Selective induction of targeted cell death and elimination by near-infrared femtosecond laser ablation

The techniques for inducing the death of specific cells in tissue has attracted attention as new methodologies for studying cell function and tissue regeneration. In this study, we show that a sequential process of targeted cell death and removal can be triggered by short-term exposure of near-infrared femtosecond laser pulses. Kinetic analysis of the intracellular accumulation of trypan blue and the assay of caspase activity revealed that femtosecond laser pulses induced immediate disturbance of plasma membrane integrity followed by apoptosis-like cell death. Yet, adjacent cells showed no sign of membrane damage and no increased caspase activity. The laser-exposed cells eventually detached from the substrate after a delay of >54 min while adjacent cells remained intact. On the base of in vitro experiments, we applied the same approach to ablate targeted single cardiac cells of a live zebrafish heart. The ability of inducing targeted cell death with femtosecond laser pulses should find broad applications that benefit from precise cellular manipulation at the level of single cells in vivo and in vitro.     

Transport-coupled ubiquitination of the borate transporter BOR1 for its boron-dependent degradation

Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.

The borate transporter BOR1 senses the boron concentration through its borate transport activity for K63-linked polyubiquitination and subsequent degradation.     

Characteristics of immobilized invertase

Five kinds of immobilized invertases (IMI)—covalently of porous glass and ion-exchange resins and ionically on ion-exchange resins—have been prepared and their kinetic characteristics for sucrose hydrolysis, such as K_m, K, pH profile, and thermal stability were studied. Comparing the values of K_m and activation energy and the entropy of IMI with those of native invertase, it was concluded that the immobilization influences not binding but kinetic specificity. The effects of the immobilization method on thermal stability were also discussed.     

Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1–4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4–28-fold and H/R ratios by 2–5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES.     

Structural requirement of sterol side chain for the silkworm growth and development

Several cholesterol analogs with modifications in the side chain were added to the artificial diet of the silkworm, and their effects on insect growth and development were determined. It was found that slight deviations of the cholesterol's side chain induced pronounced growth-retarding effects, suggesting an important functional role of the isooctane side chain of cholesterol.     

Capsule-transmitted gut symbiotic bacterium of the Japanese common plataspid stinkbug, Megacopta punctatissima.

The Japanese common plataspid stinkbug, Megacopta punctatissima, deposits small brown particles, or symbiont capsules, on the underside of the egg mass for the purpose of transmission of symbiotic bacteria to the offspring. We investigated the microbiological aspects of the bacteria contained in the capsule, such as microbial diversity, phylogenetic placement, localization in vivo, and fitness effects on the host insect. Restriction fragment length polymorphism analysis of 16S ribosomal DNA clones revealed that a single bacterial species dominates the microbiota in the capsule. The bacterium was not detected in the eggs but in the capsules, which unequivocally demonstrated that the bacterium is transmitted to the offspring of the insect orally rather than transovarially, through probing of the capsule content. Molecular phylogenetic analysis showed that the bacterium belongs to the gamma-subdivision of the Proteobacteria. In adult insects the bacterium was localized in the posterior section of the midgut. Deprivation of the bacterium from the nymphs resulted in retarded development, arrested growth, abnormal body coloration, and other symptoms, suggesting that the bacterium is essential for normal development and growth of the host insect.