Photoemission Properties of LaB6 and CeB6 Under Various Temperature and Incident Photon Energy Conditions

IPAC2016, Busan, KoreaPhotoemission properties of LaB₆ and CeB₆ were investigated at various cathode temperatures and different wavelengths of excitation laser to study for application of electron gun, especially for RF injector of infrared FEL facilities. It was found that the LaB₆ had higher photoemission property than CeB₆ at the same cathode temperature. In addition, LaB₆ can emit a measurable photoemission current being irradiated by laser with energy below work function at the cathode temperature higher than 1400 K. With increasing laser energy (over work function), a photoemission dependency on cathode temperature was getting lower. As the result, LaB₆ is revealed to have better properties than CeB₆ since LaB₆ has higher quantum efficiency than CeB₆ at same temperature

A Low Percent Ethanol Method for Immobilizing Planarians

Planarians have recently become a popular model system for the study of adult stem cells, regeneration and polarity. The system is attractive for both undergraduate and graduate research labs, since planarian colonies are low cost and easy to maintain. Also in situ hybridization, immunofluorescence and RNA-interference (RNAi) gene knockdown techniques have been developed for planarian studies. However, imaging of live worms (particularly at high magnifications) is difficult because animals are strongly photophobic; they quickly move away from light sources and out of frame. The current methods available to inhibit movement in planarians include RNAi injection and exposure to cold temperatures. The former is labor and time intensive, while the latter precludes the use of many fluorescent reporter dyes. Here, we report a simple, inexpensive and reversible method to immobilize planarians for live imaging. Our data show that a short 1 hour treatment with 3% ethanol (EtOH) is sufficient to inhibit both the fine and gross movements of Schmidtea mediterranea planarians, of the typical size used (4–6 mm), with full recovery of movement within 3–4 hours. Importantly, EtOH treatment did not interfere with regeneration, even after repeated exposure, nor lyse epithelial cells (as assayed by H&E staining). We demonstrate that a short exposure to a low concentration of EtOH is a quick and effective method of immobilizing planarians, one that is easily adaptable to planarians of all sizes and will increase the accessibility of live imaging assays to planarian researchers

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

タンパク質の抗体ラベリング技術を改良し、構造解析をアシスト --電子顕微鏡やX線結晶解析による構造決定を加速化--. 京都大学プレスリリース. 2021-04-20.Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab

18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote-specific 18S sequences

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3′-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site

Shallow water marine sediment bacterial community shifts along a natural CO2 gradient in the Mediterranean Sea off Vulcano, Italy.

The effects of increasing atmospheric CO(2) on ocean ecosystems are a major environmental concern, as rapid shoaling of the carbonate saturation horizon is exposing vast areas of marine sediments to corrosive waters worldwide. Natural CO(2) gradients off Vulcano, Italy, have revealed profound ecosystem changes along rocky shore habitats as carbonate saturation levels decrease, but no investigations have yet been made of the sedimentary habitat. Here, we sampled the upper 2 cm of volcanic sand in three zones, ambient (median pCO(2) 419 μatm, minimum Ω(arag) 3.77), moderately CO(2)-enriched (median pCO(2) 592 μatm, minimum Ω(arag) 2.96), and highly CO(2)-enriched (median pCO(2) 1611 μatm, minimum Ω(arag) 0.35). We tested the hypothesis that increasing levels of seawater pCO(2) would cause significant shifts in sediment bacterial community composition, as shown recently in epilithic biofilms at the study site. In this study, 454 pyrosequencing of the V1 to V3 region of the 16S rRNA gene revealed a shift in community composition with increasing pCO(2). The relative abundances of most of the dominant genera were unaffected by the pCO(2) gradient, although there were significant differences for some 5 % of the genera present (viz. Georgenia, Lutibacter, Photobacterium, Acinetobacter, and Paenibacillus), and Shannon Diversity was greatest in sediments subject to long-term acidification (>100 years). Overall, this supports the view that globally increased ocean pCO(2) will be associated with changes in sediment bacterial community composition but that most of these organisms are resilient. However, further work is required to assess whether these results apply to other types of coastal sediments and whether the changes in relative abundance of bacterial taxa that we observed can significantly alter the biogeochemical functions of marine sediments

Identification of N-homocysteinylated apolipoprotein AI in normal human serum

Background: In human serum, a portion of homocysteine (Hcy) exists as an N-linked form to the {varepsilon}-amino group of protein lysine residues. N-homocysteinylated proteins differ structurally and functionally from native proteins. The present study strives to develop detection and potential semi-quantification methods for N-homocysteinylated apolipoprotein AI (N-Hcy-apoAI) in human serum.Methods: Serum treated with or without cysteamine was supplied to isoelectric focusing (IEF) followed by an immunoblot using an anti-apoAI antibody. Cysteamine treatment increased the isoelectric point for N-Hcy-apoAI, but not for unmodified apoAI, due to the presence of -SH group(s) derived from Hcy and the absence of a cysteine residue in the apoAI molecule. N-Hcy-apoAI was semi-quantified from the scanned immunoblot pattern via a computer.Results: After cysteamine treatment, N-Hcy-apoAI in the serum was identified by IEF at the position with a higher pI value compared with intact apoAI. The reproducibility (between assays) of the semi-quantification method was 19.1% CV (coefficient of variation) for an average ratio 5.9% of N-Hcy-apoAI to the whole apoAI in the serum. Approximately 1.0–7.4% of apoAI was N-homocysteinylated in the serum obtained from 27 healthy subjects. Neither the ratio of N-Hcy-apoAI nor its concentration, calculated by total apoAI concentration, indicated correlation with the so-called total (free and S-linked) Hcy concentration.Conclusions: We directly found that a portion of apoAI in the serum undergoes homocysteinylation in an N-linkage manner, and used this to develop a potential semi-quantification method for N-Hcy-apoAI

Measurement and comparison of individual external doses of high-school students living in Japan, France, Poland and Belarus -- the "D-shuttle" project --

Twelve high schools in Japan (of which six are in Fukushima Prefecture), four in France, eight in Poland and two in Belarus cooperated in the measurement and comparison of individual external doses in 2014. In total 216 high-school students and teachers participated in the study. Each participant wore an electronic personal dosimeter "D-shuttle" for two weeks, and kept a journal of his/her whereabouts and activities. The distributions of annual external doses estimated for each region overlap with each other, demonstrating that the personal external individual doses in locations where residence is currently allowed in Fukushima Prefecture and in Belarus are well within the range of estimated annual doses due to the background radiation level of other regions/countries

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

細胞膜の中ではたらく特殊なタンパク質分解酵素の構造を解明 --細菌感染症の新たな治療法の開発へ期待--. 京都大学プレスリリース. 2022-08-25.Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments