Production of the Extremolyte Cyclic 2,3-Diphosphoglycerate Using Thermus thermophilus as a Whole-Cell Factory (article)

This is the final version. Available on open access from Frontiers Media via the DOI in this recordData Availability Statement: The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. Data has been deposited with the University of Exeter ORE https://doi.org/10.24378/exe.3683Osmolytes protect microbial cells against temperature, osmolarity and other stresses. The osmolyte cyclic 2,3-diphosphoglycerate, originally isolated from the thermophilic archaeon Methanothermus fervidus, naturally protects cellular proteins under extreme conditions. The biosynthetic pathway for cyclic 2,3-diphosphoglycerate has been introduced into the thermophilic bacterium Thermus thermophilus. The two enzymes in this synthetic pathway, 2-phosphoglycerate kinase and cyclic diphosphoglycerate synthetase, were incorporated into a newly designed modular BioBricks vector. The expression of this two-enzyme cascade resulted in the whole cell production of cyclic 2,3 diphosphoglycerate. In vivo production of cyclic 2,3-diphosphoglycerate was confirmed by mass spectrometry to a concentration up to 650 µM. This study demonstrates the feasibility of using this well studied thermophilic bacterium as a host in a whole-cell factory approach to produce cyclic 2,3 diphosphoglycerate. This raises the potential for commercialisation of cDPG for cosmetic and healthcare applications. Our work demonstrates the application of Thermus thermophilus as an alternative host for other high value small organic molecules of industrial interest.Biotechnology and Biological Sciences Research Council (BBSRC)University of Exete

New Thermophilic α/β Class Epoxide Hydrolases Found in Metagenomes From Hot Environments

This is the final version. Available from Frontiers Media via the DOI in this record.Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two α/β hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85°C retaining 80% of its initial activity after 3 h thermal treatment at 70°C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 Å, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs

When chefs adopt a school? An evaluation of a cooking intervention in English primary schools

This article sets out the findings from research on the impact of a, UK based, chefs in schools teaching programme on food, health, nutrition and cookery. Professional chefs link with local schools, where they deliver up to three sessions to one class over a year. The research measured the impact of a standardised intervention package and changes in food preparation and consumption as well as measuring cooking confidence. The target group was 9–11 year olds in four schools. The main data collection method was a questionnaire delivered 2 weeks before the intervention and 2 weeks afterwards. There was a group of four matched control schools. Those taking part in the intervention were enthused and engaged by the sessions and the impact measures indicated an intention to change. There were gains in skills and confidence to prepare and ask for the ingredients to be purchased for use in the home. Following the session with the chef, the average reported cooking confidence score increased from 3.09 to 3.35 (by 0.26 points) in the intervention group – a statistically significant improvement. In the control group this change was not statistically significant. Children’s average reported vegetable consumption increased after the session with the chef, with the consumption score increasing from 2.24 to 2.46 points (0.22 points) again, a statistically significant increase with no significant changes in the control group. The research highlights the need to incorporate evaluation into school cooking initiatives as the findings can provide valuable information necessary to fine-tune interventions and to ensure consistency of the healthy eating messages

Training attention control of very preterm infants: protocol for a feasibility study of the Attention Control Training (ACT)

Background Children born preterm may display cognitive, learning, and behaviour difficulties as they grow up. In particular, very premature birth (gestation age between 28 and less than 32 weeks) may put infants at increased risk of intellectual deficits and attention deficit disorder. Evidence suggests that the basis of these problems may lie in difficulties in the development of executive functions. One of the earliest executive functions to emerge around 1 year of age is the ability to control attention. An eye-tracking-based cognitive training programme to support this emerging ability, the Attention Control Training (ACT), has been developed and tested with typically developing infants. The aim of this study is to investigate the feasibility of using the ACT with healthy very preterm (VP) infants when they are 12 months of age (corrected age). The ACT has the potential to address the need for supporting emerging cognitive abilities of VP infants with an early intervention, which may capitalise on infants’ neural plasticity. Methods/design The feasibility study is designed to investigate whether it is possible to recruit and retain VP infants and their families in a randomised trial that compares attention and social attention of trained infants against those that are exposed to a control procedure. Feasibility issues include the referral/recruitment pathway, attendance, and engagement with testing and training sessions, completion of tasks, retention in the study, acceptability of outcome measures, quality of data collected (particularly, eye-tracking data). The results of the study will inform the development of a larger randomised trial. Discussion Several lines of evidence emphasise the need to support emerging cognitive and learning abilities of preterm infants using early interventions. However, early interventions with preterm infants, and particularly very preterm ones, face difficulties in recruiting and retaining participants. These problems are also augmented by the health vulnerability of this population. This feasibility study will provide the basis for informing the implementation of an early cognitive intervention for very preterm infants. Trial registration Registered Registration ID: NCT03896490. Retrospectively registered at Clinical Trials Protocol Registration and Results System (clinicaltrials.gov)

Biochemical and Structural Characterisation of a Novel D-Lyxose Isomerase From the Hyperthermophilic Archaeon Thermofilum sp.

This is the final version. Available on open access from Frontiers Media via the DOI in this recordData Availability Statement: The atomic coordinates and structure factors for the crystal structures of TsLI and its complexes have been deposited in the Protein Data Bank with the following codes: 7NZO for the native structure, 7NZP for the D-fructose bound structure, 7NZQ for the D-mannose bound structure.A novel D-lyxose isomerase has been identified within the genome of a hyperthermophilic archaeon belonging to the Thermofilum species. The enzyme has been cloned and over-expressed in Escherichia coli and biochemically characterised. This enzyme differs from other enzymes of this class in that it is highly specific for the substrate D-lyxose, showing less than 2% activity towards mannose and other substrates reported for lyxose isomerases. This is the most thermoactive and thermostable lyxose isomerase reported to date, showing activity above 95°C and retaining 60% of its activity after 60 min incubation at 80°C. This lyxose isomerase is stable in the presence of 50% (v/v) of solvents ethanol, methanol, acetonitrile and DMSO. The crystal structure of the enzyme has been resolved to 1.4–1.7 A. resolution in the ligand-free form and in complexes with both of the slowly reacting sugar substrates mannose and fructose. This thermophilic lyxose isomerase is stabilised by a disulfide bond between the two monomers of the dimeric enzyme and increased hydrophobicity at the dimer interface. These overall properties of high substrate specificity, thermostability and solvent tolerance make this lyxose isomerase enzyme a good candidate for potential industrial applications.European UnionBMBFUniversity of KielBiotechnology and Biological Sciences Research Council (BBSRC)University of Exete

Timing of immune escape linked to success or failure of vaccination

Successful vaccination against HIV should limit viral replication sufficiently to prevent the emergence of viral immune escape mutations. Broadly directed immunity is likely to be required to limit opportunities for immune escape variants to flourish. We studied the emergence of an SIV Gag cytotoxic T cell immune escape variant in pigtail macaques expressing the Mane-A*10 MHC I allele using a quantitative RT-PCR to measure viral loads of escape and wild type variants. Animals receiving whole Gag expressing vaccines completely controlled an SIVmac251 challenge, had broader CTL responses and exhibited minimal CTL escape. In contrast, animals vaccinated with only a single CTL epitope and challenged with the same SIVmac251 stock had high levels of viral replication and rapid CTL escape. Unvaccinated na&iuml;ve animals exhibited a slower emergence of immune escape variants. Thus narrowly directed vaccination against a single epitope resulted in rapid immune escape and viral levels equivalent to that of na&iuml;ve unvaccinated animals. These results emphasize the importance of inducing broadly directed HIV-specific immunity that effectively quashes early viral replication and limits the generation of immune escape variants. This has important implications for the selection of HIV vaccines for expanded human trials.<br /

Mathematics difficulties in extremely preterm children : evidence of a specific deficit in basic mathematics processing

Background: Extremely preterm (EP, <26 wk gestation) children have been observed to have poor academic achievement in comparison to their term-born peers, especially in mathematics. This study investigated potential underlying causes of this difficulty. Methods: A total of 219 EP participants were compared with 153 term-born control children at 11 y of age. All children were assessed by a psychologist on a battery of standardized cognitive tests and a number estimation test assessing children’s numerical representations. Results: EP children underperformed in all tests in comparison with the term controls (the majority of Ps < 0.001). Different underlying relationships between performance on the number estimation test and mathematical achievement were found in EP as compared with control children. That is, even after controlling for cognitive ability, a relationship between number representations and mathematical performance persisted for EP children only (EP: r = 0.346, n = 186, P < 0.001; control: r = 0.095, n = 146, P = 0.256). Conclusion: Interventions for EP children may target improving children’s numerical representations in order to subsequently remediate their mathematical skills

Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeon Methanothermus fervidus

This is the final version. Available on open access from Frontiers Media via the DOI in this recordData availability statement: The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.rcsb.org/, PDB 80RK, PDB 80RU.The enzyme cyclic di-phosphoglycerate synthetase that is involved in the production of the osmolyte cyclic 2,3-diphosphoglycerate has been studied both biochemically and structurally. Cyclic 2,3-diphosphoglycerate is found exclusively in the hyperthermophilic archaeal methanogens, such as Methanothermus fervidus, Methanopyrus kandleri, and Methanothermobacter thermoautotrophicus. Its presence increases the thermostability of archaeal proteins and protects the DNA against oxidative damage caused by hydroxyl radicals. The cyclic 2,3-diphosphoglycerate synthetase enzyme has been crystallized and its structure solved to 1.7 Å resolution by experimental phasing. It has also been crystallized in complex with its substrate 2,3 diphosphoglycerate and the co-factor ADP and this structure has been solved to 2.2 Å resolution. The enzyme structure has two domains, the core domain shares some structural similarity with other NTP-dependent enzymes. A significant proportion of the structure, including a 127 amino acid N-terminal domain, has no structural similarity to other known enzyme structures. The structure of the complex shows a large conformational change that occurs in the enzyme during catalytic turnover. The reaction involves the transfer of the γ-phosphate group from ATP to the substrate 2,3 -diphosphoglycerate and the subsequent SN2 attack to form a phosphoanhydride. This results in the production of the unusual extremolyte cyclic 2,3 -diphosphoglycerate which has important industrial applications.European Union Horizon 2020Biotechnology and Biological Sciences Research Council (BBSRC)University of ExeterEVONIK IndustriesGerman Federal Ministry of Education and Research (BMBF

A pilot randomised controlled trial of personalised care for depressed patients with symptomatic coronary heart disease in South London general practices: the UPBEAT-UK RCT protocol and recruitment.

ABSTRACT: Background: Community studies reveal people with coronary heart disease (CHD) are twice as likely to be depressed as the general population and that this co-morbidity negatively affects the course and outcome of both conditions. There is evidence for the efficacy of collaborative care and case management for depression treatment, and whilst NICE guidelines recommend these approaches only where depression has not responded to psychological, pharmacological, or combined treatments, these care approaches may be particularly relevant to the needs of people with CHD and depression in the earlier stages of stepped care in primary care settings. Methods: This pilot randomised controlled trial will evaluate whether a simple intervention involving a personalised care plan, elements of case management and regular telephone review is a feasible and acceptable intervention that leads to better mental and physical health outcomes for these patients. The comparator group will be usual general practitioner (GP) care. 81 participants have been recruited from CHD registers of 15 South London general practices. Eligible participants have probable major depression identified by a score of ≥8 on the Hospital Anxiety and Depression Scale depression subscale (HADS-D) together with symptomatic CHD identified using the Modified Rose Angina Questionnaire. Consenting participants are randomly allocated to usual care or the personalised care intervention which involves a comprehensive assessment of each participant’s physical and mental health needs which are documented in a care plan, followed by regular telephone reviews by the case manager over a 6-month period. At each review, the intervention participant’s mood, function and identified problems are reviewed and the case manager uses evidence based behaviour change techniques to facilitate achievement of goals specified by the patient with the aim of increasing the patient’s self efficacy to solve their problems. Depressive symptoms measured by HADS score will be collected at baseline and 1, 6- and 12 months post randomisation. Other outcomes include CHD symptoms, quality of life, wellbeing and health service utilisation. Discussion: This practical and patient-focused intervention is potentially an effective and accessible approach to the health and social care needs of people with depression and CHD in primary care. Trial registration: ISRCTN21615909

Change and Aging Senescence as an adaptation

Understanding why we age is a long-lived open problem in evolutionary biology. Aging is prejudicial to the individual and evolutionary forces should prevent it, but many species show signs of senescence as individuals age. Here, I will propose a model for aging based on assumptions that are compatible with evolutionary theory: i) competition is between individuals; ii) there is some degree of locality, so quite often competition will between parents and their progeny; iii) optimal conditions are not stationary, mutation helps each species to keep competitive. When conditions change, a senescent species can drive immortal competitors to extinction. This counter-intuitive result arises from the pruning caused by the death of elder individuals. When there is change and mutation, each generation is slightly better adapted to the new conditions, but some older individuals survive by random chance. Senescence can eliminate those from the genetic pool. Even though individual selection forces always win over group selection ones, it is not exactly the individual that is selected, but its lineage. While senescence damages the individuals and has an evolutionary cost, it has a benefit of its own. It allows each lineage to adapt faster to changing conditions. We age because the world changes.Comment: 19 pages, 4 figure