External Scaffolding Research Articles

The Experience of Laboratory Learning – How Do Chemistry Students Perceive Their Learning Environment?

Universities have in the recent years faced new challenges as the new generations of students enter the old institutions. We have to rethink both the physical facilities and the practices of higher education. Many of the incomers belong to the so-called digital natives who use various digital applications and mobile devices as integrated parts in their everyday lives. Their knowledge seeking and knowledge sharing activities are different from previous generations. This study stems from a need to acknowledge this gap in research on the changes in knowledge practices and their relationship to learning. The participants were university chemistry students, who were interviewed concerning their perceptions of the learning environments. The interviews revealed a variation in students’ perceptions. There was also a variation in how they felt the physical tools provided affordances for their cognition. Some students needed much more external scaffolding and procedural facilitation for their laboratory work. The results are in line with previous research on the domain-specific features in sciences. In the future, the results are going to be looked at more in-depth in relation to students’ epistemological beliefs.

Study on Application Technology of the Integral Lifting Scaffold

An integral lifting scaffold is a external scaffolding which used in high-rise building structure and outside decoration. The integral lifting scaffold gets lifting and descending function based on the cantilever scaffold and the suspended scaffold, which have the falling prevention device and the upsetting prevention device. The hook hanging on the bearing supporting frame of the elevator was connected with the scaffold bearing supporting frame, the electric hoist achieved overall lifting or descending, the signal generator and automatic console controlled synchronous lifting. It was indicated by field application that the integral lifting scaffold can integrally lift and descend, and it can shard to lift and descend in order to meet the construction requirements of stage structure and decoration.

Discs Large 1 (Dlg1) Scaffolding Protein Participates with Clathrin and Adaptator Protein Complex 1 (AP-1) in Forming Weibel-Palade Bodies of Endothelial Cells

Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network specifically require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. Discs large 1 (Dlg1) is generally considered to be a modular scaffolding protein implicated in the control of cell polarity in a large variety of cells by specific recruiting of receptors, channels, or signaling proteins to specialized zones of the plasma membrane. We propose here that in endothelial cells, Dlg1, in a complex with AP-1 and clathrin, participates in the biogenesis of WPBs. Supporting data show that Dlg1 colocalizes with microtubules, intermediate filaments, and Golgi markers. Tandem mass spectrometry experiments led to the identification of clathrin as an Dlg1-interacting partner. Interaction was confirmed by in situ proximity ligation assays. Furthermore, AP-1 and VWF immunoprecipitate and colocalize with Dlg1 in the juxtanuclear zone. Finally, Dlg1 depletion by siRNA duplexes disrupts trans-Golgi network morphology and WPB formation. Our results provide the first evidence for an unexpected role of Dlg1 in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells.

Effects of an Early Conformational Switch Defect during ϕX174 Morphogenesis Are Belatedly Manifested Late in the Assembly Pathway

C-terminal, aromatic amino acids in the ϕX174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B(-) mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B(-) assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.

Imitation by social interaction? Analysis of a minimal agent-based model of the correspondence problem

One of the major challenges faced by explanations of imitation is the “correspondence problem”: how is an agent able to match its bodily expression to the observed bodily expression of another agent, especially when there is no possibility of external self-observation? Current theories only consider the possibility of an innate or acquired matching mechanism belonging to an isolated individual. In this paper we evaluate an alternative that situates the explanation of imitation in the inter-individual dynamics of the interaction process itself. We implemented a minimal model of two interacting agents based on a recent psychological study of imitative behavior during minimalist perceptual crossing. The agents cannot sense the configuration of their own body, and do not have access to other's body configuration, either. And yet surprisingly they are still capable of converging on matching bodily configurations. Analysis revealed that the agents solved this version of the correspondence problem in terms of collective properties of the interaction process. Contrary to the assumption that such properties merely serve as external input or scaffolding for individual mechanisms, it was found that the behavioral dynamics were distributed across the model as a whole.

In Vitro Assembly of the øX174 Procapsid from External Scaffolding Protein Oligomers and Early Pentameric Assembly Intermediates

Bacteriophage øX174 morphogenesis requires two scaffolding proteins: an internal species, similar to those employed in other viral systems, and an external species, which is more typically associated with satellite viruses. The current model of øX174 assembly is based on structural and in vivo data. During morphogenesis, 240 copies of the external scaffolding protein mediate the association of 12 pentameric particles into procapsids. The hypothesized pentameric intermediate, the 12S⁎ particle, contains 16 proteins: 5 copies each of the coat, spike and internal scaffolding proteins and 1 copy of the DNA pilot protein. Assembly naïve 12S⁎ particles and external scaffolding oligomers, most likely tetramers, formed procapsid-like particles in vitro, suggesting that the 12S⁎ particle is a bona fide assembly intermediate and validating the current model of procapsid morphogenesis. The in vitro system required a crowding agent, was influenced by the ratio of the reactants and was most likely driven by hydrophobic forces. While the system reported here shared some characteristics with other in vitro internal scaffolding protein-mediated systems, it displayed unique features. These features most likely reflect external scaffolding protein-mediated morphogenesis and the øX174 procapsid structure, in which external scaffolding–scaffolding protein interactions, as opposed to coat–coat protein interactions between pentamers, constitute the primary lattice-forming contacts.

From Resistance to Stimulation: the Evolution of a Virus in the Presence of a Dominant Lethal Inhibitory Scaffolding Protein

By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks ϕX174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.

Neuroeconomics: Constructing identity

The paper asks whether neuroeconomics will make instrumental use of neuroscience to adjudicate existing disputes in economics or be more seriously transformed by neuroscience in ways that might transform economics. The paper pursues the question by asking how neuroscience constructs an understanding of individuals as whole persons. The body of the paper is devoted to examining two approaches: Don Ross's neurocellular approach to neuroeconomics and Joseph Dumit's cultural anthropological science organization approach. The accounts are used to identify boundaries on single individual explanations. Within that space Andy Clark's external scaffolding view and Nathaniel Wilcox's socially distributed cognition view are employed.

Initial Experience with a New Method of External Polyester Scaffolding for Infrainguinal Vein Grafts

This study aims to evaluate the feasibility of external polyester scaffolding in infrainguinal bypass grafting when available vein material is suboptimal due to varicosity or dilatation. Primary objectives were short-term primary patency, assisted primary patency and secondary patency. Secondary objectives were to assess the rate of graft stenoses, infections and other adverse effects related to the use of external scaffolding. A total of 50 consecutive patients were included in this prospective, multicentre, feasibility study from six centres. The indication for infrainguinal bypass was critical limb ischaemia (64%), severe claudication (34%) or popliteal aneurysm (2%). Indications for the use of the external scaffolding were varicosity of the vein graft, ectatic vein graft or the use of spliced vein grafts with segments of widely differing diameters. Duplex scanning of the graft was done perioperatively and at follow-up visits at 1, 3, 6 and 12 months after operation. Primary patency, assisted primary patency and secondary patency at 6 months were 82.3% (+/-SE 6.2%), 88.6% (+/-SE 4.8%) and 92.1% (+/-SE 4.4%), respectively. Six graft stenoses were detected in duplex surveillance. There were no infections related to polyester mesh. External scaffolding of infrainguinal vein grafts may be a promising innovation. Early results from this multicentre study show that polyester mesh is safe and feasible adjunct to peripheral revascularization enabling the use of otherwise non-optimal vein grafts with acceptable short-term patency.

Complete Virion Assembly with Scaffolding Proteins Altered in the Ability To Perform a Critical Conformational Switch

In the phiX174 procapsid, 240 external scaffolding proteins form a nonquasiequivalent lattice. To achieve this arrangement, the four structurally unique subunits must undergo position-dependent conformational switches. One switch is mediated by glycine residue 61, which allows a 30 degrees kink to form in alpha-helix 3 in two subunits, whereas the helix is straight in the other two subunits. No other amino acid should be able to produce a bend of this magnitude. Accordingly, all substitutions for G61 are nonviable but mutant proteins differ vis-à-vis recessive and dominant phenotypes. As previously reported, amino acid substitutions with side chains larger than valine confer dominant lethal phenotypes. Alone, these mutant proteins appear to have little or no biological activity but rather require the wild-type protein to interact with other structural proteins. Proteins with conservative substitutions for G61, serine and alanine, have now been characterized. Unlike the dominant lethal proteins, these proteins do not require wild-type subunits to interact with other viral proteins and cause assembly defects reminiscent of those conferred by the lethal dominant proteins in concert with wild-type subunits. Although atomic structures suggest that only a glycine residue can provide the proper torsion angle for assembly, mutants that can productively utilize the altered external scaffolding proteins were isolated, and the mutations were mapped to the coat and internal scaffolding proteins. Thus, the ability to isolate strains that could utilize the single mutant D protein species would not have been predicted from past structural analyses.

N-terminal deletions of the øX174 external scaffolding protein affect the timing and fidelity of assembly

The first α-helices of Microviridae external scaffolding proteins function as coat protein substrate specificity domains. Mutations in this helix can lengthen the lag phase before progeny production. 5′ deletion genes, encoding N-terminal deletion proteins, were constructed on plasmids and in the øX174 genome. Proteins lacking the first seven amino acids were able to rescue a nullD mutant when expressed from a plasmid. However, the lag phase before progeny production was lengthened. The øX174 mutant with the corresponding genomic gene grew very poorly. The molecular basis of the defective phenotype was complex. External scaffolding protein levels were reduced compared to wild-type and most of the viral coat protein in mutant infected cells appears to be siphoned off the assembly pathway. Second-site suppressors of the growth defects were isolated and appear to act via two different mechanisms. One class of suppressors most likely acts by altering mutant external scaffolding protein expression while the second class of suppressors appears to act on the level of protein–protein interactions.

Scaffolding Proteins Altered in the Ability To Perform a Conformational Switch Confer Dominant Lethal Assembly Defects

In the phiX174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D(1)D(2) and D(3)D(4), in a non-quasi-equivalent structure. To achieve this arrangement, alpha-helix 3 assumes two different conformations: (i) kinked 30 degrees at glycine residue 61 in subunits D(1) and D(3) and (ii) straight in subunits D(2) and D(4). Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in alpha-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein.

Being Where? Andy Clark and the Problem of Advanced Cognition

Andy Clark (1989, 1993, 1997) is a leading philosophical exponent of a view of mind as an ‘associative engine’, or connectionist pattern-completer, composed of multiple special-purpose modules that communicate in only limited ways and eschew detailed forms of internal representation. The modules, Clark and his allies argue, are both coordinated and integrated by the enviromnent, whilst ‘off-loading’ onto it by calling on external computational resources (‘external scaffolds’) to reduce cognitive load. Defenders of this position further maintain that even examples of sophisticated and distinctively human cognition, such as long-term planning or running a multi-national company, emerge from coimectionist pattern-completing brains in the ‘constraining presence of public language, culture and institutions’ (1997: 33). This constellation of ideas, Clark argues, amounts to a completely new science of mind that radically reforms ‘our whole way of thinking about intelligent behaviour’. Unfortunately, this rhetoric far outstrips the evidence: while a reasonable case can be made that external scaffolds are necessary for many types of cognition, the assertion that pattern-completion plus external scaffolding is a sufficient explanation of all human cognition has not been demonstrated. The insufficiency of the Clarkian view is particularly evident in the case of advanced cognition in the economic sphere.

Incorporation of scaffolding protein gpO in bacteriophages P2 and P4

Scaffolding proteins act as chaperones for the assembly of numerous viruses, including most double-stranded DNA bacteriophages. In bacteriophage P2, an internal scaffolding protein, gpO, is required for the assembly of correctly formed viral capsids. Bacteriophage P4 is a satellite phage that has acquired the ability to take control of the P2 genome and use the P2 capsid protein gpN to assemble a capsid that is smaller than the normal P2 capsid. This size determination is dependent on the P4 external scaffolding protein Sid. Although Sid is sufficient to form morphologically correct P4-size capsids, the P2 internal scaffolding protein gpO is required for the formation of viable capsids of both P2 and P4. In most bacteriophages, the scaffolding protein is either proteolytically degraded or exits intact from the capsid after assembly. In the P2/P4 system, however, gpO is cleaved to an N-terminal fragment, O ⁎, that remains inside the mature capsid after DNA packaging. We previously showed that gpO exhibits autoproteolytic activity, which is abolished by removal of the first 25 amino acids. Co-expression of gpN with this N-terminally truncated version of gpO leads to the production of immature P2 procapsid shells. Here, we use protein analysis and mass spectroscopy to show that P2 and P4 virions as well as procapsids isolated from viral infections contain O ⁎ and that cleavage occurs between residues 141 and 142 of gpO. By co-expression of gpN with truncated gpO proteins, we show that O ⁎ binds to gpN and retains the proteolytic activity of gpO and that the C-terminal 90 residues of gpO (residues 195–284) are sufficient to promote the formation of P2-size procapsids. Using mass spectrometry, we have also identified the head completion protein gpL in the virions.

Eliminating the Requirement of an Essential Gene Product in an Already Very Small Virus: Scaffolding Protein B-free øX174, B-free

Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage øX174 morphogenesis. The external scaffolding protein D is highly ordered in the atomic structure and proper function is very sensitive to mutation. In contrast, the internal scaffolding protein B is relatively unordered and extensive alterations do not eliminate function. Despite this genetic laxity, protein B is absolutely required for virus assembly. Thus, this system, with its complex arrangements of overlapping reading frames, can be regarded as an example of “irreducible complexity.” To address the biochemical functions of a dual scaffolding protein system and the evolution of complexity, progressive and targeted genetic selections were employed to lessen and finally eliminate B protein-dependence. The biochemical and genetic bases of adaptation were characterized throughout the analysis that led to the sextuple mutant with a B-independent phenotype, as evaluated by plaque formation in wild-type cells. The primary adaptation appears to be the over-expression of a mutant external scaffolding protein. Progeny production was followed in lysis-resistant cells. The ability to produce infectious virions does not require all six mutations. However, the lag phase before progeny production is shortened as mutations accumulate. The results suggest that the primary function of the internal scaffolding protein may be to lower the critical concentration of the external scaffolding protein needed to nucleate procapsid formation. Moreover, they demonstrate a novel mechanism by which a stringently required gene product can be bypassed, even in a system encoding only eight strictly essential proteins.

Characterization and Function of Putative Substrate Specificity Domain in Microvirus External Scaffolding Proteins

Microviruses (canonical members are bacteriophages phiX174, G4, and alpha3) are T=1 icosahedral virions with an assembly pathway mediated by two scaffolding proteins. The external scaffolding protein D plays a major role during morphogenesis, particularly in icosahedral shell formation. The results of previous studies, conducted with a cloned chimeric external scaffolding gene, suggest that the first alpha-helix acts as a substrate specificity domain, perhaps mediating the initial coat-external scaffolding protein interaction. However, the expression of a cloned gene could lead to protein concentrations higher than those found in typical infections. Moreover, its induction before infection could alter the timing of the protein's accumulation. Both of these factors could drive or facilitate reactions that may not occur under physiological conditions or before programmed cell lysis. In order to elucidate a more detailed mechanistic model, a chimeric external scaffolding gene was placed directly in the phiX174 genome under wild-type transcriptional and translational control, and the chimeric virus, which was not viable on the level of plaque formation, was characterized. The results of the genetic and biochemical analyses indicate that alpha-helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production, perhaps lowering the critical concentration of the chimeric protein required for a nucleation reaction.

RecA-DNA filament topology: The overlooked alternative of an unconventional syn–syn duplex intermediate

The helical filaments of RecA protein mediate strand exchange for homologous recombination, but the paths of the interacting DNAs have yet to be determined. Although this interaction is commonly limited to three strands, it is reasoned here that the intrinsic symmetry relationships of quadruplex topology are superior in explaining a range of observations. In particular, this topology suggests the potential of post-exchange base pairing in the unorthodox configuration of syn–syn glycosidic bonds between the nucleotide bases and the pentose rings in the sugar–phosphate backbone, which would transiently be stabilized by the external scaffolding of the RecA protein filament.

Identification of an Interacting Coat-External Scaffolding Protein Domain Required for both the Initiation of φX174 Procapsid Morphogenesis and the Completion of DNA Packaging

The phiX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven alpha-helices. The protein's core, alpha-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are phiX174, G4, and alpha3) external scaffolding proteins. On the other hand, the primary sequences of the first alpha-helices have diverged. The results of previous studies with alpha3/phiX174 chimeric external scaffolding proteins suggest that alpha-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and phiX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. phiX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.

Conformational Switching by the Scaffolding Protein D Directs the Assembly of Bacteriophage φX174

The three-dimensional structure of bacteriophage φX174 external scaffolding protein D, prior to its interaction with other structural proteins, has been determined to 3.3 Å by X-ray crystallography. The crystals belong to space group P4 12 12 with a dimer in the asymmetric unit that closely resembles asymmetric dimers observed in the φX174 procapsid structure. Furthermore, application of the crystallographic 4 1 symmetry operation to one of these dimers generates a tetramer similar to the tetramer in the icosahedral asymmetric unit of the procapsid. These data suggest that both dimers and tetramers of the D protein are true morphogenetic intermediates and can form independently of other proteins involved in procapsid morphogenesis. The crystal structure of the D scaffolding protein thus represents the state of the polypeptide prior to procapsid assembly. Hence, comparison with the procapsid structure provides a rare opportunity to follow the conformational switching events necessary for the construction of complex macromolecular assemblies.

The Functions of the N Terminus of the φX174 Internal Scaffolding Protein, a Protein Encoded in an Overlapping Reading Frame in a Two Scaffolding Protein System

φX174 utilizes two scaffolding proteins during morphogenesis, an internal protein (B) and an external protein (D). The B protein induces a conformational change in coat protein pentamers, enabling them to interact with both spike and external scaffolding proteins. While functions of the carboxyl terminus of protein B have been defined, the functions of the amino terminus remain obscure. To investigate the morphogenetic functions of the amino terminus, several 5′ deleted genes were constructed and the proteins expressed in vivo. The ΔNH 2 B proteins were assayed for the ability to complement an ochre B mutant and defects in the morphogenetic pathway were characterized. The results of the biochemical, genetic and second-site genetic analyses indicate that the amino terminus induces conformational changes in the viral coat protein and facilitates minor spike protein incorporation. Defects in conformational switching can be suppressed by substitutions in the external scaffolding protein, suggesting some redundancy of function between the two proteins.