastatic potential in mice. These results indicate that mtDNA mutations can contribute to tumor progression by enhancing the metastatic potential of tumor cells.     

Low-temperature preservation (at 4 °C) of marine rotifer Brachionus

Mass production of rotifers is essential as live food during the larval rearing season, but a major problem of rotifer culture is unpredictable culture collapse. If mass‐produced rotifers could be kept alive at low temperature for an extended period of time, they could be supplied as live food to cultured marine fish larvae without interruption. Four experiments were performed to test this possibility in six strains of Brachionus plicatilis O. F. Müller and eight strains of Brachionus rotundiformis Tschugunoff. The results showed that: (1) B. rotundiformis strains were less tolerant to 4 °C than B. plicatilis strains. Among the B. rotundiformis strains, the strains known as SS type were the most susceptible and showed the lowest survival. (2) Exchange of culture media during the incubation at 4 °C in B. plicatilis and B. rotundiformis resulted in higher survival than not changing the culture media, but there were no differences in the regression slope with or without changing the culture media. (3) Acclimation at 15 °C for 96 h for B. plicatilis and B. rotundiformis before transfer to 4 °C resulted in higher survival rates than acclimation at 10 °C. (4) The combination of frequent exchange of culture media and acclimation significantly improved the survival of B. plicatilis and B. rotundiformis compared with controls that were maintained at 4 °C without exchange of the culture media. Large‐scale trials using B. plicatilis (Kamiura strain) cultured in 30‐L tanks were conducted in a hatchery at a density of 2000–20 000 individuals mL^?1. Rotifers were transferred directly from 25 °C to 4 °C. About 50% of the rotifers at 20 000 individuals mL^?1 survived after 14 days at 4 °C. These preserved rotifers could be cultured at 20 °C, recovering within 4 days.     

Epidermal structure created by canine hair follicle keratinocytes enriched with bulge cells in a three‐dimensional skin equivalent model in vitro: implications for regenerative therapy of canine epidermis

Background – Keratinocytes in the hair follicle bulge region have a high proliferative capacity, with characteristics of epithelial stem cells. This cell population might thus be an ideal source for generating the interfollicular epidermis in a canine skin equivalent. Hypothesis/Objectives – This study was designed to determine the ability of canine hair follicle bulge cell‐enriched keratinocytes to construct canine living skin equivalents with interfollicular epidermis in vitro. Animals – Four healthy beagle dogs from a research colony. Methods – Bulge cell‐enriched keratinocytes showing keratin 15 immunoreactivity were isolated from canine hair follicles and cultured on dermal equivalent containing canine fibroblasts. Skin equivalents were subjected to histological, immunohistochemical, western blot and RT‐PCR analyses after 10–14 days of culture at the air–liquid interface. Results – The keratinocyte sheets showed an interfollicular epidermal structure comprising four to five living cell layers covered with a horny layer. Immunoreactivities for keratin 14 and desmoglein 3 were detected in the basal and immediate suprabasilar layers of the epidermis, while keratin 10 and desmoglein 1 occurred in more superficial layers. Claudin 1 immunoreactivity was seen in the suprabasalar layer of the constructed epidermis, and filaggrin monomers and loricrin were detected in the uppermost layer. Basal keratinocytes in the skin equivalent demonstrated immunoreactivity to antibodies against basement membrane zone molecules. Conclusions and clinical importance – A bulge stem cell‐enriched population from canine hair follicles formed interfollicular epidermis within 2 weeks in vitro, and thus represents a promising model for regenerative therapy of canine skin.     

Usefulness of cefovecin disk‐diffusion test for predicting mecA gene‐containing strains of Staphylococcus pseudintermedius and clinical efficacy of cefovecin in dogs with superficial pyoderma

Background –  Cefovecin has been widely used to treat skin infections in dogs. The relationship of the cefovecin disk‐diffusion test results to the presence of the mecA gene and the clinical efficacy of cefovecin have not been fully evaluated. Hypothesis/Objectives –  To determine the usefulness of an in vitro cefovecin disk‐diffusion test in predicting the presence of the mecA gene in Staphylococcus pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma. Methods –  Twenty‐six S. pseudintermedius strains isolated from 22 dogs with pyoderma were used. In vitro disk‐diffusion test results of cefovecin were compared with agar‐dilution test results, the presence of the mecA gene, and the improvement in clinical scores of dogs with superficial pyoderma at 14 days post treatment. Results –  There was a significant linear correlation (r = ?0.83) between the diameter of the obvious zone of inhibition by disk diffusion and the minimal inhibitory concentration for cefovecin (P < 0.0001). Receiver operating characteristic analysis revealed that zone diameters between 25 and 27 mm exhibited better sensitivity (92.9%) and specificity (100.0%) for detection of strains carrying the mecA gene. The mean improvement in clinical scores in dogs carrying cefovecin‐resistant strains was significantly lower than in dogs carrying cefovecin‐susceptible strains (P < 0.01). Conclusions and clinical importance –  The cefovecin disk‐diffusion test with a cut‐off value estimated in this study was valuable for predicting mecA gene carriage in S. pseudintermedius, as well as the in vivo efficacy of cefovecin therapy in dogs with superficial pyoderma caused by S. pseudintermedius.     

Dataset of CarboEastAsia and uncertainties in the CO2 budget evaluation caused by different data processing

The datasets of net ecosystem CO₂ exchange (NEE) were acquired from 21 forests, 3 grasslands, and 3 croplands in the eastern part of Asia based on the eddy covariance measurements of the international joint program, CarboEastAsia. The program was conducted by three networks in Asia, ChinaFLUX, JapanFlux, and KoFlux, to quantify, synthesize, and understand the carbon budget of the eastern part of Asia. An intercomparison was conducted for NEE estimated by three gap-filling procedures adopted by ChinaFLUX, JapanFlux, and KoFlux to test the range of uncertainty in the estimation of NEE. The overall comparison indicated good agreement among the procedures in the seasonal patterns of NEE, although a bias was observed in dormant seasons depending on the different criteria of data screening. Based on the gap-filled datasets, the magnitude and seasonality of the carbon budget were compared among various biome types, phenology, and stress conditions throughout Asia. The annual values of gross primary production and ecosystem respiration were almost proportional to the annual air temperature. Forest management, including clear-cutting, plantation, and artificial drainage, was significant and obviously affected the annual carbon uptake within the forests. Agricultural management resulted in notable seasonal patterns in the crop sites. The dataset obtained from a variety of biome types would be an essential source of knowledge for ecosystem science as well as a valuable validation dataset for modeling and remote sensing to upscale the carbon budget estimations in Asia.     

Low-cost photochemical reflectance index measurements of micropropagated plantlets using image analysis

A system for imaging the photochemical reflectance index (PRI) of micropropagated plantlet leaves was developed using a low-cost charge-coupled device (CCD) camera. The reflected light intensities of leaves at 530 and 570 nm were imaged using a monochrome CCD camera with respective band-pass filters. The reflection images were used to estimate the PRI values of the leaves. The relationships between the PRI estimated from images and a chlorophyll fluorescence parameter ΔF/Fm′, determined by pulse-amplitude modulation (PAM) chlorophyll fluorometer and representing PSII quantum yield, were investigated for several plants under various conditions to test the performance of the system. The PRI estimated from images decreased with a decline in the ΔF/Fm′ for strawberry leaves treated with high intensity light under artificial light condition and lettuce leaves treated with abscisic acid. These results suggest that the system can be used for non-destructive estimation of the PRI of leaves. The system was used to estimate the PRI of micropropagated potato leaves from outside the culture vessels. The time course of PSII quantum yield for the individual leaf could be demonstrated by the PRI estimated from images of potato plantlet leaves treated with high light. The findings suggest that the system has the potential for inexpensive, simple and efficient estimation of PRI for micropropagated plantlets.     

Method for efficiently obtaining fertilized eggs from the black scraper Thamnaconus modestus by natural spawning in captivity

To develop an efficient and stable method for enhancing spawning of black scrapers in captivity, we examined the following three factors: (1) different kinds of spawning substrate, (2) the area of the spawning substrate, (3) the grain size of the spawning substrate. The results demonstrated that sand is the optimum spawning substrate when compared to a hard polyvinyl chloride sheet, a piece of shading net, or artificial spawning grass. No difference was observed in the spawning frequency between a small sand bed of area 400?cm² and sand spread over the bottom of the tank to an area of 7085?cm², since the egg masses were spawned on the sand in a small area approximately 15?cm in diameter. In addition to the spawned eggs, the small movable sand bed is useful for subsequent harvest. No differences were observed in either the spawning frequency or the hatching rate when using sand beds with different grain sizes. In large-scale egg collection trials, an average of 33 egg masses from which an average of 403,000 larval fish were obtained. We can therefore conclude that the procedure developed in the present study is applicable to the commercial-scale seed production of black scraper.     

Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay

The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC₅₀ values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.     

Atmospheric moisture influences on conidia development in Podosphaera xanthii through host-plant morphological responses

This study investigated the effects of atmospheric moisture on conidia development in cucurbit powdery mildew fungus (Podosphaera xanthii) through host-plant responses. Cucumber (Cucumis sativus L.) seedlings were grown under a high vapour pressure deficit (VPD) (3.8 kPa) or a low VPD (0.4 kPa). When the cotyledons had expanded, spores of P. xanthii were inoculated onto the adaxial surface. Inoculated seedlings for both treatments were then placed in a growth chamber maintained at a VPD of 2.1 kPa. The density of visible P. xanthii colonies on the high-VPD-acclimated cotyledons was 0.46 to 0.85 times that of the low-VPD-acclimated cotyledons 7 days after inoculation. It is likely the post-germination behaviour of P. xanthii such as the infection and consequent hyphal development was affected because spore germination did not differ between the treatments. The percentage of adaxial epidermal leaf cells with haustoria was also lower in the seedlings which had acclimated to a higher VPD. The high-VPD-acclimated cotyledons were thicker and had greater dry mass per area. The water potential of cotyledons did not differ between the treatments, although the stomatal conductance of high-VPD-acclimated cotyledons was lower than that of low-VPD-acclimated cotyledons. From these results, we conclude that the inhibition of P. xanthii conidia development on high-VPD-acclimated cotyledons was mainly caused by changes in leaf morphological properties.