Strategies for Selection from Protein Libraries Composed of de Novo Designed Secondary Structure Modules

As more and more protein structures are determined, it has become clear that there is only a limited number of protein folds in nature. To explore whether the protein folds found in nature are the only solutions to the protein folding problem, or that a lack of evolutionary pressure causes the paucity of different protein folds found, we set out to construct protein libraries without any restriction on topology. We generated different libraries (all α-helix, all β-strand and α-helix plus β-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (α-helix, β-strand and β-turn) in various proportions, based primarily on the patterning of polar and non-polar residues. From the analysis of proteins chosen randomly from the libraries, we found that a substantial portion of pure α-helical proteins show properties similar to native proteins.Using these libraries as a starting point, we aim to establish a selection system which allows us to enrich proteins with favorable folding properties (non-aggregating, compactly folded) from the libraries. We have developed such a method based on ribosome display. This selection is based on two concepts: (1) misfolded proteins are more sensitive to proteolysis, (2) misfolded and/or aggregated proteins are more hydrophobic. We show that by applying each of these selection criteria proteins that are compactly folded and soluble can be enriched over insoluble and random coil protein

Reaction dynamics analysis of an E. coli protein translation system by computational modeling

A single enzymatic reaction can often be described by Michaelis-Menten kinetics, but once reactions are connected to one other, it becomes difficult to understand and capture a complete description of the reaction dynamics due to its high dimensionality. To elucidate the dynamic features of a biologically relevant large-scale reaction network, we constructed a computational model of minimal protein synthesis consisting of 241 components and 968 reactions that synthesize the Met-Gly-Gly (MGG) peptide based on an Escherichia coli-based reconstituted in vitro translation (IVT) system [1]. We performed a simulation using parameters collected primarily from the literature and found that the rate of MGG peptide synthesis becomes nearly constant in minutes, thus achieving a steady-state similar to experimental observations. In addition, concentration changes to 70% of the components, including intermediates, reached a plateau in a few minutes. However, the concentration change of each component exhibits several temporal plateaus, or a quasi-stationary state (QSS), before reaching the final plateau. To understand the complex dynamics, we focused on whether the components reached a QSS, mapped the arrangement of components in a QSS in the entire reaction network structure and investigated time-dependent changes. We found that components in a QSS form clusters that grow over time but not in a linear fashion and that this process involves the collapse and regrowth of clusters before the formation of a final large single cluster. These observations might commonly occur in other large-scale biological reaction networks. This developed analysis might be useful for understanding large-scale enzymatic reactions, thereby extracting the characteristics of the reaction network, including phase transitions. As the reconstituted IVT has been used for various applications inducing directed evolution of membrane proteins [2,3], the developed computational model might be useful in further enhancement of the yield of synthesized proteins using the reconstituted IVT. Please click Additional Files below to see the full abstract

高齢精神障害者の地域生活支援の現状と課題：ソーシャルワーカーへのインタビュー調査の結果から

本研究では、高齢精神障碍者の地域生活支援について、その現状や課題を調査すべくソーシャルワーカーへのインタビュー調査を実施したところ、多くのソーシャルワーカーが高齢精神障害者の地域生活支援を実際に経験し、支援に課題を感じていることが明らかとなった。その内容としては、「身体合併症とそれにともなう支援の難しさ」や障害者総合支援法と介護保険法の接続などを中心とした「制度の問題」、「当事者自身のモチベーションや意識の問題」などが確認された。先行研究でも指摘されるように「長期に渡る入院」の影響も含めて「本人の強い地域生活への不安」「表明されない（地域生活支援への）ニーズ」「根強い入院継続希望」が実際に当事者から語られる現状があることは明白であったが、一方で当時者が介護保険制度のサービス利用に拒否的であることが示唆されるなど、高齢精神障害者の地域生活支援における新たな現状や課題が明らかとなった。For this study, an interview survey of social workers was conducted to elucidate the reality and difficulties of community life support for elderly mentally handicapped people. Results showed that many social workers actually experienced community life support for them and felt some difficulties related to the support. The contents include “physical complications and the difficulties in support associated with them,” “system problems” mainly related to Services and Supports for Persons with Disabilities Act and Long-Term Care Insurance Act, and “difficulties of motivation or consciousness of the handicapped people themselves.” As previous reports have described, it has been readily apparent that a present state prevails in which handicapped people talk about “their intensive anxiety about community life,” “their needs (for community life support) life unsaid,” and “their deep-rooted hope to be hospitalized,” attributable to effects of “their long-term hospitalization.” Results of this study shed new light on the state and issues of community life support for elderly handicapped people, including the suggestion that they themselves are unwilling to receive the Nursing Care Insurance services

Effects of ribosomes on the kinetics of Qβ replication

AbstractBacteriophage Qβ utilizes some host cell translation factors during replication. Previously, we constructed a kinetic model that explains replication of long RNA molecules by Qβ replicase. Here, we expanded the previous kinetic model to include the effects of ribosome concentration on RNA replication. The expanded model quantitatively explained single- and double-strand formation kinetics during replication with various ribosome concentrations for two artificial long RNAs. This expanded model and the knowledge obtained in this study provide useful frameworks to understand the precise replication mechanism of Qβ replicase with ribosomes and to design amplifiable RNA genomes in translation-coupling systems

Quantifying epistatic interactions among the components constituting the protein translation system

In principle, the accumulation of knowledge regarding the molecular basis of biological systems should allow the development of large-scale kinetic models of their functions. However, the development of such models requires vast numbers of parameters, which are difficult to obtain in practice. Here, we used an in vitro translation system, consisting of 69 defined components, to quantify the epistatic interactions among changes in component concentrations through Bahadur expansion, thereby obtaining a coarse-grained model of protein synthesis activity. Analyses of the data measured using various combinations of component concentrations indicated that the contributions of larger than 2-body inter-component epistatic interactions are negligible, despite the presence of larger than 2-body physical interactions. These findings allowed the prediction of protein synthesis activity at various combinations of component concentrations from a small number of samples, the principle of which is applicable to analysis and optimization of other biological systems. Moreover, the average ratio of 2- to 1-body terms was estimated to be as small as 0.1, implying high adaptability and evolvability of the protein translation system

Plastic brain structure changes associated with the division of labour and ageing in termites

Division of labour is a prominent feature of social insect societies, where different castes engage in different specialised tasks. As brain differences are associated with behavioural differences, brain anatomy may be linked to caste polymorphism. Here, we show that termite brain morphology changes markedly with caste differentiation and age in the termite, Reticulitermes speratus. Brain morphology was shown to be associated with reproductive division of labour, with reproductive individuals (alates and neotenic reproductives) having larger brains than non-reproductives (workers and soldiers). Micro-computed tomography (CT) imaging and dissection observations showed that the king's brain morphology changed markedly with shrinkage of the optic lobes during their long life in the dark. Behavioural experiments showed that mature primary kings lose visual function as a result of optic lobe shrinkage. These results suggested that termites restructure their nervous systems to perform necessary tasks as they undergo caste differentiation, and that they also show flexible changes in brain morphology even after the final moult. This study showed that brain morphology in social insects is linked to caste and ageing, and that the evolution of the division of labour is underpinned by the development of diverse neural systems for specialised tasks. This article is protected by copyright. All rights reserved

PREDICTION SYSTEMS FOR BLADDER CANCER THERAPY

The present study established systems to predict the chemo‑sensitivity of muscle invasive bladder cancer (MIBC) for neoadjuvant chemotherapy (NAC) with methotrexate, vinblastine, doxorubicin plus cisplatin (M‑VAC) and carboplatin plus gemcitabine (CaG) by analyzing microarray data. The primary aim of the study was to investigate whether the clinical response would increase by combining these prediction systems. Treatment of each MIBC case was allocated into M‑VAC NAC, CaG NAC, surgery, or radiation therapy groups by their prediction score (PS), which was calculated using the designed chemo‑sensitivity prediction system. The therapeutic effect of the present study was compared with the results of historical controls (n=76 patients) whose treatments were not allocated using the chemo‑sensitivity prediction system. In addition, the overall survival between the predicted to be responder (positive PS) group and predicted to be non‑responder (negative PS) group was investigated in the present study. Of the 33 patients with MIBC, 25 cases were positive PS and 8 were negative PS. Among the 25 positive PS cases, 7 were allocated to receive M‑VAC NAC and 18 were allocated to receive CaG NAC according to the results of the prediction systems. Of the 8 negative PS cases, 3 received CaG NAC, 1 received surgery without NAC and 4 received radiation therapy. The total clinical response to NAC was 88.0% (22/25), which was significantly increased compared with the historical controls [56.6% (43/76) P=0.0041]. Overall survival of the positive PS group in the study was significantly increased compared with the negative PS group (P=0.027). In conclusion, the combination of the two prediction systems may increase the treatment efficacy for patients with MIBC by proposing the optimal NAC regimen. In addition, the positive PS group would have a better prognosis compared with the negative PS group. These results suggest that the two prediction systems may lead to the achievement of ‘precision medicine’

The royal food of termites shows king and queen specificity

シロアリの王と女王の特別食を世界初解明 --王と女王の繁殖と長寿を支えるロイヤルフード--. 京都大学プレスリリース. 2023-07-13.Society in eusocial insects is based on the reproductive division of labor, with a small number of reproductive individuals supported by a large number of non-reproductive individuals. Because inclusive fitness of all colony members depends on the survival and fertility of reproductive members, sterile members provide royals with special treatment. Here we show that termite kings and queens each receive special food of a different composition from workers. Sequential analysis of feeding processes demonstrated that workers exhibit discriminative trophallaxis, indicating their decision-making capacity in allocating food to the kings and queens. LC-MS/MS analyses of the stomodeal food and midgut contents revealed king- and queen-specific compounds including diacylglycerols and short-chain peptides. DESI-MSI analyses of ¹³C-labelled termites identified phosphatidylinositol and acetyl-L-carnitine in the royal food. Comparison of the digestive tract structure showed remarkable differences in the volume ratio of the midgut-to-hindgut among castes, indicating that digestive division of labor underlies reproductive division of labor. Our demonstration of king- and queen-specific food in termites provides insight into the nutritional system that underpins the extraordinary reproduction and longevity of royals in eusocial insects