Assembly Sites Research Articles

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

Localization of innovative technologies of industrial production as a tool for intensification of import substitution policy

The main criterion for the successful implementation of import substitution policy is the location of domestic production on the domestic territory of the state, in order to reduce the negative economic consequences of the industrial policies of unfriendly countries, with the orientation of domestic industrial enterprises towards their own intensification of production, including the production of analogous products to imported ones, for subsequent replacement of imported technologies with domestic ones and supply of domestic products to the domestic market – production and sale of own products. Localization of production is the placement of manufacturing or component industrial production facilities on its own, separate, local territory, in order to reduce the cost of products and bring them closer to assembly sites or sales markets. The term «localization of production», in the scientific literature, is represented by a special case of territorial organization (localized enterprises in separate territories) of production processes for the creation of material wealth, which has a number of features: in the organization of production, a company with a higher level of technological development forms the “conditions of the game”, organizing the distribution of production of certain products among all participants; localization is often carried out on the territory of another state, which has a lower level of development of technology and technology and a higher demand for final products in the markets of this state; An additional benefit of localization is the optimization of process logistics, bringing it closer to consumers. The goal of localization is to reduce production, transaction and logistics costs and increase the level of investment. The article presents the author's position on the development of intensification of the processes of industrial import substitution, in modern conditions, the main idea of which is the creation of localized production, a full technological cycle in the activities of Russian industrial enterprises, representing: at the initial stage, a full line of production of components and import substitution of missing parts, to meet demand in the domestic market, and in the future, the development of processes of substitution of imported technologies, the formation and use of advanced development technologies, increasing export potential and strengthening their presence of Russian industry in world markets. The work explores the essence of localization of production, which is determined by the production of parts and components manufactured in Russia, at national production facilities, using its own technologies, in order to increase the intensity of import substitution processes.

Regioselective super-assembly of Prussian blue analogue

The construction of high-asymmetrical structures demonstrates significant potential in improving the functionality and distinctness of nanomaterials, but remains a considerable challenge. Herein, we develop a one-pot method to fabricate regioselective super-assembly of Prussian blue analogue (PBA) —— a PBA anisotropic structure (PBA-AS) decorated with epitaxial modules——using a step-by-step epitaxial growth on a rapidly self-assembled cubic substrate guided by thiocyanuric acid (TCA) molecules. The epitaxial growth units manifest as diverse geometric shapes, which are predominantly concentrated on the {100}, {111}, or {100}+{111} crystal plane of the cubic substrate. The crystal plane and morphology of epitaxial module can be regulated by changing the TCA concentration and reaction temperature, enabling a high level of controllability over specific assembly sites and structures. To illustrate the advantage of the asymmetrical structure, phosphated PBA-AS demonstrates improved performance in the oxygen evolution reaction compared to simple phosphated PBA nanocube. This method offers valuable insights for designing asymmetrical nanomaterials with intricate architectures and versatile functionalities.

The molecular mechanisms regulating the assembly of the autophagy initiation complex.

The autophagy initiation complex is brought about via a highly ordered and stepwise assembly process. Two crucial signaling molecules, mTORC1 and AMPK, orchestrate this assembly by phosphorylating/dephosphorylating autophagy-related proteins. Activation of Atg1 followed by recruitment of both Atg9 vesicles and the PI3K complex I to the PAS (phagophore assembly site) are particularly crucial steps in its formation. Ypt1, a small Rab GTPase in yeast cells, also plays an essential role in the formation of the autophagy initiation complex through multiple regulatory pathways. In this review, our primary focus is to discuss how signaling molecules initiate the assembly of the autophagy initiation complex, and highlight the significant roles of Ypt1 in this process. We end by addressing issues that need future clarification.

Unsettled assemblies: performance space, infrastructure and architecture at PQ 2023

ABSTRACT The Performance Space Exhibition at the 2023 Prague Quadrennial (PQ) – titled ‘Acts of Assembly’ – explored how theatres and performance spaces operate as sites of assembly and spaces for community. It asked: How do they create connections? How do they facilitate encounters? How do they function as sites for social action and the making of culture? Focussing on the dynamics of different acts of assembly, and positing performance itself as an assemblage, the exhibition explored the productive continuities and antagonisms between more traditional theatre architectures and the broader emergent qualities of performance spaces. This essay reflects on how the exhibition expressed a vision of performance spaces as a vital social infrastructure. It suggests the concept of social infrastructure as a useful analytic for examining how performance spaces support and sustain people and practices. The essay also reflects on the unsettled positioning of theatre architecture and performance space within PQ more generally.

How the discovery of Cold Noise delayed the production of ATLAS ITk strip tracker modules by a year

The construction of the ATLAS strip tracker barrel will require the assembly of 12,000 barrel detector modules over the course of 3.5 years. In 2022, during the module pre-production phase, modules were found to display clusters of noisy channels outside required specifications when tested at operating temperatures (-35°C), called “Cold Noise”. Extensive investigations into the cause and mechanism of Cold Noise interrupted pre-production and occupied most barrel module assembly sites. This contribution presents an overview of the year-long investigations into Cold Noise, the final identification of the underlying mechanism and necessary changes for the transition to production.

경찰관기동대원의 소진 영향 요인 연구

The number of assemblies and demonstrations where police units are deployed is not decreasing, and it has been revealed that in the past 10 years, the number of cases in which riot police officers have experienced physical harm at assemblies and demonstrations has averaged about 90 people per year. Moreover, in a reality where it is difficult to quantitatively prepare riot police officers due to the abolition of the mandatory police system, if they are forced to leave their duties due to overloaded and difficult work, the gap in public security work at assembly and protest sites will become even larger. In other words, in order to maintain social order and ensure the safety of citizens, there is a need to explore factors at the organizational level to prevent riot police officers from experiencing burnout and leaving their duties. Accordingly, the purpose of this study is to statistically verify the factors that affect the burnout of riot police officers. To achieve the purpose of the study, survey data that secured representativeness through proportional quota sampling, taking into account class, age, gender, etc., were used for analysis, using the 2022 national riot squad as the population. Through a review of previous research, job stress, job satisfaction, and organizational commitment were set as independent variables and their impact on burnout was verified. As a result of the analysis, it was confirmed that job stress, job satisfaction, and organizational commitment among riot police officers all had a statistically significant impact on burnout, and that job stress in particular had the greatest impact on burnout. Based on these results, several policy recommendations were presented to reduce job stress levels and increase job satisfaction and organizational commitment.

Stabilization of the retromer complex: Analysis of novel binding sites of bis-1,3-phenyl guanylhydrazone 2a to the VPS29/VPS35 interface

The stabilization of the retromer protein complex can be effective in the treatment of different neurological disorders. Following the identification of bis-1,3-phenyl guanylhydrazone 2a as an effective new compound for the treatment of amyotrophic lateral sclerosis, in this work we analyze the possible binding sites of this molecule to the VPS35/VPS29 dimer of the retromer complex. Our results show that the affinity for different sites of the protein assembly depends on compound charge and therefore slight changes in the cell microenvironment could promote different binding states. Finally, we describe a novel binding site located in a deep cleft between VPS29 and VPS35 that should be further explored to select novel molecular chaperones for the stabilization of the retromer complex.

The readout panels for the BI-RPC project for ATLAS Phase-II upgrade

To take full advantage from the LHC upgrade to high luminosity in 2029, the ATLAS community has approved an intensive detector upgrade program that includes the construction of an additional trigger layer positioned close to the BI MDT chambers of the muon spectrometer. These chambers are RPC with thin gas gap, thin High Pressure Laminate electrodes coated with graphite on one side, and only use strips orthogonal to the beam-axis to reconstruct the eta and phi coordinates of the detector point where an ionizing particle passes. A detailed description of the construction and validation of these strip panels in China and Italy assembly sites, is presented here.

KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants.

Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.

Data-Driven Dynamic Bayesian Network Model for Safety Resilience Evaluation of Prefabricated Building Construction

Due to factors such as the availability of assembly equipment, technology, and site management level, prefabricated building construction safety accidents often occur. To ensure the safety of prefabricated buildings and effectively reduce the accident rate, the concept of resilience is introduced into the safety management of prefabricated buildings. Based on the resilience absorption capacity, adaptation capacity, recovery capacity, and optimization capacity, a comprehensive evaluation index system for the safety resilience of prefabricated buildings is established. By combining prior knowledge with structural learning and parameter learning, a dynamic Bayesian network (DBN) model is constructed to dynamically evaluate the safety resilience of prefabricated buildings. Through forward causal reasoning and backward diagnostic reasoning, the dynamic safety resilience value of prefabricated buildings and the chain of maximum failure causes are obtained. Finally, by conducting a sensitivity analysis on the target nodes, the key influencing factors of the safety resilience of prefabricated construction are identified, and improvement suggestions for enhancing resilience are proposed. The results indicate that establishing a resilience safety culture, preventing unsafe behaviors of personnel, safety management, and supervision on the construction site, emergency management actions, and building a risk management information system are crucial factors influencing the safety resilience of prefabricated buildings. The enhancement of absorption capacity has the greatest impact on the safety resilience of prefabricated buildings.

Structural restraints in the SARS-CoV-2 spike tail control its intra-cellular trafficking to virion assembly and immune presentation sites

Structural restraints in the SARS-CoV-2 spike tail control its intra-cellular trafficking to virion assembly and immune presentation sites

Proteomic analysis of SARS-CoV-2 particles unveils a key role of G3BP proteins in viral assembly

Considerable progress has been made in understanding the molecular host-virus battlefield during SARS-CoV-2 infection. Nevertheless, the assembly and egress of newly formed virions are less understood. To identify host proteins involved in viral morphogenesis, we characterize the proteome of SARS-CoV-2 virions produced from A549-ACE2 and Calu-3 cells, isolated via ultracentrifugation on sucrose cushion or by ACE-2 affinity capture. Bioinformatic analysis unveils 92 SARS-CoV-2 virion-associated host factors, providing a valuable resource to better understand the molecular environment of virion production. We reveal that G3BP1 and G3BP2 (G3BP1/2), two major stress granule nucleators, are embedded within virions and unexpectedly favor virion production. Furthermore, we show that G3BP1/2 participate in the formation of cytoplasmic membrane vesicles, that are likely virion assembly sites, consistent with a proviral role of G3BP1/2 in SARS-CoV-2 dissemination. Altogether, these findings provide new insights into host factors required for SARS-CoV-2 assembly with potential implications for future therapeutic targeting.

Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

Dicentric chromosomes are resolved through breakage and repair at their centromeres

Chromosomes with two centromeres provide a unique opportunity to study chromosome breakage and DNA repair using completely endogenous cellular machinery. Using a conditional transcriptional promoter to control the second centromere, we are able to activate the dicentric chromosome and follow the appearance of DNA repair products. We find that the rate of appearance of DNA repair products resulting from homology-based mechanisms exceeds the expected rate based on their limited centromere homology (340 bp) and distance from one another (up to 46.3 kb). In order to identify whether DNA breaks originate in the centromere, we introduced 12 single-nucleotide polymorphisms (SNPs) into one of the centromeres. Analysis of the distribution of SNPs in the recombinant centromeres reveals that recombination was initiated with about equal frequency within the conserved centromere DNA elements CDEII and CDEIII of the two centromeres. The conversion tracts range from about 50 bp to the full length of the homology between the two centromeres (340 bp). Breakage and repair events within and between the centromeres can account for the efficiency and distribution of DNA repair products. We propose that in addition to providing a site for kinetochore assembly, the centromere may be a point of stress relief in the face of genomic perturbations.

Adaptive robotic system for the inspection of aerospace slat actuator mount.

Introduction: Robotics uptake in the aerospace industry is low, mainly due to the low-volume/high-accuracy production that aerospace manufacturers require. Furthermore, aerospace manufacturing and assembly sites are often unstructured environments not specifically suitable for robots to operate in. Methods: This paper introduces a robotic visual inspection system using off-the-shelf components able to inspect the mounting holes for wing slat actuators without the need for fixed-coordinate programming; the part just needs to be left within reach of the robot. Our system sets one of the opposed pairs of mounting holes as a reference (the "datum") and then compares the tilt of all other pairs of mounting holes with respect to it. Under the assumption that any deviation in the mounting hole tilt is not systematic but due to normal manufacturing tolerances, our system will either guarantee the correct alignment of all mounting holes or highlight the existence of misaligned holes. Results and Discussion: Computer-vision tilt measurements are performed with an error of below 0.03° using custom optimization for the sub-pixel determination of the center and radius of the mounting holes. The error introduced by the robot's motion from the datum to each of the remaining hole pairs is compensated by moving back to the datum and fixing the orientation again before moving to inspect the next hole pair. This error is estimated to be approximately 0.05°, taking the total tilt error estimation for any mounting hole pair to be 0.08° with respect to the datum. This is confirmed by manually measuring the tilt of the hole pairs using a clock gauge on a calibrated table (not used during normal operation).

Chromosomal-level genome assembly and single-nucleotide polymorphism sites of black-faced spoonbill Platalea minor.

Platalea minor, or black-faced spoonbill (Threskiornithidae), is a wading bird confined to coastal areas in East Asia. Due to habitat destruction, it was classified as globally endangered by the International Union for Conservation of Nature. However, the lack of genomic resources for this species hinders the understanding of its biology and diversity, and the development of conservation measures. Here, we report the first chromosomal-level genome assembly of P.minor using a combination of PacBio SMRT and Omni-C scaffolding technologies. The assembled genome (1.24Gb) contains 95.33% of the sequences anchored to 31 pseudomolecules. The genome assembly has high sequence continuity with scaffold length N50 = 53Mb. We predicted 18,780 protein-coding genes and measured high BUSCO score completeness (97.3%). Finally, we revealed 6,155,417 bi-allelic single nucleotide polymorphisms, accounting for ∼5% of the genome. This resource offers new opportunities for studying the black-faced spoonbill and developing conservation measures for this species.

A single C-terminal residue controls SARS-CoV-2 spike trafficking and incorporation into VLPs

The spike (S) protein of SARS-CoV-2 is delivered to the virion assembly site in the ER-Golgi Intermediate Compartment (ERGIC) from both the ER and cis-Golgi in infected cells. However, the relevance and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virus-like particles (VLP) and VLP fusogenicity are determined by coatomer-dependent S delivery from the cis-Golgi and restricted by S-coatomer dissociation. Although S mimicry of the host coatomer-binding dibasic motif ensures retrograde trafficking to the ERGIC, avoidance of the host-like C-terminal acidic residue is critical for S-coatomer dissociation and therefore incorporation into virions or export for cell-cell fusion. Because this C-terminal residue is the key determinant of SARS-CoV-2 assembly and fusogenicity, our work provides a framework for the export of S protein encoded in genetic vaccines for surface display and immune activation.

Capping protein regulators of actin assembly in budding yeast.

Cellular functions of actin capping protein (CP) regulators are poorly understood. Di Pietro and colleagues (https://doi.org/10.1083/jcb.202306154) shed unprecedented light on this topic using budding yeast. Two proteins with CPI (capping protein interacting) motifs recruit CP to sites of actin assembly, while a third contributes to CP turnover.

Mec1-Rad53 Signaling Regulates DNA Damage-Induced Autophagy and Pathogenicity in Candida albicans.

DNA damage activates the DNA damage response and autophagy in C. albicans; however, the relationship between the DNA damage response and DNA damage-induced autophagy in C. albicans remains unclear. Mec1-Rad53 signaling is a critical pathway in the DNA damage response, but its role in DNA damage-induced autophagy and pathogenicity in C. albicans remains to be further explored. In this study, we compared the function of autophagy-related (Atg) proteins in DNA damage-induced autophagy and traditional macroautophagy and explored the role of Mec1-Rad53 signaling in regulating DNA damage-induced autophagy and pathogenicity. We found that core Atg proteins are required for these two types of autophagy, while the function of Atg17 is slightly different. Our results showed that Mec1-Rad53 signaling specifically regulates DNA damage-induced autophagy but has no effect on macroautophagy. The recruitment of Atg1 and Atg13 to phagophore assembly sites (PAS) was significantly inhibited in the mec1Δ/Δ and rad53Δ/Δ strains. The formation of autophagic bodies was obviously affected in the mec1Δ/Δ and rad53Δ/Δ strains. We found that DNA damage does not induce mitophagy and ER autophagy. We also identified two regulators of DNA damage-induced autophagy, Psp2 and Dcp2, which regulate DNA damage-induced autophagy by affecting the protein levels of Atg1, Atg13, Mec1, and Rad53. The deletion of Mec1 or Rad53 significantly reduces the ability of C. albicans to systematically infect mice and colonize the kidneys, and it makes C. albicans more susceptible to being killed by macrophages.