Assembly Sites Research Articles

­­Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy

ABSTRACT During macroautophagy/autophagy, precursor cisterna known as phagophores expand and sequester portions of the cytoplasm and/or organelles, and subsequently close resulting in double-membrane transport vesicles called autophagosomes. Autophagosomes fuse with lysosomes/vacuoles to allow the degradation and recycling of their cargoes. We previously showed that sequential binding of yeast Atg2 and Atg18 to Atg9, the only conserved transmembrane protein in autophagy, at the extremities of the phagophore mediates the establishment of membrane contact sites between the phagophore and the endoplasmic reticulum. As the Atg2-Atg18 complex transfers lipids between adjacent membranes in vitro, it has been postulated that this activity and the scramblase activity of the trimers formed by Atg9 are required for the phagophore expansion. Here, we present evidence that Atg9 indeed promotes Atg2-Atg18 complex-mediated lipid transfer in vitro, although this is not the only requirement for its function in vivo. In particular, we show that Atg9 function is dramatically compromised by a F627A mutation within the conserved interface between the transmembrane domains of the Atg9 monomers. Although Atg9F627A self-interacts and binds to the Atg2-Atg18 complex, the F627A mutation blocks the phagophore expansion and thus autophagy progression. This phenotype is conserved because the corresponding human ATG9A mutant severely impairs autophagy as well. Importantly, Atg9F627A has identical scramblase activity in vitro like Atg9, and as with the wild-type protein enhances Atg2-Atg18-mediated lipid transfer. Collectively, our data reveal that interactions of Atg9 trimers via their transmembrane segments play a key role in phagophore expansion beyond Atg9ʹs role as a lipid scramblase.Abbreviations:BafA1: bafilomycin A1; Cvt: cytoplasm-to-vacuole targeting; Cryo-EM: cryo-electron microscopy; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCS: membrane contact site; NBD-PE: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor Ape1; PtdIns3P: phosphatidylinositol-3-phosphate; SLB: supported lipid bilayer; SUV: small unilamellar vesicle; TMD: transmembrane domain; WT: wild type

St George, Damerham, Hampshire: an Anglo-Saxon minster church in a landscape of social power

The church of St George, Damerham, now in Hampshire but until 1895 in Wiltshire, is a former minster church. The early Romanesque church may preserve fabric from a late Anglo-Saxon building perhaps dating from shortly after the time when Æthelflæd, widow of King Eadmund (939-45), granted Damerham to Glastonbury Abbey in her will of c975-91. There is some indirect evidence that the minster at Damerham was founded in the later 9th century. Prior to this, the main minster in the region could have been at nearby Fordingbridge. What, however, is exceptional about the church is that it is adjacent to a natural knoll which appears to have formed an Anglo-Saxon assembly site of special importance: what has been termed a ‘hanging promontory’ site (Baker and Brookes, 2013a). It is probable that the minster was deliberately founded adjacent to the assembly site so that the two would project an impression of mutually reinforcing political and religious power.

Cholesterol and M2 Rendezvous in Budding and Scission of Influenza A Virus.

The cholesterol of the host cell plasma membrane and viral M2 protein plays a crucial role in multiple stages of infection and replication of the influenza A virus. Cholesterol is required for the formation of heterogeneous membrane microdomains (or rafts) in the budozone of the host cell that serves as assembly sites for the viral components. The raft microstructures act as scaffolds for several proteins. Cholesterol may further contribute to the mechanical forces necessary for membrane scission in the last stage of budding and help to maintain the stability of the virus envelope. The M2 protein has been shown to cause membrane scission in model systems by promoting the formation of curved lipid bilayer structures that, in turn, can lead to membrane vesicles budding off or scission intermediates. Membrane remodeling by M2 is intimately linked with cholesterol as it affects local lipid composition, fluidity, and stability of the membrane. Thus, both cholesterol and M2 protein contribute to the efficient and proper release of newly formed influenza viruses from the virus-infected cells.

Large scale microfluidic CRISPR screening for increased amylase secretion in yeast.

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

Mec1 regulates PAS recruitment of Atg13 via direct binding with Atg13 during glucose starvation-induced autophagy

Mec1 is a DNA damage sensor, which performs an essential role in the DNA damage response pathway and glucose starvation-induced autophagy. However, the functions of Mec1 in autophagy remain unclear. In response to glucose starvation, Mec1 forms puncta, which are recruited to mitochondria through the adaptor protein Ggc1. Here, we show that Mec1 puncta also contact the phagophore assembly site (PAS) via direct binding with Atg13. Functional analysis of the Atg13-Mec1 interaction revealed two previously unrecognized protein regions, the Mec1-Binding Region (MBR) on Atg13 and the Atg13-Binding Region (ABR) on Mec1, which mediate their mutual association under glucose starvation conditions. Disruption of the MBR or ABR impairs the recruitment of Mec1 puncta and Atg13 to the PAS, consequently blocking glucose starvation-induced autophagy. Additionally, the MBR and ABR regions are also crucial for DNA damage-induced autophagy. We thus propose that Mec1 regulates glucose starvation-induced autophagy by controlling Atg13 recruitment to the PAS.

Occupational Accidents and Need for Worker Safety in Manufacturing and High Risk Industries – An Explorative Study With Solutions

Abstract: OAs (Occupational accidents) have been responsible for fatalities and injuries in both industrialised and developing nations. Due to weak documentation and notification systems, where developing countries in particular lack reliable information on OAs, there are no worldwide standards for information on OAs. Baseline OSMs (occupational safety measures) statistics are still used in workplaces for improving worker safety notwithstanding the underreporting of accident data. 2.78 million Workers every year are estimated to pass away from OAs and other associated conditions, while another 374 million have non-fatal OAs, according to estimates from the ILO (International Labour Organization). Additionally, because manufacturing sectors are dealing with greater OAs, it is necessary to confirm links between safety performances in order to maximise productivity. Purpose: The purpose of this research was to explore the effects of workplace safety s of assembly workers in manufacturing facilities. Theoretical framework: According to studies, industries can enhance their safety cultures by focusing on five key areas: management commitment, communication, safety priority, supportive environment, and involvement. These areas in turn improve employee and equipment performance, as well as safety performance as measured by safety performance reports. Design/methodology/approach: The suggested workplace safety for this job was tested in assembly sites and put into practise. While secondary data was gathered from material found in publications, books, journals, and the internet, primary data came through questionnaires provided to management and personnel. The safety cultures of firms that did not participate in any lean events during the same time period were compared to control groups. Validity: To gauge Security Environment in the assembling enterprises in Tamilnadu, a changed rendition of the poll utilized by Corridor (2013) was utilized. This survey was chosen in light of the fact that at first, the instrument utilized by Corridor (2013) filled in as a kind of perspective. The said instrument contained 27 things. The main limit of the first estimating instrument is that the association's whose working environment wellbeing is estimated utilizing the instrument should have a comparative workplace and hierarchical construction as the steel processes that were utilized to approve the first instrument. This need was met by the associations picked for the ongoing review. Inward consistency was utilized to pass judgment on the nature of the estimating instrument, as exhorted by Cooper et al (1998) Findings: P values less than 0.05 were found for the four individual dimension scores—Management Commitment, Priority of Safety, Involvement, and Work Environment—and the overall score, indicating that the proposed framework improved workplace safety-related metrics after adoption. The improvement in the mean total scores for both groups shows that the suggested framework can increase workplace safety. Additionally, rotation times were shortened by 16.6%, space was used up by 22.2%, and stocks were cut by 25% throughout testing. Due to these enhancements, the overall level of workplace safety was greatly raised along with favourable changes in these four dimensions. Practical and social implications: However, it seems important to draw attention to the ongoing effects of the accident phenomenon on social and health systems, as well as how the development of the economic system has brought about some risk factors that can be addressed through different work practises, increased organisational well-being, and a widespread introduction of corporate welfare tools, in addition to increased controls. The introduction of hazardous work instruments and the absence of controls in particular industries are only two factors contributing to the accident statistics; high workloads are also a frequent factor, the lengthening of workdays and a "culture of performance" and productivity that, even if they do not result in an accident, raise risk margins and, thus, exacerbate the state of "work-related stress." The suggested methodology provides businesses with verified tools and specialised resources that may be applied by businesses using a sustainable and integrated approach. The recommended technique is broken down into steps that engage both workers and prevention experts. The danger of work-related stress is taken into consideration while evaluating the recognised allowances, which merely reflect the most obvious and emerging facet of a much larger issue.

Construction of broom-like Ag@N,O-C sensing interface for electrochemical detection of circulating tumor DNA using entropy-driven DNA walker

In human peripheral blood, circulating tumor DNA (ctDNA) is an excellent tumor biomarker for noninvasive diagnosis, therapy and prognosis of cancer. Herein, an innovative enzyme-free electrochemical sensor sensitized with broom-like Ag@N,O-C hierarchical nanomaterials and entropy-driven DNA walker was designed for PIK3CA E545K ctDNA assay. In this work, N, O-doped carbon layers were used to completely coat Ag nanoparticles, and then were assembled into three-dimensional broom-like nanomaterials with hierarchical structure. The application of Ag@N,O-C broom-like hierarchical nanomaterials (Ag@N,O-C BLHS) as electrode material not only facilitated electron transfer, but also increased functional active sites for a high-density assembly of DNA walking substrates. Via the entropy-driven catalytic reaction of DNA walker, one target ctDNA initiated the dissociation of abundant electroactive tags from the electrode surface, achieving a one-to-multiple amplification effect. By measuring electrochemical responses, PIK3CA E545K ctDNA was quantified with 10 aM detection limit. Additionally, the enzyme-free amplified sensing interface achieved the direct detection of ctDNA in plasma samples from breast cancer patients and healthy human. Overall, the good selectivity, reproducibility and practical application ability confirmed a promising potential of the sensor for disease diagnosis.

Regulation of RB1CC1/FIP200 stability and autophagy function by CREBBP-mediated acetylation in an intrinsically disordered region

ABSTRACT RB1CC1/FIP200 is an essential macroautophagy/autophagy protein that plays an important role in a variety of biological and disease processes through its canonical autophagy-dependent and -independent functions. However, it remains largely unknown whether post-translational modifications could regulate RB1CC1 and its associated autophagy functions. Here, we report acetylation of several lysine residues of RB1CC1 by acetyltransferase CREBBP (CREB binding protein), with K276 as the major CREBBP acetylation site. K276 is also identified as a ubiquitination site by mass spectrometry, and acetylation at this site reduces ubiquitination of RB1CC1 to inhibit its ubiquitin-dependent degradation. We also find that RB1CC1 contains an N-terminal intrinsically disordered region (IDR) capable of forming liquid-liquid phase separation (LLPS) in vitro, which may drive formation of RB1CC1 puncta with LLPS properties in cells independent of SQSTM1/p62 and other autophagy receptors CALCOCO2/NDP52, NBR1, TAX1BP1 and OPTN. Mutational analysis shows that both K276 acetylation and the N-terminal IDR containing it are important for maintaining canonical autophagy function of RB1CC1 in breast cancer cells. Our findings demonstrate regulation of RB1CC1 by a new post-translational mechanism and suggest potential therapeutic application of inducing RB1CC1 degradation through blocking K276 acetylation in the treatment of cancer and other diseases. Abbreviations: Baf-A1: bafilomycin A1; CREBBP/CBP: CREB binding protein; CHX: cycloheximide; EP300/p300: E1A binding protein p300; FRAP: fluorescence recovery after photobleaching; HADCs: histone deacetylases; IDR: intrinsically disordered region; LLPS: liquid-liquid phase separation; KAT2A/GCN5: lysine acetyltransferase 2A; KAT2B/PCAF: lysine acetyltransferase 2B; KAT5/TIP60: lysine acetyltransferase 5; KAT8/MOF: lysine acetyltransferase 8; NAM: nicotinamide; PAS: phagophore assembly site; PEG-8000: polyethylene glycol 8000; RB1CC1/FIP200: RB1 inducible coiled-coil 1; TSA: trichostatin A.

The world’s first (and probably last) autophagy video game

ABSTRACT Macroautophagy/autophagy is the process by which portions of the cytoplasm are sequestered within a transient compartment and delivered to the degradative organelle of the cell, the vacuole or lysosome. Autophagy is a fundamental cytoprotective mechanism, and defects in this process are associated with many diseases. For example, the inability to degrade certain cargo such as mitochondria may lead to neurodegenerative disorders such as Parkinson disease. Autophagic cargo can be many different things including organelles, but also proteins and protein aggregates, nucleic acids, and lipids. Much of our understanding of autophagy comes from studies in baker’s yeast, Saccharomyces cerevisiae. In that organism, autophagy begins at the phagophore assembly site (PAS), which nucleates the initial sequestering compartment, referred to as a phagophore. With the help of autophagy-related (Atg) proteins and lipid addition, the phagophore membrane expands to enclose damaged or superfluous cytoplasmic components, eventually closing into a completed double-membrane vesicle called the autophagosome. The autophagosome is delivered to the degradative organelle where it fuses, releasing the encapsulated cargo into the interior of the organelle where it is broken down into macromolecular building blocks. The resulting building blocks are released back into the cytosol for reuse. Video games are modern expressions of art incorporating illustration, animation, and mechanistic design. While often underappreciated as a scientific art form, video games can beautifully express scientific topics in a way that is both intuitive and engaging, especially to a younger audience.

Phosphatidylinositol 4,5-Bisphosphate Mediates the Co-Distribution of Influenza A Hemagglutinin and Matrix Protein M1 at the Plasma Membrane.

The fully assembled influenza A virus (IAV) has on its surface the highest density of a single membrane protein found in nature-the glycoprotein hemagglutinin (HA) that mediates viral binding, entry, and assembly. HA clusters at the plasma membrane of infected cells, and the HA density (number of molecules per unit area) of these clusters correlates with the infectivity of the virus. Dense HA clusters are considered to mark the assembly site and ultimately lead to the budding of infectious IAV. The mechanism of spontaneous HA clustering, which occurs with or without other viral components, has not been elucidated. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have previously shown that these HA clusters are interdependent on phosphatidylinositol 4,5-biphosphate (PIP2). Here, we show that the IAV matrix protein M1 co-clusters with PIP2, visualized using the pleckstrin homology domain. We find that cetylpyridinium chloride (CPC), which is a positively charged quaternary ammonium compound known for its antibacterial and antiviral properties at millimolar concentrations, disrupts M1 clustering and M1-PIP2 co-clustering at micromolar concentrations well below the critical micelle concentration (CMC). CPC also disrupts the co-clustering of M1 with HA at the plasma membrane, suggesting the role of host cell PIP2 clusters as scaffolds for gathering and concentrating M1 and HA to achieve their unusually high cluster densities in the IAV envelope.

Rab11-FIP1C Is Dispensable for HIV-1 Replication in Primary CD4+ T Cells, but Its Role Is Cell Type Dependent in Immortalized Human T-Cell Lines.

The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that small interfering RNA (siRNA)-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR-Cas9 technology was used to knock out the expression of this protein in several human T-cell lines-Jurkat E6.1, SupT1, and H9-and in primary human CD4+ T cells. FIP1C knockout caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T cells. In H9 cells, FIP1C knockout caused a cell density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knockout had no effect on HIV-1 replication. Furthermore, human T-cell leukemia virus type 1 (HTLV-1)-transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail (Y795W) implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. IMPORTANCE The incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types-human T-cell lines and primary CD4+ T cells-we used CRISPR-Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells.

Role of NS2 specific RNA binding and phosphorylation in liquid-liquid phase separation and virus assembly.

Liquid-liquid phase separation (LLPS) has assumed a prominent role in biological cell systems, where it underpins the formation of subcellular compartments necessary for cell function. We investigated the underlying mechanism of LLPS in virus infected cells, where virus inclusion bodies are formed by an RNA-binding phosphoprotein (NS2) of Bluetongue virus to serve as sites for subviral particle assembly and virus maturation. We show that NS2 undergoes LLPS that is dependent on protein phosphorylation and RNA-binding and that LLPS occurrence is accompanied by a change in protein secondary structure. Site-directed mutagenesis identified two critical arginine residues in NS2 responsible for specific RNA binding and thus for NS2-RNA complex driven LLPS. Reverse genetics identified the same residues as essential for VIB assembly in infected cells and virus viability. Our findings suggest that a specific arginine-RNA interaction in the context of a phosphorylated state drives LLPS in this, and possibly other, virus infections.

Shifting Border, Shifting Interpretation: what the Anglo-Norman Castle of Dodleston in Cheshire might be trying to tell us about the eleventh-century northern Anglo-Welsh Border

This chapter follows on from research and publication by this author on the form and placing of Anglo-Norman castles situated within the northern Anglo-Welsh medieval borderland, recently interpreted and newly termed the Irish Sea Cultural Zone (Swallow 2016). This interpretation argues for the Anglo-Normans’ reuse of pre-existing monuments dating from the prehistoric and Romano-British periods for the deliberate placing of their castle builds. Dodleston Castle was situated within the fluctuating borders of this frontier borderlands zone, and, it is argued, played a significant role in the continuity of strategic and commercial movement along the entirety of the Anglo-Welsh border and the Irish Sea Region. Within this context, and taking a cross-period and interdisciplinary research approach to re-examine the earthworks and landscape of Dodleston Castle in more detail than hitherto, the earthworks at Dodleston may reveal a meeting point of significance over millennia. It will be demonstrated, for instance, that Dodleston’s earthworks likely represent an Anglo-Saxon assembly site situated at the meeting points of important medieval administrative boundaries within the Irish Sea Cultural Zone. By considering the wider spatial significance of Dodleston beyond the temporal confines of the Anglo-Norman period, it is therefore possible to understand better, and reinterpret, the form of the castle earthworks as they exist in the landscape today.

VAMP724 and VAMP726 are involved in autophagosome formation in Arabidopsis thaliana

ABSTRACT Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis. Abbreviations: ATG, autophagy related; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; Conc A, concanamycin A; EM, electron microscopy; ER, endoplasmic reticulum; FRET, Förster/fluorescence resonance energy transfer; MS, Murashige and Skoog; MVB, multivesicular body; PAS, phagophore assembly site; PM, plasma membrane; PVC, prevacuolar compartment; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TEM, transmission electron microscopy; TGN, trans-Golgi network; WT, wild-type.

Human-centric collaborative assembly system for large-scale space deployable mechanism driven by Digital Twins and wearable AR devices

The mass personalization of spacecraft challenges the experience and skill of assembly operators, which is caused by the poor collaboration between the elements in assembly. A large number of research works show that the role of human is irreplaceable in the process of assembly, but few publications focus on improving the collaboration to reduce the strict requirement for operators. To address those challenges, this paper proposes a human-centric collaborative assembly system (HCAS) for Large-scale Space Deployable Mechanism (LSDM) that is the core supporting structure of satellite, to solve the problem of poor collaboration between human and the resources in assembly site. Three Digital Twins (DTs), i.e., assembly process DT, assembly operator DT, and assembly quality DT are developed to achieve the collaboration among the elements of human-cyber-physical in HCAS. The breakthroughs brought by the three DTs are 1) adaptive assembly process sensing based on real-virtual scenes fusion with the help of assembly process DT and wearable AR device enhances the synergy between physical system and cyber system; 2) Based on the assembly operator DT and the Knowledge Graph, the adaptive assembly tasks assignment with operator-centric is realized; 3) Dynamic tolerance allocation of LSDM assembly considering space service environment based on assembly quality DT guides the operator to adjust the process parameters in real time. In addition, in view of the special requirement of gravity compensation of LSDM assembly, the method of using Co-robot to simulate microgravity and AR to plan robot path online is included to improve the human-machine collaboration. Finally, a prototype system is developed and applied in assembly site. Application results show that the developed method and system reduce the dependence on human experience and debases the frequency of rework, which achieve the purpose of improving assembly efficiency and quality.

Surface modification of PVDF membrane via graft polymerization of acetic and acrylic acid

Surface modification of polyvinylidene fluoride (PVDF) hollow fibre membranes through simple chemical treatment is carried out to graft the surface with the presence of carboxylic acid groups. This is to provide means for TiO2 immobilisation in subsequent stages for photocatalysis application. The focus of this study was to identify the highest degree of grafting in order to provide abundant potential sites for subsequent TiO2 assembly. Both acetic and acrylic acid, at varying concentrations, were used and compared as the grafting monomer. It was observed that acrylic acid provides significantly higher grafting degree compared to acetic acid. The highest degree 238 μg/cm2 was obtained at 70% acrylic acid in water, where the surface contact angle significantly reduced to 37o. The highest grafting degree obtained using acetic acid was at 60% in water, which only achieved 31 μg/cm2. Minor improvement was also observed when the solvent for grafting solution was changed from water to toluene; the grafting degree slightly improved by 2% from 238 μg/cm2 to 243 μg/cm2. Higher grafting degree shall allow for more of TiO2 to be immobilized onto the membrane in subsequent work, hence potentially leading to better photocatalysis performance.

Wind Design of Composite Plate Shear Walls/Concrete Filled (SpeedCore) Systems

Composite steel plate shear walls/concrete filled (C-PSW/CF), also known as the SpeedCore system, are a composite solution for the design of mid- to high-rise buildings. A C-PSW/CF system consists of steel plates (web and flange plates) and an infill concrete core. The composite interaction between steel plates and concrete core is developed by either tie bars or tie bars and shear studs. Generally, in low- to mid-rise buildings (less than 15 stories), planar (uncoupled) C-PSW/CF are adequate for resisting lateral loading and deformations. Coupled C-PSW/CF become more prevalent in mid- to high-rise buildings when increased lateral stiffness is desirable. The steel parts of a SpeedCore system (steel plates, tie bars, and shear studs) are prefabricated in the shop and then transported to the site for assembly and erection. The erected empty steel module serves as falsework and formwork during construction and concrete casting, which reduces construction schedule considerably. This is a major advantage of SpeedCore systems, especially when compared with conventional reinforced concrete core systems. This paper presents the wind (nonseismic) design requirements and procedures for planar uncoupled and coupled C-PSW/CF systems. It includes a design example of a 15-story building located in Chicago, Illinois, using both uncoupled and coupled systems.

Optimization of the Stacking Plans for Precast Concrete Slab Based on Assembly Sequence

Precast concrete (PC) slabs are widely used in the assembly of concrete residential buildings. The PC slabs are manufactured at the factory and then arranged in stacks for transport to the construction site for assembly. Currently, optimization of the stacking plans for PC slabs focuses on yard-space utilization and transportation efficiency and rarely considers the assembly sequence; secondary sequencing of prefabricated elements is required during construction to meet the lifting scheme, which leads to increased construction preparation time and risk of worker injury. To enable stacking crews to generate stacking plans rapidly and systematically to improve the on-site lifting efficiency of the components, this paper proposes a storage-location allocation model with two objectives: reduce secondary-sorting workload and increase stacking stability for PC slabs. At the same time, it must match the characteristics of the problem. To prevent the solution from falling into the local optimum during the evolution of the particle swarm optimization algorithm, we introduce an elitist learning strategy, which can improve the solutions when the group converges. Finally, we verify our allocation model and optimization algorithm through example simulations. The simulation results show that, compared with the traditional method, the stacking plans generated by this method have a lower secondary-sorting workload and higher stacking stability when using the same number of storage racks.

Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe-S Cluster Biosynthesis.

Iron-sulfur (Fe-S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe-S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mössbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe-S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mössbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe-S cluster assembly process and establish that the initiation of Fe-S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.

Single‐molecule imaging reveals Tau trapping at nanometer‐sized dynamic hot spots near the plasma membrane that persists after microtubule perturbation and cholesterol depletion

Accumulation of aggregates of the microtubule‐binding protein Tau is a pathological hallmark of Alzheimer's disease. While Tau is thought to primarily associate with microtubules, it also interacts with and localizes to the plasma membrane. However, little is known about how Tau behaves and organizes at the plasma membrane of live cells. Using quantitative, single‐molecule imaging, we show that Tau exhibits spatial and kinetic heterogeneity near the plasma membrane of live cells, resulting in the formation of nanometer‐sized hot spots. The hot spots lasted tens of seconds, much longer than the short dwell time (∼ 40 ms) of Tau on microtubules. Pharmacological and biochemical disruption of Tau/microtubule interactions did not prevent hot spot formation, suggesting that these are different from the reported Tau condensation on microtubules. Although cholesterol removal has been shown to reduce Tau pathology, its acute depletion did not affect Tau hot spot dynamics. Our study identifies an intrinsic dynamic property of Tau near the plasma membrane that may facilitate the formation of assembly sites for Tau to assume its physiological and pathological functions.