Multistep Assembly Research Articles

Multistep and multivectorial assembly line library synthesis in flow

Multistep and multivectorial assembly line library synthesis in flow

An unsupervised embedding method based on streaming videos for process monitoring in repetitive production systems

Advanced production systems, such as multi-step assembly processes, predominantly comprise of repetitive operations. The repetitive manual or human–robot integrated production operations call for new real-time process management technologies such as the increasing use of sensors and the development of system intelligence. Conventional process monitoring and management methods, which are often labor-intensive, fall short in providing immediate and actionable insights. To tackle this limitation, we develop an unsupervised embedding method to automatically delineate the process into different stages and predict real-time progress information. We propose a Contrastive Variational Autoencoder as a feature extractor to adeptly embed repetitive processes into a Gaussian Mixture Model. Based on the extracted features, we propose an adaptive change-point detection and an Iterative Dynamic Time Wrapping algorithm to identify and segment multiple standardized process stages automatically. Theoretically, we establish the asymptotic optimality of the detected change-points associated with the given precision of image and feature extractors, ensuring the high-quality process stage separation and labeling. The proposed method autonomously extracts essential features encapsulating progress information from a limited set of unlabelled process videos. Through four diverse case studies including production of an actual aircraft spoiler, our method exhibits very promising performance. Specifically, it achieves an average of 98.14% accuracy in predicting production progress and 0.9202 area under the curve in predicting progress deviation across three distinct production environments. The proposed process monitoring method in repetitive production systems has the potential to significantly improve productivity, promote standardization of repetitive operations, and predict production deviations.

Formation of Linear Plasmonic Heterotrimers Using Nanoparticle Docking to DNA Origami Cages.

The fabrication of complex assemblies with interesting collective properties from plasmonic nanoparticles (NPs) is often challenging. While DNA-directed self-assembly has emerged as one of the most promising approaches to forming such complex assemblies, the resulting structures tend to have large variability in gap sizes and shapes, as the DNA strands used to organize these particles are flexible, and the polydispersity of the NPs leads to variability in these critical structural features. Here, we use a new strategy termed docking to DNA origami cages (D-DOC) to organize spherical NPs into a linear heterotrimer with a precisely defined geometrical arrangement. Instead of binding NPs to the exterior of the DNA templates, D-DOC binds the NPs to either the interior or the opening of a 3D cage, which significantly reduces the variability of critical structural features by incorporating multiple diametrically arranged capture strands to tether NPs. Additionally, such a spatial arrangement of the capture strand can work synergistically with shape complementarity to achieve tighter confinement. To assemble NPs via D-DOC, we developed a multistep assembly process that first encapsulates an NP inside a cage and then binds two other NPs to the openings. Microscopic characterization shows low variability in the bond angles and gap sizes. Both UV-vis absorption and surface-enhanced Raman scattering (SERS) measurements showed strong plasmonic coupling that aligned with predictions by electrodynamic simulations, further confirming the precision of the assembly. These results suggest D-DOC could open new opportunities in biomolecular sensing, SERS and fluorescence spectroscopies, and energy harvesting through the self-assembly of NPs into more complex 3D assemblies.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome

Dedicated assembly factors orchestrate the stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here we report cryo-electron microscopy reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, as well as how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors and reveals conceptual principles underlying human proteasome biogenesis, thus providing an explanation for many previous biochemical and genetic observations.

A genome sequence for the threatened whitebark pine.

Whitebark pine (WBP, Pinus albicaulis) is a white pine of subalpine regions in the Western contiguous United States and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola) and additional threats from mountain pine beetle (Dendroctonus ponderosae), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short reads of haploid megagametophyte tissue and Oxford Nanopore long reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gb of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gb). Approximately 87.2% (24.0 Gb) of total sequence was placed on the 12 WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the 3 subclasses of NLRs. Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo-assembled transcriptomes.

Rigid-flexible coupled origami robots via multimaterial 3D printing

Soft robots have significant advantages in flexibility and adaptability and have potential applications in the field of engineering. Unlike traditional manufacturing methods, three-dimensional (3D) printing provides a fast way to fabricate customized and multi-functional robots. However, the fabrication of soft robots requires multimaterial printers and the high-accuracy multi-step assembly process. Among them, fused deposition modeling (FDM) technology has taken the lead compared to other 3D printing methods due to its ease of use, accuracy, and repeatability. However, the FDM multimaterial printing has not been thoroughly explored. Here, we proposed a rigid and flexible material integrated printing approach based on FDM 3D printing technology and reported a cable-driven flexible pipe robot based on Yoshimura origami crease patterns. The implementations show that the robot can realize four-direction bending effectively by the corresponding drive control, which indicates the feasibility of our design and manufacturing method. The proposed approach paves an effective way to design and fabricate the rigid-flexible robot and other devices in the future.

Investigating Iron-Sulfur Proteins in Infectious Diseases: A Review of Characterization Techniques

Iron-sulfur [Fe-S] clusters, comprising coordinated iron and sulfur atoms arranged in diverse configurations, play a pivotal role in redox reactions and various biological processes. Diverse structural variants of [Fe-S] clusters exist, each possessing distinct attributes and functions. Recent discovery of [Fe-S] clusters in infectious pathogens, such as Mycobacterium tuberculosis, and in viruses, such as rotavirus, polyomavirus, hepatitis virus, mimivirus, and coronavirus, have sparked interest in them being a potential therapeutics target. Recent findings have associated these [Fe-S] cluster proteins playing a critical role in structural and host protein activity. However, for a very long time, metalloenzymes containing iron-sulfur clusters have been prone to destabilization in the presence of oxygen, which led to a delayed understanding of [Fe-S] proteins compared to other non-heme iron-containing proteins. Consequently, working with [Fe-S] proteins require specialized equipment, such as anaerobic chambers to maintain cofactor integrity, and tools like ultraviolet visible (UV-Vis) spectroscopy, mass spectrometry, X-ray crystallography, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), Mössbauer spectroscopy and electrochemical characterization. Many of these [Fe-S] cluster proteins have been misannotated as Zinc-binding proteins when purified aerobically. Moreover, the assembly of these iron-sulfur cluster cofactors have not been fully understood since it is a multi-step assembly process. Additionally, disruptions in this assembly process have been linked to human diseases. With rapid advancements in anaerobic gloveboxes and spectroscopic techniques, characterization of these [Fe-S] cluster-containing proteins that are essential for the pathogens can open up new avenues for diagnostics and therapeutics.

Multimode adaptive logic gates based on temperature-responsive DNA strand displacement.

Living organisms switch their intrinsic biological states to survive environmental turbulence, in which temperature changes are prevalent in nature. Most artificial temperature-responsive DNA nanosystems work as switch modules that transit between "ON-OFF" states, making it difficult to construct nanosystems with diverse functions. In this study, we present a general strategy to build multimode nanosystems based on a temperature-responsive DNA strand displacement reaction. The temperature-responsive DNA strand displacement was controlled by tuning the sequence of the substrate hairpin strands and the invading strands. The nanosystems were demonstrated as logic gates that performed a set of Boolean logical functions at specific temperatures. In addition, an adaptive logic gate was fabricated that could exhibit different logic functions when placed in different temperatures. Specifically, upon the same input strands, the logic gate worked as an XOR gate at 10 °C, an OR gate at 35 °C, an AND gate at 46 °C, and was reset at 55 °C. The design and fabrication of the multifunctional nanosystems would help construct advanced temperature-responsive systems that may be used for temperature-controlled multi-stage drug delivery and thermally-controlled multi-step assembly of nanostructures.

Nucleocytoplasmic transport at the crossroads of proteostasis, neurodegeneration and neuroprotection.

Nucleocytoplasmic transport comprises the multistep assembly, transport and disassembly of protein and RNA cargoes entering and exiting nuclear pores. Accruing evidence supports that impairments to nucleocytoplasmic transport are a hallmark of neurodegenerative diseases. These impairments cause dysregulations in nucleocytoplasmic partitioning and proteostasis of nuclear transport receptors and client substrates that promote intracellular deposits - another hallmark of neurodegeneration. Disturbances in liquid-liquid phase separation (LLPS) between dense and dilute phases of biomolecules implicated in nucleocytoplasmic transport promote micrometer-scale coacervates, leading to proteinaceous aggregates. This Review provides historical and emerging principles of LLPS at the interface of nucleocytoplasmic transport, proteostasis, aging and noxious insults, whose dysregulations promote intracellular aggregates. E3 SUMO-protein ligase Ranbp2 constitutes the cytoplasmic filaments of nuclear pores, where it acts as a molecular hub for rate-limiting steps of nucleocytoplasmic transport. A vignette is provided on the roles of Ranbp2 in nucleocytoplasmic transport and at the intersection of proteostasis in the survival of photoreceptor and motor neurons under homeostatic and pathophysiological environments. Current unmet clinical needs are highlighted, including therapeutics aiming to manipulate aggregation-dissolution models of purported neurotoxicity in neurodegeneration.

Enhancing photocatalytic cleavage of C[sbnd]C bonds in lignin model substrates by ternary nanocomposite of g-C3N4/rGO/CdS using rGO as electronic mediators

Depolymerizing lignin into aromatic monomers with high yield and selectivity via photocatalysis remains a challenging task. Herein, we introduced reduced graphene oxide (rGO) nanosheets as solid electronic mediators and successfully synthesized g-C3N4/rGO/CdS ternary nanocomposites using a simple multi-step assembly strategy. The photocatalysts can efficiently break the CC bond of lignin under simulated sunlight conditions. The characterization results demonstrate that the loading of CdS effectively enhances the light absorption range and accelerates the transfer of photogenerated charge carriers. Simultaneously, the introduction of rGO inhibits the recombination of electron-hole pairs. When using the β-O-4 model substrate, a 95% conversion rate could be achieved with yields of 71% benzaldehyde and 69% phenyl formate, which are 3.09 times and 3.63 times higher than that of pure g-C3N4, respectively. Our research highlights the importance of energy band structure engineering and electronic mediator design in developing efficient photocatalysts for breaking CC bonds of lignin.

Multifunctional composite foam with high strength and sound‐absorbing based on step assembly strategy for high performance electromagnetic shielding

AbstractSoft polyurethane (PU) materials exhibit excellent structural stability, sound absorption and heat insulation, and they can be used to prepare multifunctional conductive composites with electromagnetic interference (EMI) shielding function. On the basis of a multistep assembly strategy, our study constructed composite foams with sound absorption and EMI shielding properties by designing different functional layers and expanded the application field of PU foam. First, carbon black particles are compounded with PU foam to improve the mechanical properties and conductivity of pure foam. Then, an aluminum layer and a knitted fabric layer were introduced to construct composite foam samples with a sandwich structure, endowing the materials with excellent EMI shielding performance. The resultant composite foam material exhibits effective EMI shielding performance and good sound absorption performance, which are 58.33 dB and >0.3, respectively. Moreover, the composite foam material demonstrates high bursting strength and compression resistance, suggesting that it can be used under different conditions. The employment of the multistep assembly strategy provides PU foam with a flexible range of applications, offering an innovative approach for preparing multifunctional conductive composite materials.

Orthogonal Supramolecular Assemblies Using Side-Chain Functionalized Helical Poly(isocyanide)s.

Mimicking the structure of proteins using synthetic polymers requires building blocks with structural similarity and the use of various noncovalent and dynamic covalent interactions. We report the synthesis of helical poly(isocyanide)s bearing diaminopyridine and pyridine side-chains and the multistep functionalization of the polymers' side-chains using hydrogen bonding and metal coordination. The orthogonality of the hydrogen bonding and metal coordination was proved by varying the sequence of the multistep assembly. The two side-chain functionalizations are reversible through the use of competitive solvents and/or competing ligands. Throughout the assembly and disassembly, the helical conformation of the polymer backbone is sustained as proved by circular dichroism spectroscopy. These results open the possibility to incorporate helical domains into complex polymer architectures and create a helical scaffold for smart materials.

Industry 4.0-Based Framework for Real-Time Prediction of Output Power of Multi-Emitter Laser Modules during the Assembly Process

The challenges of defects in manufacturing and assembly processes in optoelectronic industry continue to persist. Defective products cause increased time to completion (cycle time), energy consumption, cost, and loss of precious material. A complex laser assembly process is studied with the aim of minimising the generation of defective laser modules. Subsequently, relevant data were gathered to investigate machine learning and artificial intelligence methods to predict the output beam power of the module during the assembly process. The assembly process was divided into a number of chain steps, where we implemented a bespoke framework of hybrid feature selection method alongside artificial neural networks (ANNs) to formulate the statistical inferences. A review of existing learning methods in manufacturing and assembly processes enabled us to select XGBoost and random forest regression (RFR) as the two methods to be compared with ANN, based on their capabilities; ANN outperformed both of them, as it avoided overfitting and scored similar test metrics in the majority of the assembly steps. The results of the proposed solution have been validated in a real production dataset, even showing good predictive capability in the early steps of the assembly process where the available information is limited. Furthermore, the transferability of the framework was validated by applying the proposed framework to another product that follows a similar assembly process. The results indicated that the proposed framework has the potential to serve as the foundation for further research on laser modules’ sophisticated and multi-step assembly lines.

Linking molecular-level RNA-protein interactions with mesoscale properties of the nucleolus.

The nucleolus is the most prominent nuclear body, and represents a condensate comprising the machinery of ribosome biogenesis, including multi-step transcription, processing, and assembly of ribosomal RNA (rRNA). These intricate steps are organized in well-characterized nucleolar substructures, with a host of RNA and proteins components that have been described in detail. However, the role of specific molecular-level interactions, such as RNA-RNA and RNA-protein, in governing nucleolar morphology and its material properties is unknown. Towards addressing this problem, we have deployed a suite of chemical biology, quantitative cellular microscopy and in vitro biophysical methods. Specifically, we use chemical perturbations to selectively and transiently inhibit distinct steps of ribosome biogenesis in cells. We then visualize and classify changes in nucleolar morphology as well as profile thermodynamics of nucleolar RNA-binding proteins (RBPs) upon treating cells with these inhibitors. Preliminary results indicate that the RBP thermodynamic profiles and morphologies are dependent on the presence or absence of specific RNA and/or RBP species in the nucleolus, suggesting that select RBP-RNA interactions may differentially contribute to nucleolar structure and function. To test this hypothesis, we are investigating material properties, dynamics, and RNA structural landscapes of in vitro nucleolar condensates with varying RNA and RBP compositions. Given that aberrant ribosome production is a hallmark of cancer and other diseases, our findings on the molecular thermodynamics of the nucleolus will pave the way towards rational therapeutic targeting.

Multistep sequence-controlled supramolecular polymerization by the combination of multiple self-assembly motifs

The precise sequence control of polymer chain is an important research topic of polymer chemistry. Although some methods such as iterative synthesis and supramolecular polymerization have been developed to fabricate sequence-controllable polymer, it is still a great challenge to consecutively prepare multiple supramolecular polymers with different sequence structures. In this work, through the reasonable utilization of assembly motifs, we integrated multiple host-guest recognitions and metal coordination interactions to prepare different sequence-controlled supramolecular polymers by a multistep assembly strategy. This research provides inspiration for the design and preparation of supramolecular polymers with different sequence structures.

From Glass to Gold: Visualization of Electrostatic Self-Assembly of Gold Nanoparticles

A multistep assembly of gold nanoparticle self-assembled monolayers (SAMs) on glass surfaces is proposed, as a third-year undergraduate experiment. The process includes self-assembly (SA) and electrostatic self-assembly (ESA) methods, which are central approaches in modern surface and materials engineering. The products are analyzed using spectrophotometry and contact angle measurements. The experiment can be completed in a standard 4 h teaching laboratory session.

Solutal-buoyancy-driven intertwining and rotation of patterned elastic sheets.

The intertwining of strands into 3D spirals is ubiquitous in biology, enabling functions from information storage to maintenance of cell structure and directed locomotion. In synthetic systems, entwined fibers can provide superior mechanical properties and act as artificial muscle or structural reinforcements. Unlike structures in nature, the entwinement of synthetic materials typically requires application of an external stimulus, such as mechanical actuation, light, or a magnetic field. Herein, we use computational modeling to design microscale sheets that mimic biology by transducing chemical energy into mechanical action, and thereby self-organize and interlink into 3D spirals, which spontaneously rotate. These flexible sheets are immersed in a fluid-filled microchamber that encompasses an immobilized patch of catalysts on the bottom wall. The sheets themselves can be passive or active (coated with catalyst). Catalytic reactions in the solution generate products that occupy different volumes than the reactants. The resulting density variations exert a force on the fluid (solutal buoyancy force) that causes motion, which in turn drives the interlinking and collective swirling of the sheets. The individual sheets do not rotate; rotation only occurs when the sheets are interlinked. This level of autonomous, coordinated 3D structural organization, intertwining, and rotation is unexpected in synthetic materials systems operating without external controls. Using physical arguments, we identify dimensionless ratios that are useful in scaling these ideas to other systems. These findings are valuable for creating materials that act as "machines", and directing soft matter to undergo self-sustained, multistep assembly that is governed by intrinsic chemical reactions.

Multi-step tandem functionalization assembly of MOFs-based hybrid polymeric films for color tuning luminescence and responsive sensing on organic vapors

Metal-organic frameworks (MOFs) are a promising class of porous crystalline materials which can be functionalized by versatile postsynthetic modification (PSM) for further practical applications. Whereas, such crystalline nature of MOFs stands as a serious obstacle to their desired shaping and manufacture for further practical applications. Herein, a multi-step tandem functionalization assembly strategy is employed to fabricate a luminescent MOF-based hybrid polymeric film to endow the luminescent MOFs with processability and applicability. In detail, Eu3+ ions are introduced into ZnMOF-5-NH2 frameworks to create a red emitting-center firstly. Next, methacrylic anhydride is incorporated to react with amino functional groups of Eu-ZnMOF-5-NH2 to obtain Eu-MOF-L with unsaturated bonds by a covalent PSM strategy. Finally, the copolymerization reaction took place between Eu-MOF-L and monomers (butyl methacrylate) which initiated by benzoyl peroxide. The polymeric hybrid film (Eu-MOF-L@PBMA film) is soft, flexible, and homogeneous. The physical characterization and the luminescence properties of these hybrid materials were studied in details. The multi-color luminescence of these hybrids and their polymeric hybrid films are tuned by optimizing the critical parameters to serve as a white-light emitting device. Furthermore, Eu-MOF-L@PBMA film is utilized for the detection of volatile organic vapors. In terms of their distinct color variation, an effective dual-readout method assists in sensing volatile organic vapors, especially organic amine vapors.

Chiral emergence in multistep hierarchical assembly of achiral conjugated polymers

Intimately connected to the rule of life, chirality remains a long-time fascination in biology, chemistry, physics and materials science. Chiral structures, e.g., nucleic acid and cholesteric phase developed from chiral molecules are common in nature and synthetic soft materials. While it was recently discovered that achiral but bent-core mesogens can also form chiral helices, the assembly of chiral microstructures from achiral polymers has rarely been explored. Here, we reveal chiral emergence from achiral conjugated polymers, in which hierarchical helical structures are developed through a multistep assembly pathway. Upon increasing concentration beyond a threshold volume fraction, dispersed polymer nanofibers form lyotropic liquid crystalline (LC) mesophases with complex, chiral morphologies. Combining imaging, X-ray and spectroscopy techniques with molecular simulations, we demonstrate that this structural evolution arises from torsional polymer molecules which induce multiscale helical assembly, progressing from nano- to micron scale helical structures as the solution concentration increases. This study unveils a previously unknown complex state of matter for conjugated polymers that can pave way to a field of chiral (opto)electronics. We anticipate that hierarchical chiral helical structures can profoundly impact how conjugated polymers interact with light, transport charges, and transduce signals from biomolecular interactions and even give rise to properties unimagined before.

Investigating the role of local DNA conformational dynamics in processive DNA polymerase holoenzyme-primer-template DNA complex formation

Single-strand-double-strand (ss-ds) DNA junctions play important roles in protein recognition and binding interactions in DNA replication and repair. The functionality of ss-dsDNA junctions is derived, at least in part, from intrinsic disorder at these sites that results in non-canonical local DNA conformations and conformational dynamics. Primer/template (P/T) DNA junctions serve as sites for the multistep assembly of the DNA polymerase/sliding clamp holoenzyme that drives DNA replication and proof-reading.