A cell-free approach to identify binding hotspots in plant immune receptors

Plant immune receptors are often difficult to express heterologously, hindering study of direct interactions between these receptors and their targets with traditional biochemical approaches. The cell-free method ribosome display (RD) enables expression of such recalcitrant proteins by keeping each nascent polypeptide chain tethered to its ribosome, which can enhance protein folding by virtue of its size and solubility. Moreover, in contrast to an in planta readout of receptor activity such as a hypersensitive response that conflates binding and signaling, RD enables direct probing of the interaction between plant immune receptors and their targets. Here, we demonstrate the utility of this approach using tomato recognition of Trichoderma viride ethylene-inducing xylanase (EIX) as a case study. Leveraging the modular nature of the tomato LeEIX2 and LeEIX1 leucine-rich repeat (LRR) receptors, we applied an entropy-informed algorithm to maximize the information content in our receptor segmentation RD experiments to identify segments implicated in EIX binding. Unexpectedly, two distinct EIX-binding hotspots were discovered on LeEIX2 and both hotspots are shared with decoy LeEIX1, suggesting that their contrasting receptor functions are not due to differential modes of ligand binding. Given that most plant immune receptors are thought to engage targets via their LRR sequences, this approach should be of broad utility in rapidly identifying their binding hotspots.

Micro- and macroevolutionary change of colony-level traits in bryozoans

The evolution of trait variation among populations of animals is difficult to study due to the many overlapping genetic and environmental influences that control phenotypic expression. In a group of animals, bryozoans, it is possible to isolate genetic contributions to phenotypic variation, due to the modular nature of bryozoan colonies. Each bryozoan colony represents a snapshot of the phenotypes that correspond to a single genotype, which can be summarized as a phenotypic distribution. We test whether these phenotypic distributions are heritable across generations of colonies in two sister species of the bryozoan Stylopoma, grown and bred in a common garden breeding experiment. We find that components of phenotypic distributions, specifically median trait values of colony members, are heritable between generations of colonies. Furthermore, this heredity has macroevolutionary importance because it correlates with the morphological distance between these two species. Because parts of phenotypic distributions are heritable, and this heritability corresponds to evolutionary divergence between species, we infer that these distributions have the potential to evolve. The evolutionary potential of these phenotypic distributions may underpin the emergence of colony-level traits, like division of labor in colonies.

A Readily-Reconfigurable Continuous-Stirred Tank Photochemical Reactor Platform

A new modular photochemical continuous-stirred tank reactor design is described, based upon the development of light-source units that can be fitted to the previously described fReactor CSTR platform. In contrast to many tubular or plate-based designs, these units are especially well-suited to handling multiphasic mixtures, exemplified here in solid-liquid and gas-liquid photochemical reactions. The use of slurries as input feeds allows for intensification of a photochemical bromination, while the modular nature of the system facilitates simple integration of downstream reaction steps, exemplified here in a continuous synthesis of an intermediate for the drug valsartan.

Role of Ring6 in the function of the E. coli MCE protein LetB

LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In Escherichia coli the LetB tunnel is formed from a stack of seven rings (Ring1 - Ring7), in which each ring is composed of a homo-hexameric assembly of MCE domains. The primary sequence of each MCE domain of the LetB protein is substantially divergent from the others, making each MCE ring unique in nature. The role of each MCE domain and how it contributes to the function of LetB is not well understood. Here we probed the importance of each MCE ring for the function of LetB, using a combination of bacterial growth assays and cryo-EM. Surprisingly, we find that ΔRing3 and ΔRing6 mutants, in which Ring3 and Ring6 have been deleted, confer increased resistance to membrane perturbing agents. Specific mutations in the pore-lining loops of Ring6 similarly confer increased resistance. A cryo-EM structure of the ΔRing6 mutant shows that despite the absence of Ring6, which leads to a shorter assembly, the overall architecture is maintained, highlighting the modular nature of MCE proteins. Previous work has shown that Ring6 is dynamic and in its closed state, may restrict the passage of substrate through the tunnel. Our work suggests that removal of Ring6 may relieve this restriction. The deletion of Ring6 combined with mutations in the pore-lining loops leads to a model for the tunnel gating mechanism of LetB. Together, these results provide insight into the functional roles of individual MCE domains and pore-lining loops in the LetB protein.

Constraint-Objective Cooperative Coevolution for Large-scale Constrained Optimization

Large-scale optimization problems and constrained optimization problems have attracted considerable attention in the swarm and evolutionary intelligence communities and exemplify two common features of real problems, i.e., a large scale and constraint limitations. However, only a little work on solving large-scale continuous constrained optimization problems exists. Moreover, the types of benchmarks proposed for large-scale continuous constrained optimization algorithms are not comprehensive at present. In this article, first, a constraint-objective cooperative coevolution (COCC) framework is proposed for large-scale continuous constrained optimization problems, which is based on the dual nature of the objective and constraint functions: modular and imbalanced components. The COCC framework allocates the computing resources to different components according to the impact of objective values and constraint violations. Second, a benchmark for large-scale continuous constrained optimization is presented, which takes into account the modular nature, as well as both imbalanced and overlapping characteristics of components. Finally, three different evolutionary algorithms are embedded into the COCC framework for experiments, and the experimental results show that COCC performs competitively.

Synthesis of Nitrile-Functionalized Polydentate N-Heterocycles as Building Blocks for Covalent Triazine Frameworks

Covalent triazine frameworks (CTFs) based on polydentate ligands are highly promising supports to anchor catalytic metal complexes. The modular nature of CTFs allows to tailor the composition, structure, and function to its specific application. Access to a broad range of chelating building blocks is therefore essential. In this respect, we extended the current available set of CTF building blocks with new nitrile-functionalized N-heterocyclic ligands. This paper presents the synthesis of the six ligands which vary in the extent of the aromatic system and the denticity. The new building blocks may help in a rational design of enhanced support materials in catalysis.

Development of an Innovative Inspection Tool for Superheater Tubes in Fossil Fuel Power Plants

Fossil fuel power plants are complex systems containing multiple components that require periodic health monitoring. Failures in these systems can lead to increased downtime for the plant, reduction of power, and significant cost for repairs. Inspections of the plant’s superheater tubes are typically manual, laborious, and extremely time-consuming. This is due to their small diameter size (between 1.3 and 7.6 cm) and the coiled structure of the tubing. In addition, the tubes are often stacked close to each other, limiting access for external inspection. This paper presents the development and testing of an electrically powered pipe crawler that can navigate inside 5 cm diameter tubes and provide an assessment of their health. The crawler utilizes peristaltic motion within the tubes via interconnected modules for gripping and extending. The modular nature of the system allows it to traverse through straight sections and multiple 90° and 180° bends. Additional modules in the system include an ultrasonic sensor for tube thickness measurements, as well as environmental sensors, a light detecting and ranging (LiDAR) sensor, and camera. These modules utilize a gear system that allows for 360° rotation and provides a means to inspect the entire internal circumference of the tubes.

Bifurcation drives the evolution of assembly-line biosynthesis

Reprogramming biosynthetic assembly-lines is a topic of intense interest. This is unsurprising as the scaffolds of most antibiotics in current clinical use are produced by such pathways. The modular nature of assembly-lines provides a direct relationship between the sequence of enzymatic domains and the chemical structure of the product, but rational reprogramming efforts have been met with limited success. To gain greater insight into the design process, we wanted to examine how Nature creates assembly-lines and searched for biosynthetic pathways that might represent evolutionary transitions. By examining the biosynthesis of the anti-tubercular wollamides, we show how whole gene duplication and neofunctionalization can result in pathway bifurcation. Importantly, we show that neofunctionalization occurs primarily through intragenomic recombination. This pathway bifurcation leads to redundancy, providing the genetic robustness required to enable large structural changes during the evolution of antibiotic structures. Should the new product be none-functional, gene loss can restore the original genotype. However, if the new product confers an advantage, depreciation and eventual loss of the original gene creates a new linear pathway. This provides the blind watchmaker equivalent to the design, build, test cycle of synthetic biology.

An Algorithm for the Broad Evaluation of Potential Matches between Actuator Concepts and Heavy-Duty Mobile Applications

In recent years, a variety of novel actuator concepts for the implements of heavy-duty mobile machines (HDMMs) has been proposed by industry and academia. Mostly, novel concepts aim at improving the typically low energy efficiency of state-of-the-art hydraulic valve-controlled actuators. However, besides energy-efficiency, many aspects that are crucial for a successful concept integration are often neglected in studies. Furthermore, most of the time, a specific HDMM is focused as an application while other HDMM types can show very different properties that might make a novel concept less suitable. In order to take more aspects and HDMM types into account when evaluating actuator concepts, this paper proposes a novel evaluation algorithm, which calculates so-called mismatch values for each potential actuator-application match, based on different problem aspects that can indicate a potential mismatch between a certain actuator concept and an HDMM. The lower the mismatch value, which depends on actuator characteristics as well as HDMM attributes, the more potential is the match. At the same time, the modular nature of the algorithm allows to evaluate a large number of possible matches at once, with low effort. For the performance demonstration of the algorithm, 36 potential matches formed out of six actuator concepts and six HDMM types are exemplarily evaluated. The resulting actuator concept ratings for the six different HDMMs are in line with general reasoning and confirm that the evaluation algorithm is a powerful tool to get a first, quick overview of a large solution space of actuator-HDMM matches. However, analyzing the limitations of the algorithm also shows that it cannot replace conventional requirements engineering and simulation studies if detailed and reliable results are required.

Computational based design and tracking of synthetic variants of Porcine circovirus reveal relations between silent genomic information and viral fitness

Viral genomes not only code the protein content, but also include silent, overlapping codes which are important to the regulation of the viral life cycle and affect its evolution. Due to the high density of these codes, their non-modular nature and the complex intracellular processes they encode, the ability of current approaches to decipher them is very limited. We describe the first computational-experimental pipeline for studying the effects of viral silent and non-silent information on its fitness. The pipeline was implemented to study the Porcine Circovirus type 2 (PCV2), the shortest known eukaryotic virus, and includes the following steps: (1) Based on the analyses of 2100 variants of PCV, suspected silent codes were inferred. (2) Five hundred variants of the PCV2 were designed to include various ‘smart’ silent mutations. (3) Using state of the art synthetic biology approaches, the genomes of these five hundred variants were generated. (4) Competition experiments between the variants were performed in Porcine kidney-15 (PK15) cell-lines. (5) The variant titers were analyzed based on novel next-generation sequencing (NGS) experiments. (6) The features related to the titer of the variants were inferred and their analyses enabled detection of various novel silent functional sequence and structural motifs. Furthermore, we demonstrate that 50 of the silent variants exhibit higher fitness than the wildtype in the analyzed conditions.