Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome

INTRODUCTION Although much effort has been devoted to studying yeast in the past few decades, our understanding of this model organism is still limited. Rapidly developing DNA synthesis techniques have made a “build-to-understand” approach feasible to reengineer on the genome scale. Here, we report on the completion of a 770-kilobase synthetic yeast chromosome II (synII). SynII was characterized using extensive Trans-Omics tests. Despite considerable sequence alterations, synII is virtually indistinguishable from wild type. However, an up-regulation of translational machinery was observed and can be reversed by restoring the transfer RNA (tRNA) gene copy number. RATIONALE Following the “design-build-test-debug” working loop, synII was successfully designed and constructed in vivo. Extensive Trans-Omics tests were conducted, including phenomics, transcriptomics, proteomics, metabolomics, chromosome segregation, and replication analyses. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium. RESULTS To efficiently construct megabase-long chromosomes, we developed an I- Sce I–mediated strategy, which enables parallel integration of synthetic chromosome arms and reduced the overall integration time by 50% for synII. An I- Sce I site is introduced for generating a double-strand break to promote targeted homologous recombination during mitotic growth. Despite hundreds of modifications introduced, there are still regions sharing substantial sequence similarity that might lead to undesirable meiotic recombinations when intercrossing the two semisynthetic chromosome arm strains. Induction of the I- Sce I–mediated double-strand break is otherwise lethal and thus introduced a strong selective pressure for targeted homologous recombination. Since our strategy is designed to generate a markerless synII and leave the URA3 marker on the wild-type chromosome, we observed a tenfold increase in URA3 -deficient colonies upon I- Sce I induction, meaning that our strategy can greatly bias the crossover events toward the designated regions. By incorporating comprehensive phenotyping approaches at multiple levels, we demonstrated that synII was capable of powering the growth of yeast indistinguishably from wild-type cells (see the figure), showing highly consistent biological processes comparable to the native strain. Meanwhile, we also noticed modest but potentially significant up-regulation of the translational machinery. The main alteration underlying this change in expression is the deletion of 13 tRNA genes. A growth defect was observed in one very specific condition—high temperature (37°C) in medium with glycerol as a carbon source—where colony size was reduced significantly. We targeted and debugged this defect by two distinct approaches. The first approach involved phenotype screening of all intermediate strains followed by a complementation assay with wild-type sequences in the synthetic strain. By doing so, we identified a modification resulting from PCRTag recoding in TSC10 , which is involved in regulation of the yeast high-osmolarity glycerol (HOG) response pathway. After replacement with wild-type TSC10 , the defect was greatly mitigated. The other approach, debugging by SCRaMbLE, showed rearrangements in regions containing HOG regulation genes. Both approaches indicated that the defect is related to HOG response dysregulation. Thus, the phenotypic defect can be pinpointed and debugged through multiple alternative routes in the complex cellular interactome network. CONCLUSION We have demonstrated that synII segregates, replicates, and functions in a highly similar fashion compared with its wild-type counterpart. Furthermore, we believe that the iterative “design-build-test-debug” cycle methodology, established here, will facilitate progression of the Sc2.0 project in the face of the increasing synthetic genome complexity. SynII characterization. ( A ) Cell cycle comparison between synII and BY4741 revealed by the percentage of cells with separated CEN2-GFP dots, metaphase spindles, and anaphase spindles. ( B ) Replication profiling of synII (red) and BY4741 (black) expressed as relative copy number by deep sequencing. ( C ) RNA sequencing analysis revealed that the significant up-regulation of translational machinery in synII is induced by the deletion of tRNA genes in synII. </jats:sec

Finite-Time Prescribed Performance Control for Spacecraft Attitude Tracking

The problem of finite-time prescribed performance control (PPC) for spacecraft attitude maneuvering is researched. To the best of the authors' knowledge, limited results have been reported. How to achieve the prescribed performance attitude tracking within a preset time interval is still an open problem, especially in the presence of inertia perturbations, external disturbances, actuator saturations, and faults. On account of this, a finite-time performance function is first constructed as the predefined boundary of tracking errors. Second, Chebyshev neural network is utilized to approximate the lumped uncertainties. Then, the Nussbaum gain technique compensating for actuator saturations and faults is incorporated into the backstepping design to develop a new fault-tolerant attitude controller. Both transient and steady-state performances (e.g., the maximum overshoot, steady-state error, and settling time) are guaranteed. Compared with the common PPC or finite-time control achievements on spacecraft attitude tracking, the setting time herein can be set in advance without relying on initial states. Finally, experiments are performed to verify the effectiveness of the solution and comparisons with related works are displayed.National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [61773108]; Natural Sciences and Engineering Research Council of CanadaNatural Sciences and Engineering Research Council of Canada (NSERC)CGIARThis work was supported in part by the National Natural Science Foundation of China under Grant 61773108, and in part by the Natural Sciences and Engineering Research Council of Canada

Finite-time adaptive fault-tolerant control for rigid spacecraft attitude tracking

Liu, Xiaoping/0000-0003-1808-4233; Jing, Yuan Wei/0000-0002-3347-2448This paper provides a new solution for the finite-time attitude maneuvers of rigid spacecraft. Uncertainties involving unknown inertial parameters, external disturbances and actuator failures are taken into account. With an effort to achieve attitude tracking despite the impact of uncertainties, a non-singular terminal sliding mode (NTSM) manifold consisting of attitude errors and angular velocity errors is first constructed. After that, a simple but efficient adaptive updating law is derived to estimate the upper bound of the lumped unknown function in the derivative of sliding surface. Combining NTSM technology and pure adaptive control, a chattering-free fault-tolerant controller is presented. The premise assumptions on uncertainties in most of the existing achievements are eliminated, which makes the controller less constrained and more practical. The rigorous proof of finite-time stability is provided and the convergent regions of tracking errors are explicitly expressed. Finally, numerical simulation is conducted to verify the effectiveness of the proposed control scheme and the comparison experiments with relevant literature demonstrate the satisfactory performances.NSERC of CanadaNatural Sciences and Engineering Research Council of Canada (NSERC); National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [61773108]NSERC of Canada; National Natural Science Foundation of China, Grant/Award Number: 6177310

A novel finite-time prescribed performance control scheme for spacecraft attitude tracking

This paper studies the issue of finite-time prescribed performance control for spacecraft attitude tracking with inertia perturbations, external disturbances, actuator saturations and faults. To the best of the authors' knowledge, limited results have been reported in the relevant literature, and how to achieve the prescribed performance attitude tracking within a preset time interval is still an open problem. A novel finite-time prescribed performance function (FTPPF) whose settling time can be arbitrarily preset is first proposed. With the FTPPF predefining the envelope of tracking-error trajectories, the original spacecraft model is transformed into a new error system. Based on the barrier Lyapunov function of converted errors, the attitude controller is derived via backstepping design. During the process, the fuzzy approximation is used to handle inertia perturbations and external disturbances, while the Nussbaum gain technique is adopted to compensate for the efficiency loss caused by actuator saturations and faults. Finally, a finite-time fault-tolerant control scheme that guarantees both transient and steady-state performances (e.g., the maximum overshoot, steady-state error, and settling time) is developed. To verify the effectiveness of the solution, numerical experiments involving comparisons with related achievements are performed. (C) 2021 Elsevier Masson SAS. All rights reserved.National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [61773108]; Natural Sciences and Engineering Research Council of CanadaNatural Sciences and Engineering Research Council of Canada (NSERC)CGIARThis work is supported by National Natural Science Foundation of China under Grant 61773108 and Natural Sciences and Engineering Research Council of Canada

Adaptive fuzzy fault-tolerant control for the attitude tracking of spacecraft within finite time

The problem of finite-time attitude-tracking control (ATC) for a rigid spacecraft subject to inertial uncertainties, external disturbances, actuator saturations and faults is addressed. First, a fast nonsingular terminal sliding mode (FNTSM) manifold is constructed to improve robustness. Second, the fuzzy logic system (FLS) is integrated into the manifold derivative to deal with the lumped unknown function. The specific assumptions about uncertainties in most of the existing achievements are no longer needed. Combining the FNTSM and fuzzy approximation techniques, an enhanced fault-tolerant control scheme is developed. Compared to almost all finite-time ATC results based on FLS or neural network, a new Proof line is proposed to prove that the approximation errors are finite-time stable instead of asymptotically stable. Therefore, the attitude controller presented herein guarantees the real finite-time stability in a complete sense. Finally, numerical experiments are conducted to verify the effectiveness of the solution, and comparisons with related works are displayed.National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [61773108]This work is supported by National Natural Science Foundation of China under Grant 61773108

Chebyshev neural network-based attitude-tracking control for rigid spacecraft with finite-time convergence

Liu, Xiaoping/0000-0003-1808-4233In this paper, the problem of finite-time attitude-tracking control for a rigid spacecraft is addressed. Uncertainties including unknown inertial parameters, external disturbances, actuator failures and saturation constraints are considered. Firstly, a smooth function which is different from the common saturation treatment is presented to deal with the actuator constraints. Secondly, a fast non-singular terminal sliding mode (FNTSM) manifold composed of tracking errors is constructed. To estimate the unknown function in the sliding surface derivative, Chebyshev neural network (CNN) is introduced and thus the strict assumptions on uncertainties in many related works are abolished. By designing the CNN adaptive laws, a new fault-tolerant control scheme is proposed such that the attitude tracking can be achieved within a limited time interval. Compared with the existing CNN-based achievements with finite-time convergence, the approximation errors are proved to be finite-time stable instead of uniformly ultimately bounded (UUB). Finally, simulation experiments are conducted to demonstrate the satisfactory tracking performance of the attitude controller.National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [61773108]; NSERC of CanadaNatural Sciences and Engineering Research Council of Canada (NSERC)This work was supported by the National Natural Science Foundation of China: [Grant Number 61773108]; the NSERC of Canada: [Grant Number Xiaoping Liu]

Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity

Lysosomes are active sites to integrate cellular metabolism and signal transduction. A collection of proteins associated with the lysosome mediate these metabolic and signaling functions. Both lysosomal metabolism and lysosomal signaling have been linked to longevity regulation; however, how lysosomes adjust their protein composition to accommodate this regulation remains unclear. Using deep proteomic profiling, we systemically profiled lysosome-associated proteins linked with four different longevity mechanisms. We discovered the lysosomal recruitment of AMP-activated protein kinase and nucleoporin proteins and their requirements for longevity in response to increased lysosomal lipolysis. Through comparative proteomic analyses of lysosomes from different tissues and labeled with different markers, we further elucidated lysosomal heterogeneity across tissues as well as the increased enrichment of the Ragulator complex on Cystinosin-positive lysosomes. Together, this work uncovers lysosomal proteome heterogeneity across multiple scales and provides resources for understanding the contribution of lysosomal protein dynamics to signal transduction, organelle crosstalk, and organism longevity