Snapshots of native pre-50S ribosomes reveal a biogenesis factor network and evolutionary specialization.

Ribosome biogenesis is a fundamental multi-step cellular process that culminates in the formation of ribosomal subunits, whose production and modification are regulated by numerous biogenesis factors. In this study, we analyze physiologic prokaryotic ribosome biogenesis by isolating bona fide pre-50S subunits from an Escherichia coli strain with the biogenesis factor ObgE, affinity tagged at its native gene locus. Our integrative structural approach reveals a network of interacting biogenesis factors consisting of YjgA, RluD, RsfS, and ObgE on the immature pre-50S subunit. In addition, our study provides mechanistic insight into how the GTPase ObgE, in concert with other biogenesis factors, facilitates the maturation of the 50S functional core and reveals both conserved and divergent evolutionary features of ribosome biogenesis between prokaryotes and eukaryotes.

Size-Selective Capturing of Exosomes Using DNA Tripods.

Fractionating and characterizing target samples are fundamental to the analysis of biomolecules. Extracellular vesicles (EVs), containing information regarding the cellular birthplace, are promising targets for biology and medicine. However, the requirement for multiple-step purification in conventional methods hinders analysis of small samples. Here, we apply a DNA origami tripod with a defined aperture of binders (e.g., antibodies against EV biomarkers), which allows us to capture the target molecule. Using exosomes as a model, we show that our tripod nanodevice can capture a specific size range of EVs with cognate biomarkers from a broad distribution of crude EV mixtures. We further demonstrate that the size of captured EVs can be controlled by changing the aperture of the tripods. This simultaneous selection with the size and biomarker approach should simplify the EV purification process and contribute to the precise analysis of target biomolecules from small samples.

Effects of Radio-Taiso on Health-related Quality of Life in Older Adults With Frailty: a Randomized Controlled Trial.

BACKGROUND: Radio-Taiso, a long-standing exercise program in Japan, could be a sustainable public health strategy for maintaining quality of life (QoL) in older adults with frailty. This study aimed to investigate whether Radio-Taiso provided greater benefits for health-related quality of life (HRQoL) and to identify the mechanisms underlying the effectiveness in this population. METHODS: A 12-week randomized controlled trial enrolled 226 older Japanese adults with pre-frailty or frailty, assessed using the modified frailty phenotype. Participants were randomly allocated to the intervention (Radio-Taiso + nutrition program) or control (nutrition program) groups. The Radio-Taiso program comprised five 60-min group sessions and daily practice at the participants' homes. The primary outcome was the change in the mental domain of HRQoL, assessed using the SF-36®. The secondary outcomes included six physical fitness items and exercise self-efficacy. RESULTS: Overall, 104 and 105 participants in the intervention and control groups, respectively, were analyzed based on the intention-to-treat principle. The median daily practice rate of Radio-Taiso was 94.1% (interquartile range, 73.2-98.8%). Although general linear models adjusted for baseline values and allocation stratification factors showed that the intervention group obtained greater benefits (adjusted mean differences) in the up-and-go (0.3; 95% confidence interval [CI], 0.1 to 0.6 s), 2-min step-in-place (-3.2; 95% CI, -6.2 to -0.2 steps) tests, and exercise self-efficacy scale (-1.4; 95% CI, -2.6 to -0.1 points) than the control group, there were no group differences in changes in the mental domain score of HRQoL. CONCLUSION: Radio-Taiso provided greater benefits for agility/dynamic balance, aerobic endurance, and exercise self-efficacy in older adults with frailty; however, these changes do not improve HRQoL.

Postoperative management using a digital drainage system for massive air leakage after pulmonary resection.

PURPOSE: To elucidate clinical outcomes using a digital drainage system (DDS) for massive air leakage (MAL) after pulmonary resection. METHODS: A total of 135 consecutive patients with pulmonary resection air leakage of > 100 ml/min on the DDS were evaluated retrospectively. In this study, MAL was defined as ≥ 1000 ml/min on the DDS. We analyzed the clinical characteristics and surgical outcomes of patients with MAL compared with non-MAL (101-999 ml/min). Using the DDS data, the duration of the air leak was plotted with the Kaplan‒Meier method and compared using the log-rank test. RESULTS: MAL was detected in 19 (14%) patients. The proportions of heavy smokers (P = 0.04) and patients with emphysematous lung (P = 0.03) and interstitial lung disease (P < 0.01) were higher in the MAL group than in the non-MAL group. The MAL group had a higher persistence rate of air leakage at 120 h after surgery than the non-MAL group (P < 0.01) and required significantly more frequent pleurodesis (P < 0.01). Drainage failure occurred in 2 (11%) and 5 (4%) patients from the MAL and non-MAL groups, respectively. Neither reoperation nor 30-day surgical mortality was observed in patients with MAL. CONCLUSIONS: MAL was able to be treated conservatively without surgery using the DDS.

[A framework for promoting and evaluating "Kayoi-no-ba" initiatives according to the Plan-Do-Check-Action cycle: The ACT-RECIPE framework].

Objective　In 2019, the Ministry of Health, Labour and Welfare emphasized the importance of promoting "Kayoi-no-ba" (or "places to go") initiatives according to the Plan-Do-Check-Action (PDCA) cycle. However, it proposed no specific promotion measures or standardized evaluation frameworks. This study is intended to propose a framework for local government officials to promote and evaluate "Kayoi-no-ba" initiatives according to the PDCA cycle.Methods　The working group (WG) conducted a narrative review of research and extracted evaluation models and indicators that could be used to create the framework. The co-researcher review committee discussed a draft framework prepared by the WG, and the WG revised it based on the discussion; this process was repeated four times. Using the completed framework, we calculated the score of the "Kayoi-no-ba" initiatives in 50 Tokyo municipalities and conducted regional correlation analyses on the relationship between the score of the "Kayoi-no-ba" initiatives in 50 Tokyo municipalities and the number of "Kayoi-no-ba" per 1,000 older persons.Results　The completed framework (named "ACT-RECIPE" by rearranging the underlined characters below) comprised the following six evaluation phases: (1) Comprehension: understanding the key lessons around disability and frailty prevention, and the necessity for "Kayoi-no-ba"; (2) Research and Planning: clarifying the current status of "Kayoi-no-ba," the strengths of the community, and the issues to be addressed through a community assessment, and developing a plan to resolve the issues; (3) Team Building and Collaboration: building a team by collaborating with organizations to solve problems; (4) Implementation: implementing the initiatives necessary to solve problems; (5) Evaluation: verifying changes in output and outcome indicators from the initiatives; and (6) Adjustment and Improvement: re-examining plans, teams, content, and goals based on the evaluation results. In these six phases, we designated 10 core items and accompanying subitems. The median score rate of the ACT-RECIPE framework in 50 municipalities was 75% for "Comprehension," 61% for "Research and Planning," 69% for "Team Building and Collaboration," 64% for "Implementation," 31% for "Evaluation," and 56% for "Adjustment and Improvement," and the mean ACT-RECIPE score rate was 57%. A significant positive correlation (rs=0.43, P=0.002) was observed between the ACT-RECIPE mean score rate and the number of "Kayoi-no-ba" per 1,000 older persons.Conclusion　We proposed the ACT-RECIPE as a framework for promoting and evaluating "Kayoi-no-ba" initiatives according to the PDCA cycle. We hope that this framework will lead to further progress in "Kayoi-no-ba" initiatives and facilitate evaluation of their effectiveness according to the PDCA cycle.

Extracellular aaRSs drive autoimmune and inflammatory responses in rheumatoid arthritis via the release of cytokines and PAD4.

OBJECTIVES: Recent studies demonstrate that extracellular-released aminoacyl-tRNA synthetases (aaRSs) play unique roles in immune responses and diseases. This study aimed to understand the role of extracellular aaRSs in the pathogenesis of rheumatoid arthritis (RA). METHODS: Primary macrophages and fibroblast-like synoviocytes were cultured with aaRSs. aaRS-induced cytokine production including IL-6 and TNF-α was detected by ELISA. Transcriptomic features of aaRS-stimulated macrophages were examined using RNA-sequencing. Serum and synovial fluid (SF) aaRS levels in patients with RA were assessed using ELISA. Peptidyl arginine deiminase (PAD) 4 release from macrophages stimulated with aaRSs was detected by ELISA. Citrullination of aaRSs by themselves was examined by immunoprecipitation and western blotting. Furthermore, aaRS inhibitory peptides were used for inhibition of arthritis in two mouse RA models, collagen-induced arthritis and collagen antibody-induced arthritis. RESULTS: All 20 aaRSs functioned as alarmin; they induced pro-inflammatory cytokines through the CD14-MD2-TLR4 axis. Stimulation of macrophages with aaRSs displayed persistent innate inflammatory responses. Serum and SF levels of many aaRSs increased in patients with RA compared with control subjects. Furthermore, aaRSs released PAD4 from living macrophages, leading to their citrullination. We demonstrate that aaRS inhibitory peptides suppress cytokine production and PAD4 release by aaRSs and alleviate arthritic symptoms in a mouse RA model. CONCLUSIONS: Our findings uncovered the significant role of aaRSs as a novel alarmin in RA pathogenesis, indicating that their blocking agents are potent antirheumatic drugs.

Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex.

Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 3' supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 3' supplementary region upon target recognition.

Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA.

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.

Systemic-pulmonary collateral supply associated with clinical severity of chronic thromboembolic pulmonary hypertension: a study using intra-aortic computed tomography angiography.

OBJECTIVES: To assess whether systemic-pulmonary collaterals are associated with clinical severity and extent of pulmonary perfusion defects in chronic thromboembolic pulmonary hypertension (CTEPH). METHODS: This prospective study was approved by a local ethics committee. Twenty-four patients diagnosed with inoperable CTEPH were enrolled between July 2014 and February 2017. Systemic-pulmonary collaterals were detected using pulmonary vascular enhancement on intra-aortic computed tomography (CT) angiography. The pulmonary enhancement parameters were calculated, including (1) Hounsfield unit differences (HUdiff) between pulmonary trunks and pulmonary arteries (PAs) or veins (PVs), namely HUdiff-PA and HUdiff-PV, on the segmental base; (2) the mean HUdiff-PA, mean HUdiff-PV, numbers of significantly enhanced PAs and PVs, on the patient base. Pulmonary perfusion defects were recorded and scored using the lung perfused blood volume (PBV) based on intravenous dual-energy CT (DECT) angiography. Pearson's or Spearman's correlation coefficients were used to evaluate correlations between the following: (1) segment-based intra-aortic CT and intravenous DECT parameters (2) patient-based intra-aortic CT parameters and clinical severity parameters or lung PBV scores. Statistical significance was set at p < 0.05. RESULTS: Segmental HUdiff-PV was correlated with the segmental perfusion defect score (r = 0.45, p < 0.01). The mean HUdiff-PV was correlated with the mean pulmonary arterial pressure (PAP) (r = 0.52, p < 0.01), cardiac output (rho = - 0.41, p = 0.05), and lung PBV score (rho = 0.43, p = 0.04). And the number of significantly enhanced PVs was correlated with the mean PAP (r = 0.54, p < 0.01), pulmonary vascular resistance (r = 0.54, p < 0.01), and lung PBV score (rho = 0.50, p = 0.01). CONCLUSIONS: PV enhancement measured by intra-aortic CT angiography reflects clinical severity and pulmonary perfusion defects in CTEPH. KEY POINTS: • Intra-aortic CT angiography demonstrated heterogeneous enhancement within the pulmonary vasculature, showing collaterals from the systemic arteries to the pulmonary circulation in CTEPH. • The degree of systemic-pulmonary collateral development was significantly correlated with the clinical severity of CTEPH and may be used to evaluate disease progression. • The distribution of systemic-pulmonary collaterals is positively correlated with perfusion defects in the lung segments in CTEPH.

Significance of systolic-phase imaging on full-phase ECG-gated CT angiography to detect intimal tears in aortic dissection.

PURPOSE: For patients with aortic dissection (AD) and intramural hematoma (IMH), the optimal cardiac phase to detect intimal tears (IT) and ulcer-like projections (ULP) on retrospective electrocardiogram (ECG)-gated computed tomography angiography (CTA) remains unclear. The purpose of this study was to compare the accuracy of retrospective ECG-gated CTA for detecting IT in AD and ULP in IMH between each cardiac phase. MATERIALS AND METHODS: A total of 75 consecutive patients with AD and IMH of the thoracic aorta were enrolled in this single-center retrospective study. The diagnostic performance to detect IT and ULP in the thoracic aortic regions (including the ascending aorta, aortic arch, and proximal and distal descending aorta) was compared in each cardiac phase on retrospective ECG-gated CTA. RESULTS: In the systolic phase (20%), the accuracy, sensitivity, and specificity to detect IT in AD was 64% (95% confidence interval [CI] 56-72%), 69% (95%CI 60-78%), and 25% (95%CI 3.3-45%), respectively. In the diastolic phase (70%), the accuracy, sensitivity, and specificity to detect IT in AD was 52% (95%CI 43-60%), 52% (95%CI 42-61%), and 50% (95%CI 25-75%), respectively. The accuracy to detect IT in AD on ECG-gated CTA was significantly higher in the systolic phase than that in the diastolic phase (P = 0.025). However, there were no differences in the accuracy (83%; 95%CI 78-89%), sensitivity (71%; 95%CI 62-80%), or specificity (100%; 95%CI 100%) to detect ULP in IMH between the cardiac cycle phases. CONCLUSION: Although it is currently recommended for routine diagnosis of AD and IMH, single-diastolic-phase ECG-gated CTA has risk to miss some IT in AD that are detectable in the systolic phase on full-phase ECG-gated CTA. This information is critical for determining the optimal treatment strategy for AD.

The Escherichia coli DnaK chaperone stimulates the α-complementation of ß-galactosidase.

pUC18 and pUC19 are well-known high copy-number plasmid vectors routinely used for DNA cloning purposes. We show here that, in Escherichia coli transformed by native pUC18, the α-complementation of ß-galactosidase (i.e., mediated by the peptide LacZα18) is intrinsically weak and slow, but is greatly stimulated by the DnaK/DnaJ/GrpE chaperone system. In contrast, the α-complementation mediated by the peptide LacZα19 (in E. coli transformed by the native pUC19) is much more efficient and therefore does not require the assistance of the DnaK chaperone machinery. The marked difference between these two LacZα peptides is reproduced in a cell-free protein expression system coupled with α-complementation. We conclude that: (i) α-complementation of ß-galactosidase is DnaK-mediated depending upon the LacZα peptide donor; (ii) DnaK, sensu stricto, is not necessary for α-complementation, but can enhance it to a great extent; (iii) this observation could be used to establish an easy and inexpensive method for screening small molecules libraries in search of DnaK inhibitors and also for deciphering the DnaK-mediated protein quality control mechanism.

[Survey of Arsenic/Heavy Metals and Pesticide Residues in Edible Insects for Human Consumption or Supplied in Japan].

Arsenic (As) and heavy metals (Cd, Hg, Pb, and Cu) and pesticide residues in 14 edible insects were investigated. The maximum levels of elements were 6.15 for As, 0.82 for Cd, 0.50 for Hg, 0.67 for Pb, and 297.7 ppm for Cu. Fenobucarb (or BPMC) has been quantified through GC- and LC-MS/MS analysis at a concentration of approximately 3 ppm. Further studies of the contaminants may help ensure the safety of edible insect consumption.

[Association between issues in activities and psychosocial health status in the social positions of older residents' community-based activities: a cross-sectional study].

Objectives　When local governments and community nurses provide support for community-based activities led by older residents, it is imperative to reduce the burden of participants in leadership positions. This study aimed to identify the issues associated with the activities by social position, such as leadership, support, and regular participation, to discuss effective support for community-based activities and to examine the association between social position, relating issues, and psychosocial health.Methods　Participants in community-based activities were recruited by the local government in Tokyo, Japan. Overall, 2,367 people from 155 activity groups from 40 municipalities responded. Social positions in the groups were defined as leaders who manage activities; supporters who support leaders; and regular members who do not have any specific role. The participants chose the issues of community-based activities from 10 items. Psychosocial health was measured by the WHO-5 well-being index (WHO-5) and Lubben social network scale-6 (LSNS-6). The relationship between recognition of issues and social positions were examined by the chi-square test. Interaction effects of social positions and issues (with or without) on the WHO-5 and LSNS-6 were investigated using a two-way analysis of covariance.Results　The final sample comprised 2,096 respondents: 174 leaders, 296 supporters, and 1,626 regular members. There was a significant association between recognition of social positions and issues in the groups: respondents who reported no issues in the activities comprised 8.6% of the leaders, 27.7% of the supporters, and 53.6% of the regular members (P<0.001). There was a large difference in recognition between roles for issues related to group management, such as the lack of management members. There was no significant interaction between these issues and social positions in either WHO-5 or LSNS-6 (P=0.729, P=0.171, respectively). The main effect of the social positions was significant in both analyses (P<0.001). The leaders and supporters showed significantly higher WHO-5 and LSNS-6 scores than regular members.Conclusion　The issues that the participants reported differed by social positions in activities. It may be effective to provide support according to the difficulty of sharing issues within the group. Regardless of whether or not the participants report issues, leaders and supporters had improved psychosocial health than regular members. The study concludes that organizational position in community-based activities may offer health benefits for older adults.

[Diversity of participants and autonomy of community residents by types of "Kayoi-no-ba": findings from a survey of urban voluntary groups in which older adults participate].

Objectives　A recent long-term care insurance policy encourages the promotion of various types of "Kayoi-no-ba," or places to go, not limited to those focusing on functional training, as resident activities are expected to keep older adults from needing care. In this study, Kayoi-no-ba were voluntary groups in which older adults participated, and their attributes were examined in terms of "diversity of participants" and "autonomy of community residents," based on the type of group.Methods　The staff from the preventing long-term care division in each of the 38 municipalities in Tokyo selected 175 voluntary groups that met three criteria: (1) more than three community residents participate in activities at least once a month, (2) participants are primarily older adults or multi-generational residents including older adults, and (3) community residents participate in group management. Representatives from 165 groups responded to the questionnaire. For the group categorization, a latent class analysis was performed using the goals and activities of the group. Participants' age, gender, and health status were used to assess diversity. The number of residents who managed and/or supported group activities and the precise role the residents played in the activities were used to assess autonomy.Results　The groups were categorized into four types: "Physical Exercises," where the primary activity was physical exercise; "Multi-purpose," which included various purposes and activities; "Social Interaction Oriented," where interaction with others was the goal, but physical exercise was not; and "Non-Interaction," where social interaction was not the goal. Participants in the Multi-purpose group ranged in age and were more likely to have health problems, such as mobility limitations, dementia, and frailty, than those in the Physical Exercises and Social Interaction Oriented groups. Moreover, the Multi-purpose group had more resident managers and supporters involved in various roles.Conclusion　The Multi-purpose group had the most diverse participants and autonomy of residents. However, the type of Kayoi-no-ba should not be considered fixed. The support system should encourage flexible changes such as adding a new activity depending on the situation and the varying needs of the residents.

Force measurements show that uL4 and uL24 mechanically stabilize a fragment of 23S rRNA essential for ribosome assembly.

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.

[Effective support based on length of participation for community-based activities led by older residents].

Objectives　Community-based activities led by older residents are important in preventing long-term care and enhancing social participation among older persons in Japan. Local governments and community nurses are required to support these activities. However, there is no knowledge about the issues of residents who participate in community-based activities, which is essential for the effective support of community-based activities. We aimed to examine the association between the length of participation and recognized issues among community-based activities.Methods　Through the local governments in Tokyo, we recruited participants from community-based activity groups; 2,367 people from 155 activity groups from 40 municipalities responded. The presence or absence of 10 items of recognized issues, such as lack of management members and lack of teachers, were examined. The number of years of participation in the activity was divided into four groups: "less than 1 year," "more than 1 year and less than 2 years," "more than 2 years and less than 4 years," and "more than 4 years." Logistic regression analysis was conducted to examine the association between the length of participation and recognized issues.Results　The number of analyzed respondents was 2,194 (14.5% were male and the average age was 76.9 years). Compared to "less than 1 year" group, the "more than 2 years and less than 4 year" group recognized "aging of group" (OR=1.92), "lack of management members" (OR=1.61), and "health condition of participants" (OR=1.47) as issues. In the "more than 4 years" group, "aging of group" (OR=3.24), "lack of management members" (OR=2.63), "lack of participants" (OR=2.12), "health condition of participants" (OR=1.95), "mannerism of activities" (OR=1.62), and "lack of place" (OR=1.48) were recognized as issues.Conclusion　Issues recognized by participants in community-based activities differed depending on the number of years they had been participating in the activity. This suggests that it is necessary to provide appropriate support taking the length of participation into account. For example, the issue of the "health condition of participants" was recognized by participants who had been participating in the activity for approximately two years. Consequently, "lack of participants" may have occurred in participants who have been participating in the activity for four years. Therefore, promoting the health management of participants from the early phase of community-based activity would be effective in preventing dropout.

The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins.

Bacterial membrane proteins are integrated into membranes through the concerted activities of a series of integration factors, including membrane protein integrase (MPIase). However, how MPIase activity is complemented by other integration factors during membrane protein integration is incompletely understood. Here, using inverted inner-membrane vesicle and reconstituted (proteo)liposome preparations from Escherichia coli cells, along with membrane protein integration assays and the PURE system to produce membrane proteins, we found that anti-MPIase IgG inhibits the integration of both the Sec-independent substrate 3L-Pf3 coat and the Sec-dependent substrate MtlA into E. coli membrane vesicles. MPIase-depleted membrane vesicles lacked both 3L-Pf3 coat and MtlA integration, indicating that MPIase is involved in the integration of both proteins. We developed a reconstitution system in which disordered spontaneous integration was precluded, which revealed that SecYEG, YidC, or both, are not sufficient for Sec-dependent and -independent integration. Although YidC had no effect on MPIase-dependent integration of Sec-independent substrates in the conventional assay system, YidC significantly accelerated the integration when the substrate amounts were increased in our PURE system-based assay. Similar acceleration by YidC was observed for MtlA integration. YidC mutants with amino acid substitutions in the hydrophilic cavity inside the membrane were defective in the acceleration of the Sec-independent integration. Of note, MPIase was up-regulated upon YidC depletion. These results indicate that YidC accelerates the MPIase-dependent integration of membrane proteins, suggesting that MPIase and YidC function sequentially and cooperatively during the catalytic cycle of membrane protein integration.

Reconstitution of 30S ribosomal subunits in vitro using ribosome biogenesis factors.

Reconstitution of ribosomes in vitro from individual ribosomal proteins provides a powerful tool for understanding the ribosome assembly process including the sequential incorporation of ribosomal proteins. However, conventional assembly methods require high-salt conditions for efficient ribosome assembly. In this study, we reconstituted 30S ribosomal subunits from individually purified ribosomal proteins in the presence of ribosome biogenesis factors. In this system, two GTPases (Era and YjeQ) facilitated assembly of a 30S subunit exhibiting poly(U)-directed polyphenylalanine synthesis and native protein synthesis under physiological conditions. This in vitro system permits a study of the assembly process and function of ribosome biogenesis factors, and it will facilitate the generation of ribosomes from DNA without using cells.

Freshwater mussels (Bivalvia: Unionidae) from the rising sun (Far East Asia): phylogeny, systematics, and distribution.

Freshwater mussels (Bivalvia: Unionidae) is a diverse family with around 700 species being widespread in the Northern Hemisphere and Africa. These animals fulfill key ecological functions and provide important services to humans. Unfortunately, populations have declined dramatically over the last century, rendering Unionidae one of the world's most imperiled taxonomic groups. In Far East Asia (comprising Japan, Korea, and Eastern Russia), conservation actions have been hindered by a lack of basic information on the number, identity, distribution and phylogenetic relationships of species. Available knowledge is restricted to studies on national and sub-national levels. The present study aims to resolve the diversity, biogeography and evolutionary relationships of the Far East Asian Unionidae in a globally comprehensive phylogenetic and systematic context. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens from across Japan, South Korea, and Russia, based on molecular (including molecular species delineation and a COI + 28S phylogeny) and comparative morphological analyses. Biogeographical patterns were then assessed based on available species distribution data from the authors and previous reference works. We revealed that Unionidae species richness in Far East Asia is 30% higher than previously assumed, counting 43 species (41 native + 2 alien) within two Unionidae subfamilies, the Unioninae (32 + 1) and Gonideinae (9 + 1). Four of these species are new to science, i.e. Beringiana gosannensissp. nov., Beringiana fukuharaisp. nov., Buldowskia kamiyaisp. nov., and Koreosolenaia sitgyensisgen. & sp. nov. We also propose a replacement name for Nodularia sinulata, i.e. Nodularia breviconchanom. nov. and describe a new tribe (Middendorffinaiini tribe nov.) within the Unioninae subfamily. Biogeographical patterns indicate that this fauna is related to that from China south to Vietnam until the Mekong River basin. The Japanese islands of Honshu, Shikoku, Kyushu, Hokkaido, and the Korean Peninsula were identified as areas of particularly high conservation value, owing to high rates of endemism, diversity and habitat loss. The genetically unique species within the genera Amuranodonta, Obovalis, Koreosolenaiagen. nov., and Middendorffinaia are of high conservation concern.

CT-guided mapping in the removal of an impalpable, radiopaque foreign body in subcutaneous tissue: a case report.

Small fragments sometimes penetrate the soft tissue, and their depth and location can be difficult to determine accurately. This case study describes localisation of a thin, short wire that had penetrated the soft tissue of a 24-year-old man's back, using computed tomography (CT) with a reference grid made with an angiographic catheter. The axial non-contrast-enhanced CT scan with the grid placed over the affected body part surveyed by the CT scout view showed that the foreign body was buried in fascia 7cm from a puncture wound. Surgical removal of the foreign body was then performed without any sequelae. CT with the use of external references that do not cause metal artefacts is a simple and helpful procedure in localising radiopaque foreign bodies.