In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology.
To reveal whether bats serve as an amplifying host for Yokose virus (YOKV), we conducted a serological survey and experimentally infected fruit bats with YOKV isolated from microbats in Japan. YOKV belongs to the Entebbe bat virus group of vector unknown group within the genus Flavivirus and family Flaviviridae. To detect antibodies against YOKV, we developed an enzyme-linked immunosorbent assay (ELISA) using biotinylated anti-bat IgG rabbit sera. Serological surveillance was conducted with samples collected in the Philippines and the sera supplied from Malaysia. One of the 36 samples from the Philippines (2.7%) and 5 of the 26 samples from Malaysia (19%) had detectable ELISA antibodies. In the experimental infections, no clinical signs of disease were observed. Moreover, no significant viral genome amplification was detected. These findings revealed that YOKV replicates poorly in the fruit bat, suggesting that fruit bats do not seem to serve as an amplifying host for YOKV.
Selective separation and sensitive detection of dissolved silicon and boron (DSi and DB) in aqueous solution was achieved by combining an electrodialytic ion isolation device (EID) as a salt remover, an ion-exclusion chromatography (IEC) column, and a corona charged aerosol detector (CCAD) in sequence. DSi and DB were separated by IEC on the H(+)-form of a cation exchange resin column using pure water eluent. DSi and DB were detected after IEC separation by the CCAD with much greater sensitivity than by conductimetric detection. The five-channel EID, which consisted of anion and cation acceptors, cathode and anode isolators, and a sample channel, removed salt from the sample prior to the IEC-CCAD. DSi and DB were scarcely attracted to the anion accepter in the EID and passed almost quantitatively through the sample channel. Thus, the coupled EID-IEC-CCAD device can isolate DSi and DB from artificial seawater and hot spring water by efficiently removing high concentrations of Cl(-) and SO4(2-) (e.g., 98% and 80% at 0.10molL(-1) each, respectively). The detection limits at a signal-to-noise ratio of 3 were 0.52μmolL(-1) for DSi and 7.1μmolL(-1) for DB. The relative standard deviations (RSD, n=5) of peak areas were 0.12% for DSi and 4.3% for DB.
Epilepsies are common neurological disorders and genetic factors contribute to their pathogenesis. Copy number variations (CNVs) are increasingly recognized as an important etiology of many human diseases including epilepsy. Whole-exome sequencing (WES) is becoming a standard tool for detecting pathogenic mutations and has recently been applied to detecting CNVs. Here, we analyzed 294 families with epilepsy using WES, and focused on 168 families with no causative single nucleotide variants in known epilepsy-associated genes to further validate CNVs using 2 different CNV detection tools using WES data. We confirmed 18 pathogenic CNVs, and 2 deletions and 2 duplications at chr15q11.2 of clinically unknown significance. Of note, we were able to identify small CNVs less than 10 kb in size, which might be difficult to detect by conventional microarray. We revealed 2 cases with pathogenic CNVs that one of the 2 CNV detection tools failed to find, suggesting that using different CNV tools is recommended to increase diagnostic yield. Considering a relatively high discovery rate of CNVs (18 out of 168 families, 10.7%) and successful detection of CNV with <10 kb in size, CNV detection by WES may be able to surrogate, or at least complement, conventional microarray analysis.