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The epigean spiders of the genus Cybaeus L. Koch, 1868 are known to have diversified in western North America and the Japanese Archipelago. To date, ~80 species of Cybaeus are known from Japan, but they have not previously been recorded from the Ryukyu Islands that harbour a diversity of endemic species. Here we describe eight new species of Cybaeus from the Ryukyu Islands, extending the range of Cybaeus southward to the central Ryukyus. Both sexes of each of the new species are described, and their phylogenetic relationships are estimated using nuclear and mitochondrial gene markers. Although Cybaeus okumurai, sp. nov. and C. kumadori, sp. nov. possess genital features that are common in the other Japanese congeners, the other six species (C. yakushimensis, sp. nov., C. kodama, sp. nov., C. amamiensis, sp. nov., C. aikana, sp. nov., C. tokunoshimensis, sp. nov., and C. hikidai, sp. nov.) are characterised by an elongated embolus and tubular spermathecae. These unique genital characteristics and the phylogeny recovered here suggest that these features evolved independently among the Japanese and Ryukyu Cybaeus species. Phylogenetic analyses highlight an unusual biogeographical pattern in which C. yakushimensis and C. kodama endemic to Yakushima Island in the northern Ryukyus are related to species distributed in the central Ryukyus. In contrast, our phylogeny suggests that C. okumurai from Tanegashima Island in the northern Ryukyus is sister to C. ashikitaensis (Komatsu, 1968), distributed in Kyushu of the Japanese Archipelago. The retreat constructs and sympatric distribution of Cybaeus found among the Ryukyus are also briefly discussed. ZooBank: urn:lsid:zoobank.org:pub:640D15AA-17F4-48EE-88B4-485CFF8FCD60

Optimisation of protein binders relies on laborious screening processes. Investigation of sequence–function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence–function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody–antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence–function relationships., 遊離型抗体の構造活性相関解析を迅速に評価可能とする新手法を開発. 京都大学プレスリリース. 2021-11-08.

Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution., 生体内タンパク質ライゲーションを用いた新規細胞表層ディスプレイ法の開発 --新しい手法によるタンパク質工学の進展--. 京都大学プレスリリース. 2021-05-28.

Summary: Unsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually override mitotic arrest by mitotic slippage. Here, we show that inactivation of TORC1 kinase elicits mitotic slippage in budding yeast and human cells. Yeast mitotic slippage was accompanied with aberrant aspects, such as degradation of the nucleolar protein Net1, release of phosphatase Cdc14, and anaphase-promoting complex/cyclosome (APC/C)-Cdh1-dependent degradation of securin and cyclin B in metaphase. This mitotic slippage caused chromosome instability. In human cells, mammalian TORC1 (mTORC1) inactivation also invoked mitotic slippage, indicating that TORC1 inactivation-induced mitotic slippage is conserved from yeast to mammalian cells. However, the invoked mitotic slippage in human cells was not dependent on APC/C-Cdh1. This study revealed an unexpected involvement of TORC1 in mitosis and provides information on undesirable side effects of the use of TORC1 inhibitors as immunosuppressants and anti-tumor drugs.

Aims Fractional flow reserve (FFR), assessed using distal coronary pressure/aortic pressure (Pd)/(Pa) ratio, functionally evaluates coronary stenosis. An assessment method without vasodilators would be helpful. A single intracoronary bolus of saline decreases Pd because of the speculated low-viscosity effect. We hypothesised that saline-induced Pd/Pa ratio (SPR) could functionally evaluate coronary stenosis. This study aimed to test the accuracy and utility of SPR for predicting FFR ≤0.80. Methods and results In 137 coronary lesions with over 50% angiographic diameter stenosis, SPR was assessed using an intracoronary bolus of saline (2 mL/s) for five heartbeats (SPR-5) and three heartbeats (SPR-3). FFR was obtained after intravenous adenosine infusion (140 µg/kg/min). There was a strong correlation between FFR and SPR-5 or SPR-3 (R=0.941 and R=0.933, respectively). Receiver operating characteristic (ROC) curve analysis demonstrated good accuracy (86.3%) for SPR-5, with a cut-off of ≤0.84 for predicting FFR ≤0.80 (area under ROC curve 0.96, specificity 94.3, sensitivity 79.9). Thirty-three lesions (24%) were located in the "grey zone" (SPR 0.83-0.88). No complications were observed in 673 SPR measurements. Conclusions SPR may accurately predict FFR and can limit adenosine use to one in four lesions. Further studies are needed to confirm the validity of SPR.

The purpose of this study was to detect porcine epidemic diarrhea virus (PEDV) subclinically infected pigs shipped from non-case farms to slaughterhouses. Systematic sampling was conducted at two slaughterhouses. A total of 1,556 blood samples were collected from 80 case and non-case farms from pigs over 6 months old. Blood samples were centrifuged to obtain sera. Serial serum dilutions were subjected to serological examination for PEDV presence using Neutralization test (NT). The cut-off titer was set at titer of 1:2 dilution and farms with at least one positive sample in duplicate were classified as PED-positive farms. Several non-case farms (9.4%, 6/64) and 100% (16/16) of the case farms were indeed positive for PEDV. The proportion of seropositive animals from case farms was 63.7%, significantly different from that of non-case farms (4.3%, P

Autophagic degradation of ribosomes is promoted by nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1). Here we show that selective autophagic degradation of ribosomes (called ribophagy) after TORC1 inactivation requires the specific autophagy receptor Atg11. Rim15 protein kinase upregulated ribophagy, while it downregulated non-selective degradation of ribosomes.

The target of rapamycin complex 1 (TORC1) protein kinase is activated by nutrients and controls nutrient uptake via the membrane trafficking of various nutrient permeases. However, its molecular mechanisms remain elusive. Cholesterol (ergosterol in yeast) in conjunction with sphingolipids forms tight-packing microdomains, "lipid rafts", which are critical for intracellular protein sorting. Here we show that a novel target of rapamycin (TOR)-interacting transcriptional activator Vhr2 is required for full expression of some ERG genes for ergosterol biogenesis and for proper sorting of the tryptophan permease Tat2 in budding yeast. Loss of Vhr2 caused sterol biogenesis disturbance and Tat2 missorting. TORC1 activity maintained VHR2 transcript and protein levels, and total sterol levels. Vhr2 was not involved in regulation of the TORC1-downstream protein kinase Npr1, which regulates Tat2 sorting. This study suggests that TORC1 regulates nutrient uptake via sterol biogenesis.

Nutrient starvation or inactivation of TORC1 induces separation of rDNA and nucleolar proteins in yeast. Mostofa et al. report that the rDNA tethering CLIP–cohibin system repositions nucleolar proteins to sites proximal to the nuclear–vacuolar junction (NVJ), where micronucleophagy occurs, whereas rDNA moves to regions distal to the NVJ., Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) in budding yeast induces nucleophagy, a selective autophagy process that preferentially degrades nucleolar components. DNA, including ribosomal DNA (rDNA), is not degraded by nucleophagy, even though rDNA is embedded in the nucleolus. Here, we show that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites. Nucleolar proteins move to sites proximal to the nuclear–vacuolar junction (NVJ), where micronucleophagy (or piecemeal microautophagy of the nucleus) occurs, whereas rDNA dissociates from nucleolar proteins and moves to sites distal to NVJs. CLIP and cohibin, which tether rDNA to the inner nuclear membrane, were required for repositioning of nucleolar proteins and rDNA, as well as effective nucleophagic degradation of the nucleolar proteins. Furthermore, micronucleophagy itself was necessary for the repositioning of rDNA and nucleolar proteins. However, rDNA escaped from nucleophagic degradation in CLIP- or cohibin-deficient cells. This study reveals that rDNA–nucleolar protein separation is important for the nucleophagic degradation of nucleolar proteins.

For maintenance of cytoplasmic protein quality control (PQC), cytoplasmic heat shock proteins (HSPs) negatively control heat shock transcriptional factor (HSF) in a negative feedback loop. However, how mitochondrial protein quality control (mtPQC) is maintained is largely unknown. Here we present evidence that HSF directly monitors mtPQC in the budding yeast Saccharomyces cerevisiae. Mitochondrial HSP70 (Ssc1) negatively regulated HSF activity. Importantly, HSF was localized not only in the nucleus but also on mitochondria. The mitochondrial localization of HSF was increased by heat shock and compromised by SSC1 overexpression. Furthermore, the mitochondrial protein translocation system downregulated HSF activity. Finally, mtPQC modulated the mtHSP genes SSC1 and MDJ1 via HSF, and SSC1 overexpression compromised mitochondrial function. These findings illustrate a model in which HSF directly monitors mtPQC.

The mitotic separase cleaves Scc1 in cohesin to allow sister chromatids to separate from each other upon anaphase onset. Separase is also required for DNA damage repair. Here, we isolated and characterized 10 temperature-sensitive (ts) mutants of separase ESP1 in the budding yeast Saccharomyces cerevisiae. All mutants were defective in sister chromatid separation at the restricted temperature. Some esp1-ts mutants were hypersensitive to the microtubule poison benomyl and/or the DNA-damaging agent bleomycin. Overexpression of securin alleviated the growth defect in some esp1-ts mutants, whereas it rather exacerbated it in others. The Drosophila Pumilio homolog MPT5 was isolated as a high-dosage suppressor of esp1-ts cells. We discuss various features of separase based on these findings. Securin positively and negatively regulates separase function. In addition, extracellular inputs affect separase function.

Spiders are usually thought to obtain their energy for growth, maintenance and reproduction from captured prey. However, not all spiders should be assumed to receive all of their energy from prey. Accumulated studies have documented nectarivory in a wide variety of hunting spiders. We report a Clubiona spider visiting flowers of the nectariferous orchid Neottianthe cucullata as an example of spider nectarivory. In addition, during the visitations by Clubiona spider, pollinia removal was observed. The impact of the Clubiona spider on the N. cucullata reproductive success is also discussed.

Target of rapamycin complex 1 (TORC1) phosphorylates autophagy-related Atg13 and represses autophagy under nutrient-rich conditions. However, when TORC1 becomes inactive upon nutrient depletion or treatment with the TORC1 inhibitor rapamycin, Atg13 dephosphorylation occurs rapidly, and autophagy is induced. At present, the phosphatases involved in Atg13 dephosphorylation remain unknown. Here, we show that two protein phosphatase 2A (PP2A) phosphatases, PP2A-Cdc55 and PP2A-Rts1, which are activated by inactivation of TORC1, are required for sufficient Atg13 dephosphorylation and autophagy induction after TORC1 inactivation in budding yeast. After rapamycin treatment, dephosphorylation of Atg13, activation of Atg1 kinase, pre-autophagosomal structure (PAS) formation and autophagy induction are all impaired in PP2A-deleted cells. Conversely, overexpression of non-phosphorylatable Atg13 suppressed defects in autophagy in PP2A mutant. This study revealed that the orchestrated action of PP2A antagonizes Atg13 phosphorylation and promotes autophagy after the inactivation of TORC1.

Etude electrochimique et cinetique a l'aide d'une methode de reduction utilisant des ions CuCl 2 - de la precipitation selective de l'or a partir d'une solution d'acide chlorhydrique contenant des ions complexes de chlorures de metaux precieux

Purpose: We evaluated the effectiveness of Closed Loop Stimulation (CLS) function by comparing heart rate variations during chorus singing in one CLS pacemaker patient and two healthy people who sing chorus regularly.Methods: Study subjects were two healthy people and a 71-year-old female sinus bradycardia patient with syncopal syndrome in whom a CLS pacemaker (Entovis DR/CLS, Biotronik, Nihon Kohden) had been implanted. Their heart rate variations while chorus singing with little body motion were simultaneously measured with 3 telemetry monitors.Results: The two healthy people had maximum heart rates of 116 and 112 bpm. The CLS patient had a maximum heart rate of 110 ppm and heart rate variation as great as that of the healthy people. Heart rate variations in the two healthy people and the CLS patient showed a moderate correlation.Conclusion: Our results suggest that in an elderly CLS patient the CLS pacemaker was able to respond to circulatory demands comparable to those of healthy people and increase its pacing rate during the same low body motion work and hobbies.

The silylene ( SiH2) density in a parallel-plate RF (13.56 MHz) discharge using monosilane ( SiH4) gas has been measured by using laser-induced fluorescence spectroscopy. The effect of Xe, Ar, He and H2 dilution gases on the SiH2 density was investigated at a total gas pressure of 40 mTorr. In all dilution cases, the SiH2 density was relatively insensitive to the SiH4 fraction (x) in the mixtures for x\gtrsim0.5; however, except in the case of H2 dilution, marked increase of the SiH2 density was observed with decreasing x for x\lesssim0.5. The production and loss mechanisms for SiH2 are discussed on the basis of the observed SiH2 density and supplementary optical emission measurements. The results indicate that, in highly diluted ( x\lesssim0.1)SiH4/Xe and SiH4/Ar mixtures, the production of SiH2 via energy transfer from excited Xe and Ar atoms to SiH4 is dominant over direct electron impact dissociation of SiH4.

Silylene radicals (SiH2) in a 40-W 40-mTorr RF (13.56 MHz) SiH4/Ar plasma were detected by use of a laser-induced-fluorescence (LIF) technique. The observed SiH2 density increased with increasing Ar partial pressure. The absolute SiH2 density, estimated from the comparison of the LIF intensity with the intensity of Rayleigh scattering caused by N2 molecules, is in the range of 109-1010 cm-3.

Since a bottom long line lies on the bottom of the sea for many hours, the bait is damaged by objective fish, small fish, a crustaceans, a cephalopods or the like and the hooked rate decreases with the lapse of time. Authors investigated the percentage of remaining bait and hooked rate of the deep sea bottom long line (the used number of hooks; 16500) 33 times to obtain the following results.1. It was concluded that squid bait had high percentage of remaining bait and was much better in bait retentivity compared with saury.2. There was a marked tendency that percentage of remaining bait was low in the water area showing a high hooked rate and the hooked rate is low in the water area showing a high percentage of remaining bait.3. The percentage of remaining bait in deep sea (200 ?? 900m) decreases with increase in depth but the reduction rate became extermely low in deep region over 600m. It is considered that percentage of remaining bait shows the relative distribution density of the benthos in that water region.4. From the percentage of remaining bait along a vertical line, it is said that the range of the vertical migration behavior of predator is very narrow.5. The difference of the hooked rate between a squid and saury was not confirmed.