The COVID University Challenge: A Hazard Analysis of Critical Control Points Assessment of the Return of Students to Higher Education Establishments.

The COVID-19 pandemic has disrupted economies and societies throughout the world since early 2020. Education is especially affected, with schools and universities widely closed for long periods. People under 25 years have the lowest risk of severe disease but their activities can be key to persistent ongoing community transmission. A challenge arose for how to provide education, including university level, without the activities of students increasing wider community SARS-CoV-2 infections. We used a Hazard Analysis of Critical Control Points (HACCP) framework to assess the risks associated with university student activity and recommend how to mitigate these risks. This tool appealed because it relies on multiagency collaboration and interdisciplinary expertise and yet is low cost, allowing rapid generation of evidence-based recommendations. We identified key critical control points associated with university student' activities, lifestyle, and interaction patterns both on-and-off campus. Unacceptable contact thresholds and the most up-to-date guidance were used to identify levels of risk for potential SARS-CoV-2 transmission, as well as recommendations based on existing research and emerging evidence for strategies that can reduce the risks of transmission. Employing the preventative measures we suggest can reduce the risks of SARS-CoV-2 transmission among and from university students. Reduction of infectious disease transmission in this demographic will reduce overall community transmission, lower demands on health services and reduce risk of harm to clinically vulnerable individuals while allowing vital education activity to continue. HACCP assessment proved a flexible tool for risk analysis in a specific setting in response to an emerging infectious disease threat. Systematic approaches to assessing hazards and risk critical control points (#HACCP) enable robust strategies for protecting students and staff in HE settings during #COVID19 pandemic.

Raising awareness of antimicrobial resistance among the general public in the UK: the role of public engagement activities.

In response to the accepted risk of emerging antimicrobial resistance, many organizations and institutions have developed and delivered events and activities designed to raise awareness of the issue and to change the behaviour of the intended audience. However, few of these events for a general public audience are documented or able to be sourced by those who might wish to repeat, adapt or modify, particularly those events that are successful. 'Insider knowledge' appears to be the best search tool. Moreover, evaluation of the success or impact of the event is rarely published. It would be useful if there were a 'hub' where descriptions of such activities could be deposited, enabling the building of a significant resource with real academic value.

Inspiring STEM undergraduates to tackle the AMR crisis.

To address the growing problem of antimicrobial resistance (AMR), it is necessary to invest in, inspire and attract future generations of scientists to this research area. Undergraduate education should be a focus for attention and efforts should be made to ensure that students are afforded opportunities to actively engage with AMR. We illustrate how as a topic AMR provides opportunities to deliver effective research-led teaching in addition to traditional teaching methods. We have used a selection of case studies to illustrate how students can be engaged with AMR using a variety of research-led approaches to develop the required skills for biology-centric students. In addition, we indicate how these skills map to the UK Quality Assurance Framework and the Vision and Change report developed by the American Association for the Advancement of Science.

Promoting microbiology education through the iGEM synthetic biology competition.

Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM, and all DNA constructs synthesized by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area.

Antimicrobial stewardship: the role of scientists?

We continue to be warned about the risk of antibiotic resistance. This campaign has targeted medicine and agriculture, asking these industries to pay attention to the risks of widespread resistance and to cut the use of antibiotics wherever possible. However, there has been little to no mention of the widespread use of antibiotics in the scientific research community. As scientists we use antibiotics and antibiotic resistance as fundamental tools for our research; almost all conventional plasmids use an antibiotic resistance gene as a selectable marker, offering us an easy method of screening. With molecular biology and genetics at the heart of many research disciplines, these tools are ubiquitous. Scientists have a responsibility to monitor and reduce our use of antibiotics. With the growth and fast advancement of synthetic biology, it is timely for us to consider other options and to teach the next generation of researchers by example how to truly value antibiotics by using them more responsibly.

The representation of scientific research in the national curriculum and secondary school pupils' perceptions of research, its function, usefulness and value to their lives.

Young people's views on what research is, how it is conducted and whether it is important, influences the decisions they make about their further studies and career choices. In this paper we report the analysis of questionnaire data with a particular focus on pupil perceptions of research in the sciences and of the scientific method. The questionnaire was a 25-item Likert Scale (1-5) distributed to seven collaborating schools. We received 2634 returns from pupils across key stages 3, 4 and 5. We also asked teachers to complete the questionnaire in order to explore how they thought their pupils would respond. We received 54 teacher responses. Statistically significant differences in the responses were identified through a chi-square test on SPSS. As what is being taught influences secondary pupil views on research we also consider how the term 'research' appears in the national curriculum for England and Wales and the three main English exam boards. The main theoretical construct that informs our analysis of the questionnaire data and the national curriculum is Angela Brew's 4-tier descriptor of perceptions of research (domino, trading, layer, journey). We use this framework in order to map what, when and how research is presented to school pupils in England and Wales. We also use this framework in order to highlight and discuss certain pupil views that emerged from the questionnaire data and which indicate areas where curriculum and pedagogy intervention may be necessary: pupils seem less confident in their understanding of research as involving the identification of a research question; and, they often see research as a means to confirm one's own opinion. They do however understand research as involving the generation of new knowledge and the collection of new data, such as interviews and questionnaires as well as laboratory work, field trips and library searches and they appear relatively confident in their statements about their ability to do research, their school experiences of research and the importance of research in their future career choice.

Twelve tips to teaching (legal and ethical aspects of) research ethics/responsible conduct of research.

Teaching research ethics is a requirement within modern health science, nursing and medical curricula. We have drawn on our experience of designing, developing and integrating the teaching of research ethics in a new, fully integrated medical school curriculum, delivered using Problem Based Learning and the recent literature relating to the teaching of research ethics to produce the following 12 Top Tips designed to encourage readers to seek opportunities to embed this teaching within a variety of curricula.

pH-dependent structures of the manganese binding sites in oxalate decarboxylase as revealed by high-field electron paramagnetic resonance.

A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site-1 and -2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site -2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pK(a). This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role.

Detection of transglucosidase-catalyzed polysaccharide synthesis on a surface in real time using surface plasmon resonance spectroscopy.

Biological systems that involve enzyme catalysis at surfaces, particularly strategically important ones that involve insoluble substrates/products such as the cell wall and the starch granule, require analyses beyond classical solution state enzymology. Using a model system, we have demonstrated the real-time measurement of transglucosidase activity on a surface using surface plasmon resonance (SPR) spectroscopy. We monitored the extension of a (partially carboxymethylated) dextran surface with alternansucrase and sucrose as a glycosyl donor. Conditions were used where surface polymer synthesis rates were a function of enzyme concentration and proportional to the extent of enzyme binding to the surface. A method to determine the turnover number of the enzyme on the surface was also developed. The presence of a new amorphous polysaccharide was observed optically, detected by lectin binding and imaged by atomic force microscopy. This surface method will have utility in a wide range of carbohydrate enzyme systems including screens.

Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282,000 by mutating an active site lid.

Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.

The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations.

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Delta162-163 or Delta162-164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Delta162-163 and S161A) strongly support the hypothesis that site 2 is purely structural.

Cloning and sequencing of two Ceriporiopsis subvermispora bicupin oxalate oxidase allelic isoforms: implications for the reaction specificity of oxalate oxidases and decarboxylases.

Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess < or =0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.

Characterization of a temperature-sensitive DNA ligase from Escherichia coli.

DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication. Several temperature-sensitive mutant strains of Escherichia coli, including strain GR501, have been described which can be complemented by functional DNA ligases. Here, it is shown that the ligA251 mutation in E. coli GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15. The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures. Compared to wild-type LigA, at 20 degrees C purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 degrees C, resulting in 60-fold lower ligation activity than wild-type LigA. It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA. The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase.

SAD at home: solving the structure of oxalate decarboxylase with the anomalous signal from manganese using X-ray data collected on a home source.

Oxalate decarboxylase (OxdC) from Bacillus subtilis is a hexamer containing two manganese ions per 43.6 kDa subunit. A single highly redundant data set collected at a medium resolution of 2 A on an in-house X-ray source was sufficient to solve the structure by the single-wavelength anomalous diffraction (SAD) method using the anomalous signal from the manganese ions. The experimentally phased electron-density map was of high quality, enabling 96% of the amino-acid sequence to be automatically traced using ARP/wARP. Further analysis showed that only half of the original raw data were required for successful structure solution. Manganese currently occurs in approximately 2% of PDB entries. A brief survey suggests that several of these structures could also have been determined using manganese SAD. Moreover, the ability of manganese to substitute for other more commonly occurring divalent metal ions may indicate that the use of Mn SAD could have much wider application.

A closed conformation of Bacillus subtilis oxalate decarboxylase OxdC provides evidence for the true identity of the active site.

Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659-7669). The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 3(10) helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Site-directed mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.

Bacillus subtilis YxaG is a novel Fe-containing quercetin 2,3-dioxygenase.

The Bacillus subtilis genome contains genes for three hypothetical proteins belonging to the bicupin family, two of which we have previously shown to be Mn(II)-dependent oxalate decarboxylases. We have now shown that the third, YxaG, exhibits quercetin 2,3-dioxygenase activity and that it contains Fe ions. This contrasts with the eukaryotic enzyme which contains a Cu ion. YxaG is the first prokaryotic carbon monoxide-forming enzyme that utilises a flavonol to be characterised and is only the second example of a prokaryotic dioxygenolytic carbon monoxide-forming enzyme known to contain a cofactor. It is proposed to rename the B. subtilis gene qdoI.