Biology subjects

Kurosawa, Y.

Publications and source records attributed to Kurosawa, Y..

3 recordsLinked to original sources

bioRxiv2024

PURIFICATION METHOD FOR ADENO-ASSOCIATED VIRAL VECTOR SEROTYPE 9 USING CERAMIC HYDROXYAPATITE CHROMATOGRAPHY AND ITS QUALIFICATION

Adeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors that comprise only genome-packaged full particles. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic protocols and optional methods for evaluating purified AAV vectors are also described.

molecular biology↗

bioRxiv2021

Prospective isolation of mouse and human hematopoietic stem cells using Plexin domain containing 2

Numerous strategies exist to isolate hematopoietic stem cells (HSCs) using complex combinations of markers and flow cytometry. However, robust identification of HSCs using imaging techniques is substantially more challenging which has prompted the recent development of HSC reporter mice. To date, none of the molecules used in these reporters have been useful for human HSC identification. Here we report that PLXDC2 is a useful marker for both mouse and human HSCs. Using a green fluorescent protein (GFP) knock-in at the Plxdc2 locus in mice (hereafter denoted as Plxdc2-GFP), we showed that Plxdc2-GFP is highly expressed in HSCs with 1 in 2.8 Plxdc2-GFP+CD150+ cells giving long-term multi-lineage reconstitution in transplantation. Moreover, we developed a novel human PLXDC2 antibody and showed that human PLXDC2+ HSCs have stronger long-term multilineage reconstitution ability compared with PLXDC2- HSCs in a xenograft model. Thus, our study identifies PLXDC2 as a highly relevant molecule in HSC identification, potentially allowing greater purity and live in vivo tracking of these cells. SummaryTo date, few molecules are available for isolation of HSCs across species. The present study shows that PLXDC2 is a highly useful molecule for isolation of HSCs, which works across mouse and human.

cell biology↗

bioRxiv2019

\"Low-cost\" initial burst of root development in whole Fagus crenata seedlings: The key to survival?

Terrestrial plants are rooted in one place, and therefore their metabolism must be flexible to adapt to continuously changing environments. This flexibility is probably influenced by the divergent metabolic traits of plant organs. However, direct measurements on organ-specific metabolic rates are particularly scarce and little is known about their roles in determining whole-individual meatabolism. To reveal this on seedlings of Fagus crenata, which is one of the most widespread dominant genus in temperate deciduous broad leaf forests in the circum-polar Northern Hemisphere, we measured respiration, fresh mass and surface area for total leaves, stems and roots of 55 individuals in two years from germination and analyzed their relationships with individual metabolism. Proportion of roots to whole plant in mass increased from approximately 17% to 74%, and that in surface area increased from about 11% to 82% in the two years. Nonetheless, the increment of the proportion of root respiration to whole-plant respiration was from 9.2% to only 40%, revealing that the increment in mass and surface area of roots was much larger than the increment in energetic cost. As a result, only the roots showed a substantial decline in both respiration/surface area and respiration/mass among the three organs; roots had about 90% decline in their respiration/surface area, and 84% decline in their respiration/mass, while those in leaves and stems were relatively constant. The low-cost and rapid root development is specific to the two years after germination and would be effective for avoiding water and nutrient deficit, and possibly helps seedling survival. This drastic shift in structure and function with efficient energy use in developmental change from seeds to seedlings may underpin the establishment of F. crenata forests. We discuss significance of lowering energetic cost for various individual organisms to effectively acquire resources from a wide perspective of view.

plant biology↗