FRET-Based Conformational Sensor for the m1 Muscarinic Cholinergic Receptor

Signaling behaviors of G-protein-mediated signaling are the outcome of a regulated cycle of GTP binding and hydrolysis. Binding of GTP, which activates G proteins, is promoted by an agonist-activated receptor; and hydrolysis of bound GTP, which deactivates G proteins, is accelerated by a GTPase-activating protein (GAP). These two processes need to be coordinately regulated to achieve fast turning on and off of signaling with robust signal output. I developed an optical conformational sensor for the m1 muscarinic cholinergic receptor (M1), a prototypical G protein-coupled receptor (GPCR), to study how receptor activity is regulated by the coordinated action of agonist, G protein and GAP. To create the sensor, I adopted an underlying design originally developed by the Lohse group. The sensor exploits intramolecular fluorescence resonance energy transfer, FRET, to monitor activation-associated conformational changes in intracellular loop 3 of the receptor. In the sensor, a CFP FRET donor and a labeling site for FlAsH (fluorescein-based biarsenical dye) FRET acceptor are engineered into the M1 receptor at the C terminus and loop 3, respectively. The development proceeded through several distinct optimization steps that probably reflect general considerations for developing such sensors for class A GPCRs. After optimizing the labeling conditions to approach stoichiometric derivatization by FlAsH, I found that the fluorescence response of the sensor depended on: (1) the location of the FlAsH labeling site in loop 3; (2) the length of the C-terminal region, which apparently acts as a lever arm, prior to placement of the CFP; and (3) the choice among circularly permuted CFP moieties. Finally, based on a homology-modeled structure of the M1 receptor, placement of the FlAsH site and the length of its flexible linkers were re-optimized to prevent interference with binding of the sensor to G?q. The sensor retained essentially wild-type agonist binding and signaling activity of the M1 receptor in living cells and cell membranes. Fluorescence responses of the sensor to muscarinic agonists paralleled their cellular efficacies. The sensor in living cells faithfully reported agonist-driven conformational change of the M1 receptor. Therefore, the FRET-based sensor proves to be a useful tool to investigate the mechanisms by which conformational dynamics of the M1 receptor is regulated by agonist in living cells and membranes. Effects of G?q on the conformation of the M1 receptor could not be determined because stable interaction between the M1 receptor and G?q could not be detected in cells or cell membranes either by fluorescence change or by agonist binding affinity; this is also true for wild-type receptor. Although the sensor reconstituted in phospholipid vesicles retained wild-type agonist binding and signaling function, fluorescence response to agonist was not detected in the vesicles. I demonstrated that solubilization of the sensor denatured a substantial fraction of the sensor, resulting in low fractional ligand binding activity of in vitro labeled sensor and thus artifactually low fluorescence response to agonist

Robust Tracking Control Using Fuzzy Disturbance Observer for Wheeled Mobile Robots with Skidding and Slipping

This paper proposes a robust tracking controller based on the Fuzzy Disturbance Observer (FDO) for a Wheeled Mobile Robot (WMR) with unknown skidding and slipping. The proposed method provides disturbance-free techniques for stability analysis. In our previous work [ 1 ], we proposed an extended state-observer approach to robust tracking control for wheeled mobile robots with skidding and slipping. Even though satisfying performances were shown and the proposed method was verified in [ 1 ], the derivatives of disturbance should go to zero as time passes in order to guarantee performance. This is a very critical assumption. The method proposed in this paper overcomes this problem using universal approximation with a fuzzy model. Thus, the condition that disturbance should disappear with time is not required anymore. Furthermore, the proposed method can be used more widely than that shown in the previous work. This is guaranteed by a Lyapunov-theory-based stability analysis, and performance is verified by simulation results. </jats:p

Reliable Decision from Multiple Subtasks through Threshold Optimization: Content Moderation in the Wild

Social media platforms struggle to protect users from harmful content through content moderation. These platforms have recently leveraged machine learning models to cope with the vast amount of user-generated content daily. Since moderation policies vary depending on countries and types of products, it is common to train and deploy the models per policy. However, this approach is highly inefficient, especially when the policies change, requiring dataset re-labeling and model re-training on the shifted data distribution. To alleviate this cost inefficiency, social media platforms often employ third-party content moderation services that provide prediction scores of multiple subtasks, such as predicting the existence of underage personnel, rude gestures, or weapons, instead of directly providing final moderation decisions. However, making a reliable automated moderation decision from the prediction scores of the multiple subtasks for a specific target policy has not been widely explored yet. In this study, we formulate real-world scenarios of content moderation and introduce a simple yet effective threshold optimization method that searches the optimal thresholds of the multiple subtasks to make a reliable moderation decision in a cost-effective way. Extensive experiments demonstrate that our approach shows better performance in content moderation compared to existing threshold optimization methods and heuristics.Comment: WSDM2023 (Oral Presentation

Variation block-based genomics method for crop plants

BACKGROUND: In contrast with wild species, cultivated crop genomes consist of reshuffled recombination blocks, which occurred by crossing and selection processes. Accordingly, recombination block-based genomics analysis can be an effective approach for the screening of target loci for agricultural traits. RESULTS: We propose the variation block method, which is a three-step process for recombination block detection and comparison. The first step is to detect variations by comparing the short-read DNA sequences of the cultivar to the reference genome of the target crop. Next, sequence blocks with variation patterns are examined and defined. The boundaries between the variation-containing sequence blocks are regarded as recombination sites. All the assumed recombination sites in the cultivar set are used to split the genomes, and the resulting sequence regions are termed variation blocks. Finally, the genomes are compared using the variation blocks. The variation block method identified recurring recombination blocks accurately and successfully represented block-level diversities in the publicly available genomes of 31 soybean and 23 rice accessions. The practicality of this approach was demonstrated by the identification of a putative locus determining soybean hilum color. CONCLUSIONS: We suggest that the variation block method is an efficient genomics method for the recombination block-level comparison of crop genomes. We expect that this method will facilitate the development of crop genomics by bringing genomics technologies to the field of crop breeding

Exchange between Escherichia coli polymerases II and III on a processivity clamp

Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, β. Single-molecule experiments reveal that the interactions of Pol II and Pol III with β allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a β-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork

Pattern graph tracking-based stock price prediction using big data

Stock price forecasting is the most difficult field owing to irregularities. However, because stock prices sometimes show similar patterns and are determined by a variety of factors, we propose determining similar patterns in historical stock data to achieve daily stock prices with high prediction accuracy and potential rules for selecting the main factors that significantly affect the price, while simultaneously considering all factors. This study is intended at suggesting a new complex methodology that finds the optimal historical dataset with similar patterns according to various algorithms for each stock item and provides a more accurate prediction of daily stock price. First, we use a Dynamic Time Warping algorithm to find patterns with the most similar situation adjacent to a current pattern. Second, we select the determinants most affected by the stock price using feature selection based on Stepwise Regression Analysis. Moreover, we generate an artificial neural network model with selected features as training data for predicting the best stock price. Finally, we use Jaro–Winkler distance with Symbolic Aggregate approXimation (SAX) as a prediction accuracy measure to verify the accuracy of our model

Translation, Cultural Adaptation, and Validation of a Korean Version of the Information Needs in Cardiac Rehabilitation Scale

Objective To translate and culturally adapt the Information Needs in Cardiac Rehabilitation (INCR) questionnaire into Korean and perform psychometric validation. Methods The original English version of the INCR, in which patients are asked to rate the importance of 55 topics, was translated into Korean (INCR-K) and culturally adapted. The INCR-K was tested on 101 cardiac rehabilitation (CR) participants at Kangwon National University Hospital and Seoul National University Bundang Hospital in Korea. Structural validity was assessed using principal component analysis, and Cronbach’s alpha of the areas was computed. Criterion validity was assessed by comparing information needs according to CR duration and knowledge sufficiency according to receipt of education. Half of the participants were randomly selected for 1 month of re-testing to assess their responsiveness. Results Following cognitive debriefing, the number of items was reduced to 41 and ratings were added to assess participants’ sufficient knowledge of each item. The INCR-K structure comprised eight areas, each with sufficient internal consistency (Cronbach’s alpha>0.7). Criterion validity was supported by significant differences in mean INCR-K scores based on CR duration and knowledge sufficiency ratings according to receipt of education (p<0.05). Information needs and knowledge sufficiency ratings increased after 1 month of CR, thus supporting responsiveness (p<0.05). Conclusion The INCR-K demonstrated adequate face, content, cross-cultural, structural, and criterion validities, internal consistency, and responsiveness. Information needs changed with CR, such that multiple assessments of information needs may be warranted as rehabilitation progresses to facilitate patient-centered education

Variation block-based genomics method for crop plants

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.Abstract Background In contrast with wild species, cultivated crop genomes consist of reshuffled recombination blocks, which occurred by crossing and selection processes. Accordingly, recombination block-based genomics analysis can be an effective approach for the screening of target loci for agricultural traits. Results We propose the variation block method, which is a three-step process for recombination block detection and comparison. The first step is to detect variations by comparing the short-read DNA sequences of the cultivar to the reference genome of the target crop. Next, sequence blocks with variation patterns are examined and defined. The boundaries between the variation-containing sequence blocks are regarded as recombination sites. All the assumed recombination sites in the cultivar set are used to split the genomes, and the resulting sequence regions are termed variation blocks. Finally, the genomes are compared using the variation blocks. The variation block method identified recurring recombination blocks accurately and successfully represented block-level diversities in the publicly available genomes of 31 soybean and 23 rice accessions. The practicality of this approach was demonstrated by the identification of a putative locus determining soybean hilum color. Conclusions We suggest that the variation block method is an efficient genomics method for the recombination block-level comparison of crop genomes. We expect that this method will facilitate the development of crop genomics by bringing genomics technologies to the field of crop breeding