Production of viral vectors using recombinase-mediated cassette exchange

DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector

Generalized Reciprocity Relations in Solar Cells with Voltage-Dependent Carrier Collection: Application to p-i-n Junction Devices

Two reciprocity theorems are important for fundamental understanding of the solar cell operation and applications to device evaluation: (1) the carrier-transport reciprocity connecting the dark-carrier injection with the short-circuit photocarrier collection and (2) the optoelectronic reciprocity connecting the electroluminescence with the photovoltaic quantum efficiency at short circuit. These theorems, however, fail in devices with thick depletion regions such as p-i-n junction solar cells. By properly linearizing the carrier-transport equation in such devices, we report that the dark-carrier injection is related to the photocarrier collection efficiency at the operating voltage, not at short circuit as suggested in the original theorem. This leads to the general form of the optoelectronic reciprocity relation connecting the electroluminescence with the voltage-dependent quantum efficiency, providing a correct interpretation of the optoelectronic properties of p-i-n junction devices. We also discuss the validity of the well-known relation between the open-circuit voltage and the external luminescence efficiency. The impact of illumination intensity and device parameters on the validity of the reciprocity theorems is quantitatively investigated

Potential Role of the Intratumoral Microbiota in Prognosis of Head and Neck Cancer

The tumor microbiome, a relatively new research field, affects tumor progression through several mechanisms. The Cancer Microbiome Atlas (TCMA) database was recently published. In the present study, we used TCMA and The Cancer Genome Atlas and examined microbiome profiling in head and neck squamous cell carcinoma (HNSCC), the role of the intratumoral microbiota in the prognosis of HNSCC patients, and differentially expressed genes in tumor cells in relation to specific bacterial infections. We investigated 18 microbes at the genus level that differed between solid normal tissue (n = 22) and primary tumors (n = 154). The tissue microbiome profiles of Actinomyces, Fusobacterium, and Rothia at the genus level differed between the solid normal tissue and primary tumors of HNSCC patients. When the prognosis of groups with rates over and under the median for each microbe at the genus level was examined, rates for Leptotrichia which were over the median correlated with significantly higher overall survival rates. We then extracted 35 differentially expressed genes between the over- and under-the-median-for-Leptotrichia groups based on the criteria of >1.5 fold and p < 0.05 in the Mann–Whitney U-test. A pathway analysis showed that these Leptotrichia-related genes were associated with the pathways of Alzheimer disease, neurodegeneration-multiple diseases, prion disease, MAPK signaling, and PI3K-Akt signaling, while protein–protein interaction analysis revealed that these genes formed a dense network. In conclusion, probiotics and specific antimicrobial therapy targeting Leptotrichia may have an impact on the prognosis of HNSCC.Hamada M., Inaba H., Nishiyama K., et al. Potential Role of the Intratumoral Microbiota in Prognosis of Head and Neck Cancer. International Journal of Molecular Sciences 24, 15456 (2023); https://doi.org/10.3390/ijms242015456

Comparing the Osteogenic Potential and Bone Regeneration Capacities of Dedifferentiated Fat Cells and Adipose-Derived Stem Cells In Vitro and In Vivo: Application of DFAT Cells Isolated by a Mesh Method

Background: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). Method: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. Results: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p Conclusion: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies

Depth profiles of radioactive cesium in soil using a scraper plate over a wide area surrounding the Fukushima Dai-ichi Nuclear Power Plant, Japan

AbstractDuring the Fukushima Dai-ichi Nuclear Power Plant (NPP) accident, radioactive cesium was released in the environment and deposited on the soils. Depth profiles of radioactive cesium in contaminated soils provide useful information not only for radiation protection and decontamination operations but also for geoscience and radioecology studies. Soil samples were collected using a scraper plate three times between December 2011 and December 2012 at 84 or 85 locations within a 100-km radius of the Fukushima Dai-ichi NPP. In most of the obtained radioactive cesium depth profiles, it was possible to fit the concentration to a function of mass depth as either an exponential or hyperbolic secant function. By using those functions, following three parameters were estimated: (i) relaxation mass depth β (g cm−2), (ii) effective relaxation mass depth βeff (g cm−2), which is defined for a hyperbolic secant function as the relaxation mass depth of an equivalent exponential function giving the same air kerma rate at 1 m above the ground as the inventory, and (iii) 1/10 depth L1/10 (cm), at which the soil contains 90% of the inventory. The average β value (wet weight) including ones by hyperbolic secant function in December 2012, was 1.29 times higher than that in December 2011. In fact, it was observed that depth profiles at some study sites deviated from the typical exponential distributions over time. These results indicate the gradual downward migration of radioactive cesium in the soils. The L1/10 values in December 2012 were summarized and presented on a map surrounding the Fukushima Dai-ichi NPP, and the average value of L1/10 was 3.01 cm (n = 82) at this time. It was found that radioactive cesium remained within 5 cm of the ground surface at most study sites (71 sites). The sech function can also be used to estimate the downward migration rate V (kg m−2 y−1). The V values in December 2012 (n = 25) were in good agreement with those found by a realistic approach using a diffusion and migration model. Almost all values ranged between 1.7 and 9.6 kg m−2 y−1 in this study