Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis>)

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant ‘McIntosh Wijcik’, which was discovered as a bud mutation from ‘McIntosh’, exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in ‘McIntosh Wijcik’ but not in ‘McIntosh’. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in ‘McIntosh Wijcik’ is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.     

Affinity Improvement of a Cancer-Targeted Antibody through Alanine-Induced Adjustment of Antigen-Antibody Interface

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Antifibrotic effect of methylated quercetin derivatives on TGFβ-induced hepatic stellate cells

Quercetin (QCT) and isorhamnetin (ISO), natural flavonoids, were both shown to possess antifibrotic activity in in vivo and in vitro models of hepatic fibrosis. Although ISO is a direct metabolite of QCT differing by a methyl group, it has been reported to be absorbed more adequately and eliminated slower than QCT after oral administration. Our aim of the study was to investigate biological effect of mono-methylated QCT derivatives against fibrosis using rat hepatic stellate cells (HSC-T6). All test derivatives were synthesized from QCT. HSC-T6 cells were induced by TGFβ and treated with derivatives followed by cell proliferation assay, immunofluorescence staining of αSMA, and gene expression analysis of fibrosis markers. All compounds showed a dose- and time-dependent antiproliferation effect. ISO, 3-O-methylquercetin (3MQ), and rhamnetin (RHA) reduced αSMA mRNA; 3MQ prevented the augmentation of collagen I mRNA; and compounds, except azaleatin and 3MQ, reduced Timp1 mRNA expression in TGFβ-induced HSCs. In conclusion, each compound had singular effect against different features of fibrosis depending on the position of methyl group although the further mechanism of action of compounds during fibrosis development remains to be investigated. These findings suggest that antifibrotic effect of quercetin can be enhanced by adding methyl group on functionally important position.     

Heart rate and electroencephalogram changes caused by finger acupressure on planta pedis

Preliminary experiments were carried out to investigate the feasibility of using an electroencephalogram and heart rates to evaluate the efficacy of finger acupressure on the key points of planta pedis (both soles). Continuous electroencephalograms were recorded from 19 electrodes based on the International 10-20 electrode placement system on 22 university students (21+/-2.3 years). Spectral power changes were obtained at each electrode site. The power of the alpha1 frequency range (8-10 Hz) increased slightly during acupressure although no statistical significance was observed, while heart rates decreased in all subjects (p<0.05). Cerebral cortex asymmetry in the spectral power changes was not clearly observed during the right and left sole acupressure. This preliminary study suggests that a classification of subjects is necessary in understanding brain wave data during acupressure on soles.     

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.     

Norlichexanthone produced by cultured endolichenic fungus induced from Pertusaria laeviganda and its antioxidant activity

Endolichenic fungi, nonobligate microfungi that live in lichen, are promising as new bioresources of pharmacological compounds. We found that norlichexanthone isolated from the endolichenic fungus in Pertusaria laeviganda exhibited high antioxidant activity. Norlichexanthone produced by endolichenic fungus had the antioxidant activity with same level of ascorbic acid. This is the first report of high antioxidant activity of norlichexanthone.

Abbreviations: AAPH: 2,2?-azobis (2-methylpropionamidine) dihydrochloride; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FL: fluorescein sodium salt; HPLC-PDA: high-performance liquid chromatography with photodiode array; LC-ESI-MS: liquid chromatography with electrospray ionization mass spectrometry; ORAC: oxygen radical absorbance capacity; PB: phosphate buffer; ROS: reactive oxygen species; TLC: thin-layer chromatography     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.