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While the movement for open research has gained momentum in recent years, there remain concerns about the broader commitment to openness in knowledge production and dissemination. Increasingly, universities are under pressure to transform themselves to engage with the wider community and to be more inclusive. Open knowledge institutions (OKIs) provide a framework that encourages universities to act with the principles of openness at their centre; not only should universities embrace digital open access (OA), but also lead actions in cultivating diversity, equity, transparency and positive changes in society. This leads to questions of whether we can evaluate the progress of OKIs and what are potential indicators for OKIs. As an exploratory study, this article reports on the collection and analysis of a list of potential OKI indicators. Data for these indicators are gathered for 43 Australian universities. The indicators provide high-dimensional and complex signals about university performances. They show evidence of large disparities in characteristics such as Indigenous employment and gender equity, and a preference for repository-mediated OA across Australian universities. We demonstrate use of the OKI evaluation framework to categorise these indicators into three platforms of diversity, communication and coordination. The analysis provides new insights into the Australian open knowledge landscape and ways of mapping different paths of OKIs., Competing Interests: The authors declare that they have no competing interests., (© 2021 Huang et al.)

Background: Inaccurate citations are erroneous quotations or instances of paraphrasing of previously published material that mislead readers about the claims of the cited source. They are often unaddressed due to underreporting, the inability of peer reviewers and editors to detect them, and editors' reluctance to publish corrections about them. In this paper, we propose a new tool that could be used to tackle their circulation., Methods: We provide a review of available data about inaccurate citations and analytically explore current ways of reporting and dealing with these inaccuracies. Consequently, we make a distinction between publication (i.e., first occurrence) and circulation (i.e., reuse) of inaccurate citations. Sloppy reading of published items, literature ambiguity and insufficient quality control in the editorial process are identified as factors that contribute to the publication of inaccurate citations. However, reiteration or copy-pasting without checking the validity of citations, paralleled with lack of resources/motivation to report/correct inaccurate citations contribute to their circulation., Results and Discussion: We propose the development of an online annotation tool called "MyCites" as means with which to mark and map inaccurate citations. This tool allows ORCID users to annotate citations and alert authors (of the cited and citing articles) and also editors of journals where inaccurate citations are published. Each marked citation would travel with the digital version of the document (persistent identifiers) and be visible on websites that host peer-reviewed articles (journals' websites, Pubmed, etc.). In the future development of MyCites, challenges such as the conditions of correct/incorrect-ness and parties that should adjudicate that, and, the issue of dealing with incorrect reports need to be addressed., Competing Interests: Competing interestsNot applicable., (© The Author(s) 2020.)

The proportion of research outputs published in open access journals or made available on other freely-accessible platforms has increased over the past two decades, driven largely by funder mandates, institutional policies, grass-roots advocacy, and changing attitudes in the research community. However, the relative effectiveness of these different interventions has remained largely unexplored. Here we present a robust, transparent and updateable method for analysing how these interventions affect the open access performance of individual institutes. We studied 1,207 institutions from across the world, and found that, in 2017, the top-performing universities published around 80-90% of their research open access. The analysis also showed that publisher-mediated (gold) open access was popular in Latin American and African universities, whereas the growth of open access in Europe and North America has mostly been driven by repositories., Competing Interests: CH, CN, RH, LM, KW, AO, CB No competing interests declared, (© 2020, Huang et al.)

Peer review of research articles is a core part of our scholarly communication system. In spite of its importance, the status and purpose of peer review is often contested. What is its role in our modern digital research and communications infrastructure? Does it perform to the high standards with which it is generally regarded? Studies of peer review have shown that it is prone to bias and abuse in numerous dimensions, frequently unreliable, and can fail to detect even fraudulent research. With the advent of Web technologies, we are now witnessing a phase of innovation and experimentation in our approaches to peer review. These developments prompted us to examine emerging models of peer review from a range of disciplines and venues, and to ask how they might address some of the issues with our current systems of peer review. We examine the functionality of a range of social Web platforms, and compare these with the traits underlying a viable peer review system: quality control, quantified performance metrics as engagement incentives, and certification and reputation. Ideally, any new systems will demonstrate that they out-perform current models while avoiding as many of the biases of existing systems as possible. We conclude that there is considerable scope for new peer review initiatives to be developed, each with their own potential issues and advantages. We also propose a novel hybrid platform model that, at least partially, resolves many of the technical and social issues associated with peer review, and can potentially disrupt the entire scholarly communication system. Success for any such development relies on reaching a critical threshold of research community engagement with both the process and the platform, and therefore cannot be achieved without a significant change of incentives in research environments., Competing Interests: Competing interests: JPT works for ScienceOpen; TRH works for OpenAIRE.

Peer review of research articles is a core part of our scholarly communication system. In spite of its importance, the status and purpose of peer review is often contested. What is its role in our modern digital research and communications infrastructure? Does it perform to the high standards with which it is generally regarded? Studies of peer review have shown that it is prone to bias and abuse in numerous dimensions, frequently unreliable, and can fail to detect even fraudulent research. With the advent of web technologies, we are now witnessing a phase of innovation and experimentation in our approaches to peer review. These developments prompted us to examine emerging models of peer review from a range of disciplines and venues, and to ask how they might address some of the issues with our current systems of peer review. We examine the functionality of a range of social Web platforms, and compare these with the traits underlying a viable peer review system: quality control, quantified performance metrics as engagement incentives, and certification and reputation. Ideally, any new systems will demonstrate that they out-perform and reduce the biases of existing models as much as possible. We conclude that there is considerable scope for new peer review initiatives to be developed, each with their own potential issues and advantages. We also propose a novel hybrid platform model that could, at least partially, resolve many of the socio-technical issues associated with peer review, and potentially disrupt the entire scholarly communication system. Success for any such development relies on reaching a critical threshold of research community engagement with both the process and the platform, and therefore cannot be achieved without a significant change of incentives in research environments., Competing Interests: Competing interests: JPT works for ScienceOpen and is the founder of paleorXiv; DG is on the Editorial Board of Journal of Open Research Software and RIO Journal; TRH and LM work for OpenAIRE; LM works for Aletheia; DM is a co-founder of RIO Journal, on the Editorial Board of PLOS Computational Biology and on the Board of WikiProject Med; DRB is the founder of engrXiv and the Journal of Open Engineering; KN, DSK, and CRM are on the Editorial Board of the Journal of Open Source Software; DSK is an academic editor for PeerJ Computer Science; CN and DPD are the President and Vice-President of FORCE11, respectively.

Peer review of research articles is a core part of our scholarly communication system. In spite of its importance, the status and purpose of peer review is often contested. What is its role in our modern digital research and communications infrastructure? Does it perform to the high standards with which it is generally regarded? Studies of peer review have shown that it is prone to bias and abuse in numerous dimensions, frequently unreliable, and can fail to detect even fraudulent research. With the advent of web technologies, we are now witnessing a phase of innovation and experimentation in our approaches to peer review. These developments prompted us to examine emerging models of peer review from a range of disciplines and venues, and to ask how they might address some of the issues with our current systems of peer review. We examine the functionality of a range of social Web platforms, and compare these with the traits underlying a viable peer review system: quality control, quantified performance metrics as engagement incentives, and certification and reputation. Ideally, any new systems will demonstrate that they out-perform and reduce the biases of existing models as much as possible. We conclude that there is considerable scope for new peer review initiatives to be developed, each with their own potential issues and advantages. We also propose a novel hybrid platform model that could, at least partially, resolve many of the socio-technical issues associated with peer review, and potentially disrupt the entire scholarly communication system. Success for any such development relies on reaching a critical threshold of research community engagement with both the process and the platform, and therefore cannot be achieved without a significant change of incentives in research environments., Competing Interests: Competing interests: JPT works for ScienceOpen and is the founder of paleorXiv; TRH and LM work for OpenAIRE; LM works for Aletheia; DM is a co-founder of RIO Journal, on the Editorial Board of PLOS Computational Biology and on the Board of WikiProject Med; CN and DPD are the President and Vice-President of FORCE11, respectively.

Increasingly, preprints are at the center of conversations across the research ecosystem. But disagreements remain about the role they play. Do they "count" for research assessment? Is it ok to post preprints in more than one place? In this paper, we argue that these discussions often conflate two separate issues, the history of the manuscript and the status granted it by different communities. In this paper, we propose a new model that distinguishes the characteristics of the object, its "state", from the subjective "standing" granted to it by different communities. This provides a way to discuss the difference in practices between communities, which will deliver more productive conversations and facilitate negotiation, as well as sharpening our focus on the role of different stakeholders on how to collectively improve the process of scholarly communications not only for preprints, but other forms of scholarly contributions., Competing Interests: Competing interests: DP is employed by, and owns stock in, Research Square LLP, a company that provides editorial services for authors and publishers. JL and GB are employed by Crossref, a provider of scholarly metadata.

Competing Interests: The author is an employee of PLOS whose salary is supported by PLOS income derived from the publication of Open Access papers.

Background: The electronic laboratory notebook (ELN) has the potential to replace the paper notebook with a marked-up digital record that can be searched and shared. However, it is a challenge to achieve these benefits without losing the usability and flexibility of traditional paper notebooks. We investigate a blog-based platform that addresses the issues associated with the development of a flexible system for recording scientific research., Methodology/principal Findings: We chose a blog-based approach with the journal characteristics of traditional notebooks in mind, recognizing the potential for linking together procedures, materials, samples, observations, data, and analysis reports. We implemented the LabTrove blog system as a server process written in PHP, using a MySQL database to persist posts and other research objects. We incorporated a metadata framework that is both extensible and flexible while promoting consistency and structure where appropriate. Our experience thus far is that LabTrove is capable of providing a successful electronic laboratory recording system., Conclusions/significance: LabTrove implements a one-item one-post system, which enables us to uniquely identify each element of the research record, such as data, samples, and protocols. This unique association between a post and a research element affords advantages for monitoring the use of materials and samples and for inspecting research processes. The combination of the one-item one-post system, consistent metadata, and full-text search provides us with a much more effective record than a paper notebook. The LabTrove approach provides a route towards reconciling the tensions and challenges that lie ahead in working towards the long-term goals for ELNs. LabTrove, an electronic laboratory notebook (ELN) system from the Smart Research Framework, based on a blog-type framework with full access control, facilitates the scientific experimental recording requirements for reproducibility, reuse, repurposing, and redeployment.

Puroindolines (Pins) and purothionins (Pths) are basic, amphiphilic, cysteine-rich wheat proteins that play a role in plant defense against microbial pathogens. This study examined the co-adsorption and sequential addition of Pins (Pin-a, Pin-b, and a mutant form of Pin-b with Trp-44 to Arg-44 substitution) and β-purothionin (β-Pth) model anionic lipid layers using a combination of surface pressure measurements, external reflection FTIR spectroscopy, and neutron reflectometry. Results highlighted differences in the protein binding mechanisms and in the competitive binding and penetration of lipid layers between respective Pins and β-Pth. Pin-a formed a blanket-like layer of protein below the lipid surface that resulted in the reduction or inhibition of β-Pth penetration of the lipid layer. Wild-type Pin-b participated in co-operative binding with β-Pth, whereas the mutant Pin-b did not bind to the lipid layer in the presence of β-Pth. The results provide further insight into the role of hydrophobic and cationic amino acid residues in antimicrobial activity.

Studying the structure of protein-lipid complexes, be they in vesicles, planar bilayers, monolayers, or nanodiscs, poses two particular challenges. Firstly such complexes are often dynamic. Secondly we need to resolve the lipid and protein structures within the complex. Neutron scattering is well placed to help in both respects since it deals with molecules in large, complex, dynamic structures and can easily differentiate between different molecular species. This comes from the great penetrating power of neutrons and their sensitivity to the difference between hydrogen (H) and deuterium (D). Both membrane proteins and lipids can be produced with varying degrees of deuteration, thus allowing us to dissect complexes with great accuracy. Two main scattering techniques are immediately applicable to the study of protein-lipid interactions. Neutron reflection exploits the constructive interference, which occurs when neutrons are reflected from different points in a layer. An everyday example is the rainbow of colors reflected from an oil film on water, which result from varying film thickness and the angle of reflection. Neutrons because of their short wavelengths (4-15 Å) and H/D sensitivity can, in reflectometry mode, provide accurate cross sections of lipid monolayers and bilayers. Small-angle neutron scattering (SANS) can resolve the structures of protein-lipid complexes if they are present as homogeneous dispersions. This is easiest with detergent micelles, but increasingly methods are being developed whereby vesicles, nanodiscs, etc., can be resolved. Again the ability to deuterate proteins and lipids enables SANS to resolve the inner structure of big, dynamic, lipid-protein complexes. The recent introduction of advanced neutron beam lines means that the technique is now within the grasp of a broad cross section of researchers.

The plant defence proteins α1- and α2-purothionin (Pth) are type 1 thionins from common wheat (Triticum aestivum). These highly homologous proteins possess characteristics common amongst antimicrobial peptides and proteins, that is, cationic charge, amphiphilicity and hydrophobicity. Both α1- and α2-Pth possess the same net charge, but differ in relative hydrophobicity as determined by C18 reversed phase HPLC. Brewster angle microscopy, X-ray and neutron reflectometry, external reflection FTIR and associated surface pressure measurements demonstrated that α1 and α2-Pth interact strongly with condensed phase 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) monolayers at the air/liquid interface. Both thionins disrupted the in-plane structure of the anionic phospholipid monolayers, removing lipid during this process and both penetrated the lipid monolayer in addition to adsorbing as a single protein layer to the lipid head-group. However, analysis of the interfacial structures revealed that the α2-Pth showed faster disruption of the lipid film and removed more phospholipid (12%) from the interface than α1-Pth. Correlating the protein properties and lipid binding activity suggests that hydrophobicity plays a key role in the membrane lipid removal activity of thionins.

The indolines and thionins are basic, amphiphilic and cysteine-rich proteins found in cereals; puroindoline-a (Pin-a) and β-purothionin (β-Pth) are members of these families in wheat (Triticum aestivum). Pin-a and β-Pth have been suggested to play a significant role in seed defence against microbial pathogens, making the interaction of these proteins with model bacterial membranes an area of potential interest. We have examined the binding of these proteins to lipid monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) using a combination of neutron reflectometry, Brewster angle microscopy, and infrared spectroscopy. Results showed that both Pin-a and β-Pth interact strongly with condensed phase DPPG monolayers, but the degree of penetration was different. β-Pth was shown to penetrate the lipid acyl chain region of the monolayer and remove lipids from the air/liquid interface during the adsorption process, suggesting this protein may be able to both form membrane spanning ion channels and remove membrane phospholipids in its lytic activity. Conversely, Pin-a was shown to interact mainly with the head-group region of the condensed phase DPPG monolayer and form a 33 Å thick layer below the lipid film. The differences between the interfacial structures formed by these two proteins may be related to the differing composition of the Pin-a and β-Pth hydrophobic regions.

The self-assembly in solution of puroindoline-a (Pin-a), an amphiphilic lipid binding protein from common wheat, was investigated by small angle neutron scattering, dynamic light scattering and size exclusion chromatography. Pin-a was found to form monodisperse prolate ellipsoidal micelles with a major axial radius of 112 ± 4.5 Å and minor axial radius of 40.4 ± 0.18 Å. These protein micelles were formed by the spontaneous self-assembly of 38 Pin-a molecules in solution and were stable over a wide pH range (3.5-11) and at elevated temperatures (20-65 °C). Pin-a micelles could be disrupted upon addition of the non-ionic surfactant dodecyl-β-maltoside, suggesting that the protein self-assembly is driven by hydrophobic forces, consisting of intermolecular interactions between Trp residues located within a well-defined Trp-rich domain of Pin-a., (© The Owner Societies 2011)

Background: Directed protein evolution has been used to modify protein activity and research has been carried out to enhance the production of high quality mutant libraries. Many theoretical approaches suggest that allowing a population to undergo neutral selection may be valuable in directed evolution experiments., Findings: Here we report on an investigation into the value of neutral selection in a classical model system for directed evolution, the conversion of the E. coli β-glucuronidase to a β-galactosidase activity. We find that neutral selection, i.e. selection for retaining glucuronidase activity, can efficiently identify the majority of sites of mutation that have been identified as beneficial for galactosidase activity in previous experiments. Each variant demonstrating increased galactosidase activity identified by our neutral drift experiments contained a mutation at one of four sites, T509, S557, N566 or W529. All of these sites have previously been identified using direct selection for beta galactosidase activity., Conclusions: Our results are consistent with others that show that a neutral selection approach can be effective in selecting improved variants. However, we interpret our results to show that neutral selection is, in this case, not a more efficient approach than conventional directed evolution approaches. However, the neutral approach is likely to be beneficial when the resulting library can be screened for a range of related activities. More detailed statistical studies to resolve the apparent differences between this system and others are likely to be a fruitful avenue for future research.

In this work, the kinetics and dissociation constant for the binding of a biotin-modified oligonucleotide to microparticle-immobilized avidin were measured. Avidin has been immobilized by both covalent coupling and bioaffinity capture to a surface prefunctionalized with biotin. The measured rate and equilibrium dissociation constants of avidin immobilized by these different methods have been compared with those for nonimmobilized avidin. We found that immobilization resulted in both a decrease in the rate of binding and an increase in the rate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7 ± 3 × 10(-12) M, higher than the value measured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitude larger than values for the wild-type avidin-biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, which resulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed little variation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings are critical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin-biotin interaction.

A more flexible nucleotide building block for the synthesis of new DNA based porphyrin-zipper arrays is described. Changing the rigid acetylene linker between the porphyrin substituent and the 2'-deoxyuridine to a more flexible propargyl amide containing linkage leads in part to an increased duplex stability. The CD spectra reveal different electronic interactions between the porphyrins depending on the type of linker used. Molecular modelling suggests large variation of the relative orientation of the porphyrins within the major groove of the DNA. The porphyrins can be metallated post-synthetically with different metals as shown with zinc, cobalt and copper. The spectroscopic features do not alter drastically upon metallation apart from the CD spectra, and the stability of the metal complex is highly dependent on the nature of the metal. As shown by CD spectroscopy, the zinc porphyrin is rapidly demetallated at high temperatures. Globular structure determination using SAXS indicates that a molecular assembly comprised of a two to four helical bundle dominates in solution at higher concentrations (≥50 μM) which is not observed by spectroscopy at lower concentrations (≤1 μM).

Competing Interests: The authors have declared that no competing interests exist.

The means we use to record the process of carrying out research remains tied to the concept of a paginated paper notebook despite the advances over the past decade in web based communication and publication tools. The development of these tools offers an opportunity to re-imagine what the laboratory record would look like if it were re-built in a web-native form. In this paper I describe a distributed approach to the laboratory record based which uses the most appropriate tool available to house and publish each specific object created during the research process, whether they be a physical sample, a digital data object, or the record of how one was created from another. I propose that the web-native laboratory record would act as a feed of relationships between these items. This approach can be seen as complementary to, rather than competitive with, integrative approaches that aim to aggregate relevant objects together to describe knowledge. The potential for the recent announcement of the Google Wave protocol to have a significant impact on realizing this vision is discussed along with the issues of security and provenance that are raised by such an approach.

We present the theory of thermal equivalence in the framework of the Peyrard-Bishop model and some of its anharmonic variants. The thermal equivalence gives rise to a melting index τ which maps closely the experimental DNA melting temperatures for short DNA sequences. We show that the efficient calculation of the melting index can be used to analyse the parameters of the Peyrard-Bishop model and propose an improved set of Morse potential parameters. With this new set we are able to calculate some of the experimental melting temperatures to ± 1.2 °C. We review some of the concepts of sequencing probe design and show how to use the melting index to explore the possibilities of gene coverage by tuning the model parameters.

Quasi-elastic neutron scattering (QENS) has been used to study the deviation from Debye-law harmonic behavior in lyophilized and hydrated apoferritin, a naturally occurring, multisubunit protein. Whereas analysis of the measured mean squared displacement (msd) parameter reveals a hydration-dependent inflection above 240 K, characteristic of diffusive motion, a hydration-independent inflection is observed at 100 K. The mechanism responsible for this low-temperature anharmonic response is further investigated, via analysis of the elastic incoherent neutron scattering intensity, by applying models developed to describe side-group motion in glassy polymers. Our results suggest that the deviation from harmonic behavior is due to the onset of methyl group rotations which exhibit a broad distribution of activated processes ( E a,ave = 12.2 kJ.mol (-1), sigma = 5.0 kJ x mol (-1)). Our results are likened to those reported for other proteins.

Background: Sequencing by hybridisation is an effective method for obtaining large amounts of DNA sequence information at low cost. The efficiency of SBH depends on the design of the probe library to provide the maximum information for minimum cost. Long probes provide a higher probability of non-repeated sequences but lead to an increase in the number of probes required whereas short probes may not provide unique sequence information due to repeated sequences. We have investigated the effect of probe length, use of reference sequences, and thermal filtering on the design of probe libraries for several highly variable target DNA sequences., Results: We designed overlapping probe libraries for a range of highly variable drug target genes based on known sequence information and develop a formal terminology to describe probe library design. We find that for some targets these libraries can provide good coverage of a previously unseen target whereas for others the coverage is less than 30%. The optimal probe length varies from as short at 12 nt to as large as 19 nt and depends on the sequence, its variability, and the stringency of thermal filtering. It cannot be determined from inspection of an example gene sequence., Conclusions: Optimal probe length and the optimal number of reference sequences used to design a probe library are highly target specific for highly variable sequencing targets. The optimum design cannot be determined simply by inspection of input sequences or of alignments but only by detailed analysis of the each specific target. For highly variable sequences, shorter probes can in some cases provide better information than longer probes. Probe library design would benefit from a general purpose tool for analysing these issues. The formal terminology developed here and the analysis approaches it is used to describe will contribute to the development of such tools.

Small angle scattering can provide unique structural information on the shape, domain organisation, and interactions of biomacromolecules in solution. Small angle neutron scattering (SANS) combined with deuterium labelling makes it possible to define the positions of specific components within a complex while small angle X-ray scattering (SAXS) provides more precise data on the overall shape. Here I review four recent publications, three of which were presented at the Neutrons in Biology meeting at the STFC Rutherford Appleton Laboratory in July 2007, that utilise SANS, SAXS, and complementary techniques to define the solution structure of large multidomain proteins and macromolecular complexes. These four papers emphasise the critical importance of sample quality and characterisation as well as the important role played by complementary techniques in building structural models based on small angle scattering data. They show the ability of SANS and SAXS in determining solution structures provides an important complementary structural technique for large, flexible, and glycosylated proteins where high resolution structural techniques, such as crystallography and NMR, cannot be applied.

Microparticles incorporating micrometer-sized diffractive bar codes have been modified with oligonucleotides and immunoglobulin Gs to enable DNA hybridization and immunoassays. The bar codes are manufactured using photolithography of a chemically functional commercial epoxy photoresist (SU-8). When attached by suitable linkers, immobilized probe molecules exhibit high affinity for analytes and fast reaction kinetics, allowing detection of single nucleotide differences in DNA sequences and multiplexed immunoassays in <45 min. Analysis of raw data from assays carried out on the diffractive microparticles indicates that the reproducibility and sensitivity approach those of commercial encoding platforms. Micrometer-sized particles, imprinted with several superimposed diffraction gratings, can encode many million unique codes. The high encoding capacity of this technology along with the applicability of the manufactured bar codes to multiplexed assays will allow accurate measurement of a wide variety of molecular interactions, leading to new opportunities in diverse areas of biotechnology such as genomics, proteomics, high-throughput screening, and medical diagnostics.

Neutron radiation offers significant advantages for the study of biological molecular structure and dynamics. A broad and significant effort towards instrumental and methodological development to facilitate biology experiments at neutron sources worldwide is reviewed.

Background: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein., Methodology: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours., Conclusions: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.

SU-8 is an epoxy-novolac resin and a well-established negative photoresist for microfabrication and microengineering. The photopolymerized resist is an extremely highly crosslinked polymer showing outstanding chemical and physical robustness with residual surface epoxy groups amenable for chemical functionalization. In this paper we describe, for the first time, the preparation and surface modification of SU-8 particles shaped as microbars, the attachment of appropriate linkers, and the successful application of these particles to multistep solid-phase synthesis leading to oligonucleotides and peptides attached in an unambiguous manner to the support surface.

During chromosome synthesis in Escherichia coli, replication forks are blocked by Tus bound Ter sites on approach from one direction but not the other. To study the basis of this polarity, we measured the rates of dissociation of Tus from forked TerB oligonucleotides, such as would be produced by the replicative DnaB helicase at both the fork-blocking (nonpermissive) and permissive ends of the Ter site. Strand separation of a few nucleotides at the permissive end was sufficient to force rapid dissociation of Tus to allow fork progression. In contrast, strand separation extending to and including the strictly conserved G-C(6) base pair at the nonpermissive end led to formation of a stable locked complex. Lock formation specifically requires the cytosine residue, C(6). The crystal structure of the locked complex showed that C(6) moves 14 A from its normal position to bind in a cytosine-specific pocket on the surface of Tus.

Several methods for ultra high-throughput DNA sequencing are currently under investigation. Many of these methods yield very short blocks of sequence information (reads). Here we report on an analysis showing the level of genome sequencing possible as a function of read length. It is shown that re-sequencing and de novo sequencing of the majority of a bacterial genome is possible with read lengths of 20-30 nt, and that reads of 50 nt can provide reconstructed contigs (a contiguous fragment of sequence data) of 1000 nt and greater that cover 80% of human chromosome 1.

The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process.

Intein-mediated ligation provides a site-specific method for the attachment of molecular probes to proteins. The method is inherently flexible with regard to either the protein sequence or the attached probe, but practical difficulties have limited the widespread use of this valuable labeling system for the attachment of small- to medium-sized molecules. We report herein studies to improve the efficiency and practical application of these reactions, including the assembly of plasmids for the expression of target-intein fusion proteins and the analysis of their reaction with a fluorescent cysteine derivative under a range of conditions. Optimal ligation of the fluorophore to the target protein is critically dependent on the degree of oxidation of the fluorescent cysteine derivative. Efficient ligation has been achieved with freshly prepared fluorescent cysteine derivative under rigorously anaerobic conditions. Similar ligation yields have also been achieved using more practically convenient conditions including anaerobic reaction with addition of thiophenol, or aerobic reaction with the further addition of tricarboxyethylphosphine.

Directed molecular evolution and combinatorial methodologies are playing an increasingly important role in the field of protein engineering. The general approach of generating a library of partially randomized genes, expressing the gene library to generate the proteins the library encodes and then screening the proteins for improved or modified characteristics has successfully been applied in the areas of protein-ligand binding, improving protein stability and modifying enzyme selectivity. A wide range of techniques are now available for generating gene libraries with different characteristics. This review will discuss these different methodologies, their accessibility and applicability to non-expert laboratories and the characteristics of the libraries they produce. The aim is to provide an up to date resource to allow groups interested in using directed evolution to identify the most appropriate methods for their purposes and to guide those moving on from initial experiments to more ambitious targets in the selection of library construction techniques. References are provided to original methodology papers and other recent examples from the primary literature that provide details of experimental methods.

Oligophrenin-1 is a recently discovered Rho-GTPase activating protein, mutation of which is associated with X-linked mental retardation. Since little is known about the cellular localization of oligophrenin-1 in central and peripheral neurons, we investigated its expression by RT-PCR and immunochemical analysis. Oligophrenin-1 immunoreactivity was found in glial cells forming myelin sheaths in the vagus nerve, sciatic nerve and dorsal roots of guinea-pig, rat and human, in chromaffin cells of the adrenal medulla, and in chromaffin cells associated with sympathetic ganglia. No immunoreactivity was detected in sympathetic neurons, in glial cells surrounding these neurons, in optic nerve or in spinal cord myelin. The full length cDNA sequence was determined from guinea-pig sciatic nerve. The translated amino acid sequence was 99% identical to the published human oligophrenin-1 sequence. Western blotting revealed two protein forms which were expressed to different relative extents in different tissues. A 91 kDa form was predominant in extracts of sciatic nerve whereas a 36 kDa form was relatively more abundant in adrenal medulla and brain. Greater amounts of the full length oligophrenin-1 protein occurred in the sciatic nerve of adult rats, compared with P2 rats, which reflects the development of myelination. The presence of multiple forms does not appear to be due to alternative mRNA splicing since RT-PCR products amplified from a variety of tissues were identical and only a single mRNA transcript of 7.4 kb was identified by Northern analysis. These findings demonstrate that a major site of oligophrenin-1 expression is peripheral myelin.

Hyperpolarisation-activated non-specific cation currents (Ih currents) are important for the regulation of cell excitability. These currents are carried by channels of the hyperpolarisation-activated nucleotide-gated (HCN) family, of which there are four known subtypes. In the enteric nervous system (ENS), the Ih current is prominent in AH neurons. We investigated the expression and localization of HCN isoforms in the ENS of mice, rats and guinea-pigs. HCN1, HCN2 and HCN4 were expressed in enteric neurons. Immunoreactivity for HCN1 was observed on neuronal cell membranes of Dogiel type II neurons in rat and mouse. HCN2 channel immunoreactivity occurred in the majority of enteric neurons in the guinea-pig, rat and mouse. Immunoreactivity for HCN4 protein was revealed on the cell membranes of many neurons, including Dogiel type II neurons, in the guinea-pig. HCN4 was expressed by glial cells in guinea-pig. There was no evidence of HCN3 channel protein in any species with either immunohistochemistry or Western analysis. RT-PCR (polymerase chain reaction) using mouse HCN primers revealed mRNA for all four channels in the longitudinal muscle plus myenteric plexus of mouse distal colon. Sequencing confirmed the identity of the mRNA. Quantitative PCR demonstrated that HCN2 was the most highly expressed HCN channel subtype in the myenteric plexus of mouse distal colon. HCN1 and HCN4 were expressed at lower levels. HCN3 subtype mRNA was 0.2% of HCN2. We used intracellular recording to identify neurons having Ih currents and intracellular dye filling to locate the neurons for the immunohistochemical determination of channel expression. AH neurons with Ih currents were HCN2 and HCN4 channel positive. There was no correlation between the magnitude of the Ih and intensity of channel immunoreactivity. Our results indicate that HCN1, 2 and 4 genes and protein are expressed in the ENS. AH/Dogiel type II neurons, which have a prominent Ih, express HCN2 and 4 in guinea-pig and HCN1 and 2 in mouse and rat.

The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication. To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (K(D)) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB. The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined. The K(D) measured for the binding of wild type and His(6)-Tus to TerB was 0.5 nM in 250 mM KCl. Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences. Three of these substitutions (K89A, R198A, and Q250A) increased K(D) by 200-300-fold, whereas the A173T substitution increased K(D) by 4000-fold. Only the R198A substitution had a significant effect on binding to the nonspecific sequences. The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein.

Recent evidence suggests that functional diversity of vascular smooth muscle is produced in part by a differential expression of ion channels. The aim of the present study was to examine the role of Ca(2+)-activated K(+) channels (K(Ca) channels) in the expression of smooth muscle cell functional phenotype. We found that smooth muscle cells exhibiting a contractile function express predominantly large-conductance ( approximately 200 pS) K(Ca) (BK) channels. In contrast, proliferative smooth muscle cells express predominantly K(Ca) channels exhibiting a much smaller conductance ( approximately 32 pS). These channels are blocked by low concentrations of charybdotoxin (10 nmol/L) but, unlike BK channels, are insensitive to iberiotoxin (100 nmol/L). To determine the molecular identity of this K(+) channel, we cloned a 1.9-kb cDNA from an immature-phenotype smooth muscle cell cDNA library. The cDNA contains an open reading frame for a 425 amino acid protein exhibiting sequence homology to other K(Ca) channels, in particular with mIK1 and hIK1. Expression in oocytes gives rise to a K(+)-selective channel exhibiting intermediate-conductance (37 pS at -60 mV) and potent activation by Ca(2+) (K(d) 120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance K(Ca) channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these K(Ca) channels in functionally distinct smooth muscle cell types suggests that K(Ca) channels play a role in defining the physiological properties of vascular smooth muscle.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P4) and thereby regulates cellular transformation. To investigate the role Ins(1,4,5)P3-mediated Ca2+ oscillations play in cellular transformation, we studied Ins(1,4,5)P3-mediated Ca2+ responses in cells underexpressing the 43 kDa 5-phosphatase. Chronic reduction in 43 kDa 5-phosphatase enzyme activity resulted in a 2.6-fold increase in the resting Ins(1,4,5)P3 concentration and a 4.1-fold increase in basal intracellular Ca2+. The increased Ins(1,4,5)P3 levels resulted in partial emptying (40%) of the Ins(1,4,5)P3-sensitive Ca2+ store, however, store-operated Ca2+ influx remained unchanged. In addition, Ins(1,4,5)P3 receptors were chronically down-regulated in unstimulated cells, as shown by a 53% reduction in [3H]Ins(1,4,5)P3 binding to microsomal receptor sites. Agonist stimulation with endothelin-1 resulted in the rapid rise and fall of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels, with no significant differences in the rates of hydrolysis of these second messengers in antisense- or vector-transfected cells. These studies indicate, in contrast to its predicted action, the 43 kDa 5-phosphatase does not metabolise Ins(1, 4,5)P3 and Ins(1,3,4,5)P4 post agonist stimulation. Cells with decreased 43 kDa 5-phosphatase activity exhibited spontaneous Ca2+ oscillations in the absence of any agonist stimulation, and increased sensitivity and amplitude of intracellular Ca2+ responses to both high and low dose endothelin-1 stimulation. We conclude the 43 kDa 5-phosphatase exerts a profound influence on Ins(1,4, 5)P3-induced Ca2+ spiking, both in the unstimulated cell and following agonist stimulation. We propose the enhanced Ca2+ oscillations may mediate cellular transformation in cells underexpressing the 43 kDa 5-phosphatase.

1. Endothelins regulate cell function by interacting with two classes of cell surface receptors, ETA and ETB receptors. Both receptor types are members of the heptahelical transmembrane-spanning receptor superfamily and couple via G-proteins to multiple intracellular effectors. 2. Many of the cellular responses induced by endothelins are mediated by changes in cytoplasmic Ca2+ concentration. Stimulation of inositol 1,4,5-trisphosphate (IP3) formation promotes release of Ca2+ from intracellular stores via IP3-sensitive Ca2+ channels. This mechanism accounts for the initial transient peak of the Ca2+ elevation. The entry of Ca2+ across the plasma membrane through multiple types of Ca2+ channels is responsible for the sustained phase of Ca2+ elevation and, together, both mechanisms regulate cell function. 3. Endothelin-mediated Ca2+ signals vary markedly in duration, spatial organization and temporal pattern. The elevations in Ca2+ are sustained, transient or oscillatory and occur either globally or are localized to discrete spatial domains. These different Ca2+ signals, which are dependent on the availability of specific ion channels, control distinct cellular functions. Ryanodine-sensitive Ca2+ release channels may be important in determining the organization of the Ca2+ signal. 4. Endothelin-induced Ca2+ elevations near the plasma membrane stimulate the opening of Ca(2+)-dependent K+ and Cl- channels. These channels are key regulators of membrane potential and, consequently, regulate the activity of voltage-dependent Ca2+ influx pathways. 5. Endothelin regulates the growth and differentiation of cells. It markedly potentiates the mitogenic response of other growth factors, an effect that involves activation of the mitogen-activated protein kinase cascade and induction of early response genes. 6. Finally, the vascular actions of endothelin are influenced by the relative expression of specific ion channels, the spatial and temporal pattern of the Ca2+ signal and the cellular composition of the vascular wall.

In vascular smooth muscle, pertussis toxin (PT) inhibits thrombin-induced Ca2+ release by a mechanism independent of its effect on IP3 formation. Thus, the possibility of a direct role of G alpha i proteins in regulating IP3-sensitive Ca2+ release was investigated by examining whether G alpha i proteins are associated with the IP3 receptor complex. Purified microsomal membranes were prepared and separated by sucrose density gradient centrifugation. The relative density of [3H]-IP3 binding sites between the microsomal fractions was inversely related to the distribution of the plasma membrane marker. The relative distribution of G alpha i3 determined by immunoblotting was closely correlated with the density of [3H]-IP3 binding. Levels of G alpha i2 were more evenly distributed with highest levels present in plasma membrane-enriched fractions. IP3 receptor immunoprecipitated from triton-solubilized microsomal membranes contained G alpha i3 immunoreactivity. To determine whether G alpha i proteins influence IP3-induced Ca2+ release, the effect of PT on Ca2+ release from digitonin-permeabilized cell suspensions using Fluo-3 was examined. Exposure to PT (0.1 microgram/ml, 5 min) attenuated the initial rate of IP3 (1 microM)-induced Ca2+ release. Together, these findings are consistent with the hypothesis that a heterotrimeric G alpha i protein directly regulates IP3-dependent Ca2+ release.