Expression analysis of type I ARF small GTPases ARF1-3 during mouse brain development.

BACKGROUND: ARF (ADP-ribosylation factor) GTPases are major regulators of intracellular trafficking, and classified into 3 groups (Type I - III), among which the type I group members, ARF1 and 3, are responsible genes for neurodevelopmental disorders. METHODS: In this study, we analysed the expression of Type I ARFs ARF1-3 during mouse brain development using biochemical and morphological methods. RESULTS: Western blotting analyses revealed that ARF1-3 are weakly expressed in the mouse brain at embryonic day 13 and gradually increase until postnatal day 30. ARF1-3 appear to be abundantly expressed in various telencephalon regions. Biochemical fractionation studies detected ARF1-3 in the synaptosome fraction of cortical neurons containing both pre- and post-synapses, however ARF1-3 were not observed in post-synaptic compartments. In immunohistochemical analyses, ARF1-3 appeared to be distributed in the cytoplasm and dendrites of cortical and hippocampal neurons as well as in the cerebellar molecular layer including dendrites of Purkinje cells and granule cell axons. Immunofluorescence in primary cultured hippocampal neurons revealed that ARF1-3 are diffusely distributed in the cytoplasm and dendrites with partial colocalization with a pre-synaptic marker, synaptophysin. CONCLUSIONS: Overall, our results support the notion that ARF1-3 could participate in vesicle trafficking both in the dendritic shaft (excluding spines) and axon terminals (pre-synaptic compartments).

Specific inhibition of oncogenic RAS using cell-permeable RAS-binding domains.

Oncogenic RAS proteins, common oncogenic drivers in many human cancers, have been refractory to conventional small-molecule and macromolecule inhibitors due to their intracellular localization and the lack of druggable pockets. Here, we present a feasible strategy for designing RAS inhibitors that involves intracellular delivery of RAS-binding domain (RBD), a nanomolar-affinity specific ligand of RAS. Screening of 51 different combinations of RBD and cell-permeable peptides has identified Pen-cRaf-v1 as a cell-permeable pan-RAS inhibitor capable of targeting both G12C and non-G12C RAS mutants. Pen-cRaf-v1 crosses the cell membrane via endocytosis, competitively inhibits RAS-effector interaction, and thereby exerts anticancer activity against several KRAS-mutant cancer cell lines. Moreover, Pen-cRaf-v1 exhibits excellent activity comparable with a leading pan-RAS inhibitor (BI-2852), as well as high target specificity in transcriptome analysis and alanine mutation analysis. These findings demonstrate that specific inhibition of oncogenic RAS, and possibly treatment of RAS-mutant cancer, is feasible by intracellular delivery of RBD.

Temporal muscle thickness and area with various characteristics data of the patients with aneurysmal subarachnoid hemorrhage who underwent endovascular coiling.

These data present the characteristics of patients with subarachnoid hemorrhage who underwent endovascular coiling. We retrospectively collected data from the medical records of Iwaki City Medical Center including physiological symptoms, laboratory data, radiological data on admission, and modified Rankin Scale scores at 6 months. Our article entitled "Temporal Muscle as an Indicator of Sarcopenia is Independently Associated with Hunt and Kosnik Grade on Admission and the Modified Rankin Scale at 6 Month of Patients with Subarachnoid Hemorrhage Treated by Endovascular Coiling" was based on these data [1]. We previously reported similar small dataset of elderly patients with subarachnoid hemorrhage who underwent surgical clipping [2], [3]. However, remarkably, this is the largest and the first dataset on temporal muscle thickness or area of patients of all ages with subarachnoid hemorrhage who underwent endovascular coiling, not surgical clipping.

Temporal Muscle as an Indicator of Sarcopenia is Independently Associated with Hunt and Kosnik Grade on Admission and the Modified Rankin Scale Score at 6 Months of Patients with Subarachnoid Hemorrhage Treated by Endovascular Coiling.

OBJECTIVE: Sarcopenia is defined as the loss of skeletal muscle mass and is considered an important factor for clinical outcomes in various diseases. Recent studies have shown that temporal muscle thickness (TMT) and area (TMA) can be novel indicators of sarcopenia. We examined clinical characteristics, including TMT and TMA, of patients with subarachnoid hemorrhage (SAH) treated by endovascular coiling. METHODS: A retrospective analysis of 298 patients with SAH who were treated with endovascular coiling from 2009 to 2019 was conducted. Their premorbid modified Rankin Scale (mRS) score was 0-2. The association between the factors and Hunt and Kosnik (H-K) grades on admission and that between the clinical variables and mRS scores 6 months after the operation were analyzed. RESULTS: In all 298 patients with SAH, Fisher group 4 and TMA <200 mm2 were independently associated with H-K grade III-V on admission in the multivariate analysis. In 254 patients with H-K grades I-III on admission, age, H-K grade III, presence of ventriculoperitoneal shunt, presence of postoperative complications, and TMA <200 mm2 were independent factors related to poor outcomes in the multivariate analysis. CONCLUSIONS: The H-K grade on admission was independently associated with TMA. The mRS score 6 months after aneurysm treatment in patients with H-K grades I-III was also independently associated with TMA. Sarcopenia could be one of a few modifiable factors that prevent severe symptoms of SAH and improve outcomes after coiling by strengthened nutrition and physical activity.

Structural and functional characterization of fast-cycling RhoF GTPase.

Ras superfamily GTPases are molecular switches that cycle between GDP-bound inactive state and GTP-bound active state to control many signaling pathways. Emerging evidence suggests that several Ras superfamily GTPases, including RhoF, do not follow the classical GDP/GTP exchange cycle; they act as constitutively active GTP-bound proteins due to their fast activities of GDP/GTP exchange (termed as 'fast-cycling' GTPases). To understand the molecular basis of the fast-cycling GTPases, we generated a GTPase active recombinant RhoF and examined its function and structure. Two point mutations in the switch I/II regions (Q77L and P45S, corresponding to Q61L and P29S of Rac1) significantly reduced the GTPase activity of RhoF, suggesting a conserved mechanism of GTP hydrolysis between RhoF and other RAS superfamily GTPases. However, in contrary to the previous evidence, RhoF represented a slow GDP/GTP exchange activity that dissociates GDP very slowly on a day-to-week time scale, in our experiment using fluorescently labeled GDP. The slow GDP dissociation was accelerated by Mg2+ chelation and canonical fast-cycling mutations, F44L (corresponding to F28L of Rac1) and P45S. NMR and dynamic light scattering data revealed a multimeric structure of RhoF that can switch between different conformations depending on the GTP/GDP-bound state. Overall, our study suggests that (1) RhoF shares a conserved mechanism of GTP hydrolysis with other RAS superfamily GTPases, but (2) RhoF adopts a unique multimeric structure. Our study also argues that (3) the emerging concept of the fast-cycling GTPases for RhoF should be validated using an alternative assay that does not rely on fluorescently labeled GDP (251 words).

Rapid and reliable species identification of wild mushrooms by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).

Mushrooms are a favourite natural food in many countries. However, some wild species cause food poisoning, sometimes lethal, due to misidentification caused by confusing fruiting bodies similar to those of edible species. The morphological inspection of mycelia, spores and fruiting bodies have been traditionally used for the identification of mushrooms. More recently, DNA sequencing analysis has been successfully applied to mushrooms and to many other species. This study focuses on a simpler and more rapid methodology for the identification of wild mushrooms via protein profiling based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). A preliminary study using 6 commercially available cultivated mushrooms suggested that a more reproducible spectrum was obtained from a portion of the cap than from the stem of a fruiting body by the extraction of proteins with a formic acid-acetonitrile mixture (1 + 1). We used 157 wild mushroom-fruiting bodies collected in the centre of Hokkaido from June to November 2014. Sequencing analysis of a portion of the ribosomal RNA gene provided 134 identifications of mushrooms by genus or species, however 23 samples containing 10 unknown species that had lower concordance rate of the nucleotide sequences in a BLAST search (less than 97%) and 13 samples that had unidentifiable poor or mixed sequencing signals remained unknown. MALDI-TOF MS analysis yielded a reproducible spectrum (frequency of matching score ≥ 2.0 was ≥6 spectra from 12 spectra measurements) for 114 of 157 samples. Profiling scores that matched each other within the database gave correct species identification (with scores of ≥2.0) for 110 samples (96%). An in-house prepared database was constructed from 106 independent species, except for overlapping identifications. We used 48 wild mushrooms that were collected in autumn 2015 to validate the in-house database. As a result, 21 mushrooms were identified at the species level with scores ≥2.0 and 5 mushrooms at the genus level with scores ≥1.7, although the signals of 2 mushrooms were insufficient for analysis. The remaining 20 samples were recognized as "unreliable identification" with scores <1.7. Subsequent DNA analysis confirmed that the correct species or genus identifications were achieved by MALDI-TOF MS for the 26 former samples, whereas the 18 mushrooms with poorly matched scores were species that were not included in the database. Thus, the proposed MALDI-TOF MS coupled with our database could be a powerful tool for the rapid and reliable identification of mushrooms; however, continuous updating of the database is necessary to enrich it with more abundant species.

Identification of Mushroom Species by Automated rRNA Intergenic Spacer Analysis (ARISA) and Its Application to a Suspected Case of Food Poisoning with Tricholoma ustale.

Automated rRNA intergenic spacer analysis (ARISA), a method of microbiome analysis, was evaluated for species identification of mushrooms based on the specific fragment sizes. We used 51 wild mushroom-fruiting bodies collected in the centre of Hokkaido and two cultivated mushrooms. Samples were hot-air-dried and DNA were extracted by a beads beating procedure. Sequencing analysis of portions of the rRNA gene (rDNA) provided 33 identifications of mushrooms by genus or species. The results of ARISA identification based on the combination of the fragment sizes corresponding to two inter spacer regions (ITS2 and ITS1) of rDNA within±0.1% accuracy showed that 27 out of the 33 species had specific fragment sizes differentiated from other species. The remaining 6 species formed 3 pairs that showed overlapping fragment sizes. In addition, within-species polymorphisms were observed as 1 bp differences among 32 samples of 13 species. ARISA was applied to investigate a case of suspected food poisoning in which the mushroom was thought to be a toxic Kakishimeji. The morphological identification of the mushroom was ambiguous since the remaining sample lacked a part of the fruiting body. Further, yeast colonies had grown on the surface of the fruiting body during storage. The ARISA fragment size of the mushroom showed 7 bp difference from that of the candidate toxic mushroom. Although ARISA could be a useful tools for estimation of mushroom species, especially in case where the fruiting bodies have deteriorated or been processed, further studies are necessary for reliable identification. For example, it may be necessary to adopt more informative genes which could provide clearer species-specific polymorphisms than the ITS regions.