Assembly Steps Research Articles

Anion-Guided Hierarchical Assembly of Heterometallic Clusters.

Anions are important as templates for the construction of many structurally sophisticated and functional supramolecular architectures. Deciphering the specific role of such templates remains a great challenge due to the unique characteristics of anions. Herein, three heterometallic Ce-Ni clusters, namely, [Ce24Ni36L36(μ4-OAc)6(OH)52(Cl)8]Cl6·110H2O (Ce24Ni36, OAc = acetate ion, L = N-methyliminodiacetate), [Ce17Ni27L27(μ6-C2O4)6(OH)15(Cl)16(H2O)19](ClO4)2Cl6·45H2O (Ce17Ni27), and [Ce21Ni30L30(μ6-C2O4)2(μ4-OAc)(OH)32(Cl)15(H2O)14](ClO4)4Cl7·55H2O (Ce21Ni30), were obtained and structurally characterized; the formation of these cage-like clusters was assisted, respectively, by acetate, oxalate, and a combination of the two as templates. Structural analyses generate key information on the anion-directed hierarchical assembly, starting from a primary building block of {CeNi3} to two anion-specific secondary building blocks (SBUs)─the tetragonal {Ce4Ni8(OAc)} and the pentagonal {Ce5Ni9(C2O4)}. Further assembly steps are guided by these curved SBUs, affording a truncated octahedral Ce24Ni36 and a lantern-like Ce17Ni27, with acetate and oxalate being the sole template, respectively. The combined use of acetate and oxalate produced Ce21Ni30, a cluster with an unprecedented structure viewable as a hybrid of a truncated octahedron and a dodecahedron.

Multi-Step Assembly of an RNA-Liposome Nanoparticle Formulation Revealed by Real-Time, Single-Particle Quantitative Imaging.

Self-assembly plays a critical role in nanoparticle-based applications. However, it remains challenging to monitor the self-assembly of multi-component nanomaterials at a single-particle level, in real-time, with high throughput, and in a model-independent manner. Here, multi-color fluorescence microscopy is applied to track the assembly of both liposomes and mRNA simultaneously in clinical mRNA-based cancer immunotherapy. Imaging reveals that the assembly occurs in discrete steps: initially, RNA adsorbs onto the liposomes; then, the RNA-coated liposomes cluster into heterogeneous structures ranging from sub-micrometer to tens of micrometers. The clustering process is consistent with a Smoluchowski model with a Brownian diffusion kernel. The transition between the two steps of assembly is determined by the orientation of RNA-mediated interactions. Given the facile application of this approach and the ubiquity of the components studied, the imaging and analysis in this work are readily applied to monitor multi-component assembly of diverse nanomaterials.

An efficient assembly method for a viral genome based on T7 endonuclease Ⅰ-mediated error correction

Gene synthesis is an enabling technology that supports the development of synthetic biology. The existing approaches for de novo gene synthesis generally have tedious operation, low efficiency, high error rates, and limited product lengths, being difficult to support the huge demand of synthetic biology. The assembly and error correction are the keys in gene synthesis. This study first designed the oligonucleotide sequences by reasonably splitting the virus genome of approximately 10 kb by balancing the parameters of sequence design software ability, PCR amplification ability, and assembly enzyme assembly ability. Then, two-step PCR was performed with high-fidelity polymerase to complete the de novo synthesis of 3.0 kb DNA fragments, and error correction reactions were performed with T7 endonuclease Ⅰ for the products from different stages of PCR. Finally, the virus genome was assembled by 3.0 kb DNA fragments from de novo synthesis and error correction and then sequenced. The experimental results showed that the proposed method successfully produced the DNA fragment of about 10 kb and reduced the probability of large fragment mutations during the assembly process, with the lowest error rate reaching 0.36 errors/kb. In summary, this study developed an efficient de novo method for synthesizing a viral genome of about 10 kb with T7 endonuclease Ⅰ-mediated error correction. This method enabled the synthesis of a 10 kb viral genome in one day and the correct plasmid of the viral genome in five days. This study optimized the de novo gene synthesis process, reduced the error rate, simplified the synthesis and assembly steps, and reduced the cost of viral genome assembly.

Chemical and temporal manipulation of early steps in protein assembly tunes the structure and intermolecular interactions of protein-based materials.

The Drosophila intrinsically disordered protein Ultrabithorax (Ubx) undergoes a series of phase transitions, beginning with noncovalent interactions between apparently randomly organized monomers, and evolving over time to form increasingly ordered coacervates. This assembly process ends when specific dityrosine covalent bonds lock the monomers in place, forming macroscale materials. Inspired by this hierarchical, multistep assembly process, we analyzed the impact of protein concentration, assembly time, and subphase composition on the early, noncovalent stages of Ubx assembly, which are extremely sensitive to their environment. We discovered that in low salt buffers, we can generate a new type of Ubx material from early coacervates using 5-fold less protein, and 100-fold less assembly time. Comparison of the new materials with standard Ubx fibers also revealed differences in the extent of wrinkling on the fiber surface. A new image analysis technique based on autocorrelation of scanning electron microscopy (SEM) images was developed to quantify these structural differences. These differences extend to the molecular level: new materials form more dityrosine covalent cross-links per monomer, but without requiring the specific tyrosine residues necessary for crosslinking previously established materials. We conclude that varying the assembly conditions represents a facile and inexpensive process for creating new materials. Most new biopolymers are created by changing the composition of the monomers or the method used to drive assembly. In contrast, in this study we used the same monomers and assembly approach, but altered the assembly time and chemical environment to create a new material with unique properties.

Structures and mRNP remodeling mechanism of the TREX-2 complex.

mRNAs are packaged with proteins into messenger ribonucleoprotein complexes (mRNPs) in the nucleus. mRNP assembly and export are of fundamental importance for all eukaryotic gene expression. Before export to the cytoplasm, mRNPs undergo dynamic remodeling governed by the DEAD-box helicase DDX39B (yeast Sub2). DDX39B/Sub2 primarily functions in the nucleus and leaves the mRNP prior to export through the nuclear pore complex; however, the underlying mechanisms remain elusive. Here, we identify the conserved TREX-2 complex as the long-sought factor that facilitates DDX39B/Sub2 to complete the mRNP remodeling cycle. Our crystallographic and cryoelectron microscopy (cryo-EM) analyses demonstrate that TREX-2 modulates the activities of DDX39B/Sub2 through multiple interactions. Critically, a conserved "trigger loop" from TREX-2 splits the two RecA domains of DDX39B/Sub2 and promotes the removal of DDX39B/Sub2 from mRNP. Our findings suggest that TREX-2 coordinates with DDX39B/Sub2 and the human export receptor NXF1-NXT1 (yeast Mex67-Mtr2) to complete the final steps of nuclear mRNP assembly.

The Yeast Optogenetic Toolkit (yOTK) for Spatiotemporal Control of Gene Expression in Budding Yeast.

Optogenetic systems utilize genetically encoded light-sensitive proteins to control cellular processes such as gene expression and protein localization. Like most synthetic systems, generation of an optogenetic system with desirable properties requires multiple design-test-build cycles. A yeast optogenetic toolkit (yOTK) allows rapid assembly of optogenetic constructs using Modular Cloning, or MoClo. In this protocol, we describe how to assemble, integrate, and test optogenetic systems in the budding yeast Saccharomyces cerevisiae. Generating an optogenetic system requires the user to first define the structure of the final construct and identify all basic parts and vectors required for the construction strategy, including light-sensitive proteins that need to be domesticated into the toolkit. The assembly is then defined following a set of standard rules. Multigene constructs are assembled using a series of one-pot assembly steps with the identified parts and vectors and transformed into yeast. Screening of the transformants allows optogenetic systems with optimal properties to be selected.

Semblans: automated assembly and processing of RNA-seq data.

Recent advancements in parallel sequencing methods have precipitated a surge in publicly available short-read sequence data. This has encouraged the development of novel computational tools for the de novo assembly of transcriptomes from RNA-seq data. Despite the availability of these tools, performing an end-to-end transcriptome assembly remains a programmatically involved task necessitating familiarity with best practices. Aside from quality control steps, including error correction, adapter trimming, and chimera filtration needing to be correctly used, moving data between programs often requires manual reformatting or restructuring, which can further impede throughput. Here, we introduce Semblans, a tool for streamlining the assembly process that efficiently and consistently produces high-quality transcriptome assemblies. Semblans abstracts the key quality control, reconstitution, and postprocessing steps of transcriptome assembly from raw short-read sequences to annotated coding sequences. Evaluating its performance against previously assembled transcriptomes on the basis of assembly quality, we find that Semblans produced higher quality assemblies for 98 of the 101 short-read runs tested. Semblans is written in C++ and runs on Unix-compliant operating systems. Source code, documentation, and compiled binaries are hosted under the GNU General Public License at https://github.com/gladshire/Semblans.

Stereo‐ and Chemoselective Enrichment of sp3 Character in Post‐Ugi Morpholines

AbstractWe conducted a systematic study on synthesis and modification of morpholine core using Ugi adducts derived from propiolic acid and glycolaldehyde dimer. The assembly step involved triphenylphosphine‐promoted 6‐exo‐dig umpolung oxa‐Michael cyclization of Ugi‐derived hydroxypropargylamides yielding 2‐methylenemorpholin‐3‐one derivatives. Subsequent heterogeneous catalytic hydrogenation of the exocyclic double bond proceeded in a highly diastereoselective fashion establishing a relative cis configuration in the resulting sp3‐enriched morpholinone scaffold. Finally, chemoselective amide reduction with lithium aluminum hydride (LAH) allowed to obtain fully saturated morpholines while leaving the more sterically hindered exocyclic amide moiety intact. Taken together these findings outline the strategy for controlled reduction of the inherent rigidity of post‐Ugi scaffolds.

Application of Time and Motion Study in the Assembly of A Formula 1 Car Using Lego® Blocks: Analysis of Time Study and Standart Time

Objective: The aim of this study is to analyze the figures provided by calculations related to standard time, applying the steps of Time and Motion Study in the use of LEGO® blocks for the assembly of a Formula 1 car. Theoretical Framework: This study is based on the theories of time study and standard time determination, which are essential tools in the Time and Motion Study. Using LEGO® blocks as a study tool provides a controlled and standardized environment for applying these theories, facilitating the observation and measurement of times and movements. Method: The development of this work was based on applied and experimental research. Applied research, as described by Vergara (2007), is an approach focused on generating knowledge with practical application, aimed at solving specific problems. In addition, experimental research, also according to Vergara (2007), is characterized by the rigorous control of the variables involved, allowing the observation of the effects of an independent variable on a dependent variable. Results and Discussion: The results showed that the application of time study and standard time determination is effective in assembling a LEGO ® Formula 1 car. The detailed analysis allowed calculating the time required for each assembly step. The total standard time found for the complete assembly of the car was 507.2 seconds, demonstrating the feasibility of using LEGO® blocks for time and motion studies. Research Implications: This study contributes theoretically by demonstrating the practical application of time study and standard time determination theories in the assembly of simple products such as a LEGO car. Methodologically, it provides a detailed procedure for applying these techniques in assembly processes, which can be replicated in other contexts. Managerial/Social Contributions: Managerially, this study offers insights on how to improve productivity and reduce waste in assembly processes, even in simple operations. Socially, the application of these techniques can be adapted to educational and training programs, promoting planning and operational efficiency skills among participants.

Scaling up Solid State Battery Research at Fraunhofer FFB

All-solid-state batteries (ASSBs) hold immense promise for revolutionizing the future of energy storage due to their enhanced safety and higher potential energy density. Within the context of the federally funded FoFeBat project dedicated to production research for batteries, this abstract outlines the overarching goals and collaborative strategies of the Fraunhofer Research Institution for Battery Cell Production FFB in advancing ASSB production.In five designated so-called innovation laboratories, which are run in close collaboration with some of the leading academic battery research institutions in Germany, including Universities of Muenster, Giessen and Aachen, as well as Research Center Juelich (FZJ), Fraunhofer FFB addresses the problems in scaling up solid state battery and solid electrolyte production. The planned research activities include upscaling the synthesis of polymer-, oxide- and thiophosphate-based solid electrolytes to the kg-scale. The in-house produced materials are used to study the production process of ASSB electrodes with different cathode active materials and how different forms of pressure application (warm-isostatic pressing, calendaring) influence the resulting electrode composite. This includes the evaluation of the influence of different binder materials and solvents on the production process of the electrodes. For the cell assembly, a prototype-scale production line will be established, where the different sheets (electrodes, separator) will be combined, and environmental influences can be studied by the application of sealed mini environments at the different assembly steps. The finalized cells are electrochemically characterized to study the influence of different operating conditions (temperature, pressure) on the cell performance. In addition to this, safety evaluation of the different materials and the fully assembled cells is part of this project. The establishment of the innovation laboratories for ASSB research provide the unique opportunity to test the acquired parameters on the pilot scale production line of the Fraunhofer FFB, which will help to evaluate the transferability of the different production steps into a full-scale lithium-ion-battery production line at FFB.Scaling up these processing steps will produce challenges that are currently being investigated by various companies without the possibility of accessing the acquired knowledge. Solving these challenges and making the obtained knowledge publicly available through scientific journal publications, will help speed up the development of solid-state batteries.

Model‐Based Exploration of Web Guiding Behavior for a Novel Battery Cell Stacking Process

Innovative production processes are required to meet the rapidly growing demand for batteries and the associated material trends. In particular, the process step of stack assembly using new machine concepts promises a high potential for optimization. However, these concepts are untested in practice. The use of digital models makes it possible to develop corresponding optimization approaches. This article presents the development of a model for the web guiding systems of electrodes and its integration into an overall machine model for a novel machine concept. Finally, optimization approaches are identified by determining appropriate controller parameters for the web guiding systems.

Localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus to thylakoid subdomains in Arabidopsis.

Thylakoid membranes in chloroplasts and cyanobacteria harbor the multisubunit protein complexes that catalyze the light reactions of photosynthesis. In plant chloroplasts, the thylakoid membrane system comprises a highly organized network with several subcompartments that differ in composition and morphology: grana stacks, unstacked stromal lamellae, and grana margins at the interface between stacked and unstacked regions. The localization of components of the photosynthetic apparatus among these subcompartments has been well characterized. However, less is known about the localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus, the partitioning of proteins between two recently resolved components of the traditional margin fraction (refined margins and curvature), and the effects of light on these features. In this study, we analyzed the partitioning of numerous thylakoid biogenesis and repair factors among grana, curvature, refined margin, and stromal lamellae fractions of Arabidopsis thylakoid membranes, comparing the results from illuminated and dark-adapted plants. Several proteins previously shown to localize to a margin fraction partitioned in varying ways among the resolved curvature and refined margin fractions. For example, the ALB3 insertase and FtsH protease involved in photosystem II (PSII) repair were concentrated in the refined margin fraction, whereas TAT translocon subunits and proteins involved in early steps in photosystem assembly were concentrated in the curvature fraction. By contrast, two photosystem assembly factors that facilitate late assembly steps were depleted from the curvature fraction. The enrichment of the PSII subunit OE23/PsbP in the curvature fraction set it apart from other PSII subunits, supporting the previous conjecture that OE23/PsbP assists in PSII biogenesis and/or repair. The PSII assembly factor PAM68 partitioned differently among thylakoid fractions from dark-adapted plants and illuminated plants and was the only analyzed protein to convincingly do so. These results demonstrate an unanticipated spatial heterogeneity of photosystem biogenesis and repair functions in thylakoid membranes and reveal the curvature fraction to be a focal point of early photosystem biogenesis.

Structural profiles of the full phagocyte NADPH oxidase unveiled by combining computational biology and experimental knowledge

The phagocyte NADPH oxidase (NOX2) is an enzyme, crucial for innate immune defense, producing reactive oxygen species necessary for pathogen destruction. Its activation requires the assembly of soluble proteins (p47phox, p40phox, p67phox, and Rac) with the membrane-bound flavocytochrome b558 (cytb558). We combined circular-dichroism analyses, with decades of experimental data, to filter structural models of the NADPH oxidase complex generated by the artificial intelligence program AlphaFold2 (AF2). The predicted patterns tend to closely resemble the active states of the proteins, as shown by the compact structure of the cytb558, whose dehydrogenase domain is stabilized closer to the membrane. The modeling of the interaction of p47phox with cytb558, which is the initial assembly and activation steps of the NADPH oxidase, enables us to describe how the C-terminus of p47phox interacts with the cytb558. Combining the AF2 cytb558 -p47phox model and its classical molecular dynamics simulations, we highlighted new hydrophobic lipid insertions of p47phox, particularly at residues Trp80-Phe81 of its PX domain. The AF2 models also revealed the implications of intrinsically disordered regions, such as the fragment between the PX domain and the SH3 regions of p47phox, in ensuring distant protein-protein and membrane-protein interactions. Finally, the AF2 prediction of the cytb558-Trimera model highlighted the importance of leaving Rac1 as a separate protein to reach an active state of the NADPH oxidase complex. Altogether, our step-by-step approach provides a structural model of the active complex showing how disordered regions and specific lipid and protein interactions can enable and stabilize the multi-subunit assembly.

A piezoelectric driven amphibious microrobot capable of fast and controllable movement

Abstract Due to its excellent adaptability to the environment and flexibility in narrow spaces, amphibious microrobots have become an important research direction recently. This study proposes an amphibious microrobot driven by piezoelectric actuators with a body length of 4.5 cm and a mass of 1.4 g. The microrobot consists of two active front legs, two passive rear legs, two caudal fins, and a support frame. Each front leg and each caudal fin are designed as structures integrated with their respective piezoelectric actuators. The microrobot has a tilted body, and the ground exerts an oblique upward impact force that makes it jump forward when its front legs swing backwards. The opposite swing of the two caudal fins generates propulsion for swimming. The components of the microrobot are manufactured based on the monolithic laminate process. The monolithic front actuator-leg and monolithic actuator-fin both emerge from a multi-layer material laminate. The support frame is designed and fabricated as a monolithic structure to improve assembly accuracy and reduce redundant assembly steps. The manufactured microrobot demonstrates its flexible and fast amphibious movements. Its maximum land walking speed reaches 15.3 cm/s and its turning speed reaches 48.2 degrees per second. The microrobot has a maximum payload capacity of 5 g moving on land. When the front legs and caudal fins work simultaneously, its underwater swimming speed reaches 9.1 cm/s, and the maximum turning speed is 20.5 degrees per second. The microrobot also confirms a maximum payload of 3 g during its underwater movement.

Label-free electrochemical immunosensor for rapid and highly sensitive detection of Brucella abortus in serum

Accurate and efficient detection of Brucella is crucial to control the disease and mitigate its impact on animal health and human safety. In this study, a simple and rapid label-free electrochemical immunosensor for the detection of Brucella abortus was fabricated based on the principle of current decrease after specific capture of the bacteria. A certain proportion of multi-walled carbon nanotubes (MWCNTs), dopamine hydrochloride (DA), and gold nanoparticles (AuNPs) were modified on the surface of a gold disc electrode. Then, Brucella abortus antibody and bovine serum albumin (BSA) were immobilized in sequence to complete the assembly of the sensor. The electrodes from various assembly steps were characterized by a cyclic voltammetry (CV) curve. The immunosensor exhibited high sensitivity with a wide linear range between 10 CFU/mL and 108 CFU/mL. Additionally, the sensor displayed good repeatability, stability, and interference resistance. The recovery rates for low, medium, and high concentrations of bacteria were all within the range of 90 % to 100 %, indicating a high level of accuracy. The method established in this study can provide reference and assistance for early diagnosis of Brucella abortus in clinical practice.

A fatty acid-ordered plasma membrane environment is critical for Ebola virus matrix protein assembly and budding

Plasma membrane (PM) domains and order phases have been shown to play a key role in the assembly, release, and entry of several lipid-enveloped viruses. In the present study, we provide a mechanistic understanding of the Ebola virus (EBOV) matrix protein VP40 interaction with PM lipids and their effect on VP40 oligomerization, a crucial step for viral assembly and budding. VP40 matrix formation is sufficient to induce changes in the PM fluidity. We demonstrate that the distance between the lipid headgroups, the fatty acid tail saturation, and the PM order are important factors for the stability of VP40 binding and oligomerization at the PM. The use of FDA-approved drugs to fluidize the PM destabilizes the viral matrix assembly leading to a reduction in budding efficiency. Overall, these findings support an EBOV assembly mechanism that reaches beyond lipid headgroup specificity by using ordered PM lipid regions independent of cholesterol.

The Assembly of HTLV-1-How Does It Differ from HIV-1?

Retroviral assembly is a highly coordinated step in the replication cycle. The process is initiated when the newly synthesized Gag and Gag-Pol polyproteins are directed to the inner leaflet of the plasma membrane (PM), where they facilitate the budding and release of immature viral particles. Extensive research over the years has provided crucial insights into the molecular determinants of this assembly step. It is established that Gag targeting and binding to the PM is mediated by interactions of the matrix (MA) domain and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This binding event, along with binding to viral RNA, initiates oligomerization of Gag on the PM, a process mediated by the capsid (CA) domain. Much of the previous studies have focused on human immunodeficiency virus type 1 (HIV-1). Although the general steps of retroviral replication are consistent across different retroviruses, comparative studies revealed notable differences in the structure and function of viral components. In this review, we present recent findings on the assembly mechanisms of Human T-cell leukemia virus type 1 and highlight key differences from HIV-1, focusing particularly on the molecular determinants of Gag-PM interactions and CA assembly.

The role of an ontology-based knowledge backbone in a circular factory

Abstract In a circular factory, new products are produced reusing parts from used products, as well as newly manufactured parts. The production system consists of disassembly, testing as well as assembly steps. Due to the unforeseeable conditions of the used parts, the complexity of such a circular factory is challenging. This paper contributes a concept of an ontology-based knowledge backbone to master the challenges of such a circular factory. The concept addresses the representation of knowledge especially taking into account uncertainty, how to design queries and means to detect similarities and analogies. Furthermore, the role of research data management with automatized workflows as a supplier for FAIR data is elaborated.

Conjugative transfer of the IncN plasmid pKM101 is mediated by dynamic interactions between the TraK accessory factor and TraI relaxase.

Conjugative dissemination of mobile genetic elements (MGEs) among bacteria is initiated by assembly of the relaxosome at the MGE's origin-of-transfer (oriT) sequence. A critical but poorly defined step of relaxosome assembly involves recruitment of the catalytic relaxase to its DNA strand-specific nicking site within oriT. Here, we present evidence by AlphaFold modeling, affinity pulldowns, and in vivo site-directed photocrosslinking that the TraK Ribbon-Helix-Helix DNA-binding protein recruits TraI to oriT through a dynamic interaction in which TraI's C-terminal unstructured domain (TraICTD) wraps around TraK's C-proximal tetramerization domain. Upon relaxosome assembly, conformational changes disrupt this contact, and TraICTD instead self-associates as a prerequisite for relaxase catalytic functions or substrate engagement with the transfer channel. These findings delineate key early-stage processing reactions required for conjugative dissemination of a model MGE.

Tail assembly interference is a common strategy in bacterial antiviral defenses

Many bacterial immune systems recognize phage structural components to activate antiviral responses, without inhibiting the function of the phage component. These systems can be encoded in specific chromosomal loci, known as defense islands, and in mobile genetic elements such as prophages and phage-inducible chromosomal islands (PICIs). Here, we identify a family of bacterial immune systems, named Tai (for ‘tail assembly inhibition’), that is prevalent in PICIs, prophages and P4-like phage satellites. Tai systems protect their bacterial host population from other phages by blocking the tail assembly step, leading to the release of tailless phages incapable of infecting new hosts. To prevent autoimmunity, some Tai-positive phages have an associated counter-defense mechanism that is expressed during the phage lytic cycle and allows for tail formation. Interestingly, the Tai defense and counter-defense genes are organized in a non-contiguous operon, enabling their coordinated expression.