The structural mechanism of dimeric DONSON in replicative helicase activation.

The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients' mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.

DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation.

Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Our data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. Here we show that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients.

Optimisation of GraPhage13 macro-dispersibility via understanding the pH-dependent ionisation during self-assembly: towards the manufacture of graphene-based nanodevices.

GraPhage13 aerogels (GPAs) are micro-porous structures generated through the self-assembly of graphene oxide (GO) and M13 bacteriophage. As GPA fabrication involves the aggregation of GO and M13 in aqueous solution, we aim to understand its dispersibility across a wide pH range. Herein, a novel technique has been developed to relate the ionisation of functional groups to the surface charge, offering insights into the conditions required for GPA fabrication and the mechanism behind its self-assembly. The aggregation of GO and M13 was observed between pH 2-6 and exhibited dependence on the surface charge of the resulting aggregate with the M13 bacteriophage identified as the primary factor contributing to this, whilst originating from the ionisation of its functional groups. In contrast, GO exhibited a lesser impact on the surface charge due to the deprotonation of its carboxylic, enolic and phenolic functional groups at pH 6 and above, which falls outside the required pH range for aggregation. These results enhance our understanding of the GPA self-assembly mechanism, the conditions required for their fabrication and the optimal processability, laying the foundation towards its broad range of applications and the subsequent manufacture of graphene-based nanodevices.

Pre-lab video demonstrations to enhance students' laboratory experience in a first-year chemical engineering class.

The limited capabilities of teaching laboratories, combined with an increasing number of students enrolled in university, require constant augmentation of instructional approaches. By enhancing laboratory demonstrations with digital technology, these structural issues can be addressed while at the same time enhancing student understanding and learning. Our case study focuses on the fermentation lab part of the Reaction Equilibria and Thermodynamics (RET) module, a first-year chemical engineering course at the University of Birmingham. Video demonstrations were used to introduce students to the laboratory set-ups and walk them through each step and technique. The video demonstrations allowed the students to attend the in-person lab sessions having established knowledge and understanding of the processes involved and the outcomes desired, which decreased the burden on the facilities and the staff. A knowledge-based quiz and a student survey conducted at the end of the module showed that the pre-lab videos encouraged more active participation in the laboratory sessions and reinforced learning. Approximately 70% of the students polled in the first survey conducted within this project felt more confident going into the laboratory sessions after watching the pre-lab videos and attempting the knowledge quiz, while 92% of the students polled in the second survey judged the pre-lab video sessions as beneficial to them. Overall, the teaching method has the potential to improve student participation and access, boost confidence and learning, and provided a more structured and flexible approach to laboratory learning outcomes.

The safety of nanomaterials in food production and packaging.

Nanotechnology involves developing, characterising, and applying structures ranging in size from 1 to 100 nm. As a key advanced technology, it has contributed to a substantial impact across engineering, medicine, agriculture and food. With regards to their application in food, nanomaterials posses the ability to lead the quantitative and qualitative development of high-quality, healthier, and safer foods by outperforming traditional food processing technologies for increasing shelf life and preventing contaminations. Although rapid progress has been made in nanotechnology in food products, the toxicity of nanoparticles and nanomaterials is not very well known. As a result, nanomaterials are potentially toxic, therefore, considering the constantly increasing employment in food science, they need to be further characterised, and their use must be better regulated. We may face a crisis of nanotoxicity if the molecular mechanisms by which nanoparticles and nanomaterials interact with food and within living organisms is not fully understood. Food safety can be guaranteed only if we are thoroughly aware of nanomaterial properties and potential toxicity. Therefore, it is urgently necessary to have in the food sector a regulatory system capable of managing nanofood risks and nanotechnology, considering the health effects of food processing techniques based on nanotechnology. This present review discusses the impact and role nanotechnology play in food science. The specific application of Nanomaterials in food science, their advantages and disadvantages, the potential risk for human health and the analysis to detect nanocomponents are also highlighted.

Graphene Oxide and Biomolecules for the Production of Functional 3D Graphene-Based Materials.

Graphene and its derivatives have been widely employed in the manufacturing of novel composite nanomaterials which find applications across the fields of physics, chemistry, engineering and medicine. There are many techniques and strategies employed for the production, functionalization, and assembly of graphene with other organic and inorganic components. These are characterized by advantages and disadvantages related to the nature of the specific components involved. Among many, biomolecules and biopolymers have been extensively studied and employed during the last decade as building blocks, leading to the realization of graphene-based biomaterials owning unique properties and functionalities. In particular, biomolecules like nucleic acids, proteins and enzymes, as well as viruses, are of particular interest due to their natural ability to self-assemble via non-covalent interactions forming extremely complex and dynamic functional structures. The capability of proteins and nucleic acids to bind specific targets with very high selectivity or the ability of enzymes to catalyse specific reactions, make these biomolecules the perfect candidates to be combined with graphenes, and in particular graphene oxide, to create novel 3D nanostructured functional biomaterials. Furthermore, besides the ease of interaction between graphene oxide and biomolecules, the latter can be produced in bulk, favouring the scalability of the resulting nanostructured composite materials. Moreover, due to the presence of biological components, graphene oxide-based biomaterials are more environmentally friendly and can be manufactured more sustainably compared to other graphene-based materials assembled with synthetic and inorganic components. This review aims to provide an overview of the state of the art of 3D graphene-based materials assembled using graphene oxide and biomolecules, for the fabrication of novel functional and scalable materials and devices.

Use of a model to understand the synergies underlying the antibacterial mechanism of H2O2-producing honeys.

Honey has been valued as a powerful antimicrobial since ancient times. However, the understanding of the underlying antibacterial mechanism is incomplete. The complexity and variability of honey composition represent a challenge to this scope. In this study, a simple model system was used to investigate the antibacterial effect of, and possible synergies between, the three main stressors present in honey: sugars, gluconic acid, and hydrogen peroxide (H2O2), which result from the enzymatic conversion of glucose on honey dilution. Our results demonstrated that the synergy of H2O2 and gluconic acid is essential for the antibacterial activity of honey. This synergy caused membrane depolarization, destruction of the cell wall, and eventually growth inhibition of E. coli K-12. The presence of H2O2 stimulated the generation of other long-lived ROS in a dose-dependent manner. Sugars caused osmosis-related morphological changes, however, decreased the toxicity of the H2O2/gluconic acid. The susceptibility of catalase and general stress response sigma factor mutants confirmed the synergy of the three stressors, which is enhanced at higher H2O2 concentrations. By monitoring cellular phenotypic changes caused by model honey, we explained how this can be bactericidal even though the antimicrobial compounds which it contains are at non-inhibitory concentrations.

Improvements in the production of purified M13 bacteriophage bio-nanoparticle.

M13 bacteriophage is a well-established versatile nano-building block, which can be employed to produce novel self-assembled functional materials and devices. Sufficient production and scalability of the M13, often require a large quantity of the virus and thus, improved propagation methods characterised by high capacity and degree of purity are essential. Currently, the 'gold-standard' is represented by infecting Escherichia coli cultures, followed by precipitation with polyethylene glycol (PEG). However, this is considerably flawed by the accumulation of contaminant PEG inside the freshly produced stocks, potentially hampering the reactivity of the individual M13 filaments. Our study demonstrates the effectiveness of implementing an isoelectric precipitation procedure to reduce the residual PEG along with FT-IR spectroscopy as a rapid, convenient and effective analytic validation method to detect the presence of this contaminant in freshly prepared M13 stocks.

Nanomechanics of graphene oxide-bacteriophage based self-assembled porous composites.

Graphene oxide, integrated with the filamentous bacteriophage M13, forms a 3D large-scale multifunctional porous structure by self-assembly, with considerable potential for applications. We performed Raman spectroscopy under pressure on this porous composite to understand its fundamental mechanics. The results show that at low applied pressure, the [Formula: see text] bonds of graphene oxide stiffen very little with increasing pressure, suggesting a complicated behaviour of water intercalated between the graphene layers. The key message of this paper is that water in a confined space can have a significant impact on the nanostructure that hosts it. We introduced carbon nanotubes during the self-assembly of graphene oxide and M13, and a similar porous macro-structure was observed. However, in the presence of carbon nanotubes, pressure is transmitted to the [Formula: see text] bonds of graphene oxide straightforwardly as in graphite. The electrical conductivity of the composite containing carbon nanotubes is improved by about 30 times at a bias voltage of 10 V. This observation suggests that the porous structure has potential in applications where good electrical conductivity is desired, such as sensors and batteries.

Determination and characterisation of the surface charge properties of the bacteriophage M13 to assist bio-nanoengineering.

To truly understand the mechanisms behind the supramolecular self-assembly of nanocomponents, the characterisation of their surface properties is crucial. M13 emerged as a practical nanocomponent for bio-nanoassemblies of functional materials and devices, and its popularity is increasing as time goes by. The investigation performed in this study provides important information about the surface charge and the surface area of M13 determined through the comparison of structural data and the measurement of ζ-potential at pH ranging between 2 and 11. The developed methodologies along with the experimental findings can be subsequently exploited as a novel and convenient prediction tool of the total charge of modified versions of M13. This, in turn, will facilitate the design of the self-assembly strategies which would combine the virus building block with other micro and nano components via intermolecular interactions.

Multifunctional graphene oxide-bacteriophage based porous three-dimensional micro-nanocomposites.

Graphene, since its successful exfoliation and characterisation has been continuously drawing extensive research interests due to its potential for a broad range of applications ranging from energy, microelectronics, through polymer fillers and sensors to environmental and biomedical devices. Exploitation of its unique chemical and physical properties for the manufacturing of functional materials, requires careful structural control and scaling-up into three-dimensional morphologies. Here, a facile method is established to create and control the bottom-up self-assembly of graphene oxide nano-sheets via unprecedented integration with a highly versatile bio-ingredient, the filamentous bacteriophage M13, into hierarchical, three-dimensional, porous sponges of GraPhage13. This study explores the interplay of the GraPhage13 structure formation and studies the mechanisms that give rise to the controllable self-assembly. The straightforward fabrication of robust hierarchical micro-nano-architectures further lays a platform for applications in energy storage and conversion, catalysis and sensing.

Dye Aggregate-Mediated Self-Assembly of Bacteriophage Bioconjugates.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

Supramolecular self-assembly of graphene oxide and metal nanoparticles into stacked multilayers by means of a multitasking protein ring.

Graphene oxide (GO) is rapidly emerging worldwide as a breakthrough precursor material for next-generation devices. However, this requires the transition of its two-dimensional layered structure into more accessible three-dimensional (3D) arrays. Peroxiredoxins (Prx) are a family of multitasking redox enzymes, self-assembling into ring-like architectures. Taking advantage of both their symmetric structure and function, 3D reduced GO-based composites are hereby built up. Results reveal that the "double-faced" Prx rings can adhere flat on single GO layers and partially reduce them by their sulfur-containing amino acids, driving their stacking into 3D multi-layer reduced GO-Prx composites. This process occurs in aqueous solution at a very low GO concentration, i.e. 0.2 mg ml(-1). Further, protein engineering allows the Prx ring to be enriched with metal binding sites inside its lumen. This feature is exploited to both capture presynthesized gold nanoparticles and grow in situ palladium nanoparticles paving the way to straightforward and "green" routes to 3D reduced GO-metal composite materials.