A prebiotic basis for ATP as the universal energy currency.

ATP is universally conserved as the principal energy currency in cells, driving metabolism through phosphorylation and condensation reactions. Such deep conservation suggests that ATP arose at an early stage of biochemical evolution. Yet purine synthesis requires 6 phosphorylation steps linked to ATP hydrolysis. This autocatalytic requirement for ATP to synthesize ATP implies the need for an earlier prebiotic ATP equivalent, which could drive protometabolism before purine synthesis. Why this early phosphorylating agent was replaced, and specifically with ATP rather than other nucleoside triphosphates, remains a mystery. Here, we show that the deep conservation of ATP might reflect its prebiotic chemistry in relation to another universally conserved intermediate, acetyl phosphate (AcP), which bridges between thioester and phosphate metabolism by linking acetyl CoA to the substrate-level phosphorylation of ADP. We confirm earlier results showing that AcP can phosphorylate ADP to ATP at nearly 20% yield in water in the presence of Fe3+ ions. We then show that Fe3+ and AcP are surprisingly favoured. A wide range of prebiotically relevant ions and minerals failed to catalyse ADP phosphorylation. From a panel of prebiotic phosphorylating agents, only AcP, and to a lesser extent carbamoyl phosphate, showed any significant phosphorylating potential. Critically, AcP did not phosphorylate any other nucleoside diphosphate. We use these data, reaction kinetics, and molecular dynamic simulations to infer a possible mechanism. Our findings might suggest that the reason ATP is universally conserved across life is that its formation is chemically favoured in aqueous solution under mild prebiotic conditions.

The limits of metabolic heredity in protocells.

The universal core of metabolism could have emerged from thermodynamically favoured prebiotic pathways at the origin of life. Starting with H2 and CO2, the synthesis of amino acids and mixed fatty acids, which self-assemble into protocells, is favoured under warm anoxic conditions. Here, we address whether it is possible for protocells to evolve greater metabolic complexity, through positive feedbacks involving nucleotide catalysis. Using mathematical simulations to model metabolic heredity in protocells, based on branch points in protometabolic flux, we show that nucleotide catalysis can indeed promote protocell growth. This outcome only occurs when nucleotides directly catalyse CO2 fixation. Strong nucleotide catalysis of other pathways (e.g. fatty acids and amino acids) generally unbalances metabolism and slows down protocell growth, and when there is competition between catalytic functions cell growth collapses. Autocatalysis of nucleotide synthesis can promote growth but only if nucleotides also catalyse CO2 fixation; autocatalysis alone leads to the accumulation of nucleotides at the expense of CO2 fixation and protocell growth rate. Our findings offer a new framework for the emergence of greater metabolic complexity, in which nucleotides catalyse broad-spectrum processes such as CO2 fixation, hydrogenation and phosphorylation important to the emergence of genetic heredity at the origin of life.

Prebiotic Synthesis of Aspartate Using Life's Metabolism as a Guide.

A protometabolic approach to the origins of life assumes that the conserved biochemistry of metabolism has direct continuity with prebiotic chemistry. One of the most important amino acids in modern biology is aspartic acid, serving as a nodal metabolite for the synthesis of many other essential biomolecules. Aspartate's prebiotic synthesis is complicated by the instability of its precursor, oxaloacetate. In this paper, we show that the use of the biologically relevant cofactor pyridoxamine, supported by metal ion catalysis, is sufficiently fast to offset oxaloacetate's degradation. Cu2+-catalysed transamination of oxaloacetate by pyridoxamine achieves around a 5% yield within 1 h, and can operate across a broad range of pH, temperature, and pressure. In addition, the synthesis of the downstream product ß-alanine may also take place in the same reaction system at very low yields, directly mimicking an archaeal synthesis route. Amino group transfer supported by pyridoxal is shown to take place from aspartate to alanine, but the reverse reaction (alanine to aspartate) shows a poor yield. Overall, our results show that the nodal metabolite aspartate and related amino acids can indeed be synthesised via protometabolic pathways that foreshadow modern metabolism in the presence of the simple cofactor pyridoxamine and metal ions.

Biophysical Interactions Underpin the Emergence of Information in the Genetic Code.

The genetic code conceals a 'code within the codons', which hints at biophysical interactions between amino acids and their cognate nucleotides. Yet, research over decades has failed to corroborate systematic biophysical interactions across the code. Using molecular dynamics simulations and NMR, we have analysed interactions between the 20 standard proteinogenic amino acids and 4 RNA mononucleotides in 3 charge states. Our simulations show that 50% of amino acids bind best with their anticodonic middle base in the -1 charge state common to the backbone of RNA, while 95% of amino acids interact most strongly with at least 1 of their codonic or anticodonic bases. Preference for the cognate anticodonic middle base was greater than 99% of randomised assignments. We verify a selection of our results using NMR, and highlight challenges with both techniques for interrogating large numbers of weak interactions. Finally, we extend our simulations to a range of amino acids and dinucleotides, and corroborate similar preferences for cognate nucleotides. Despite some discrepancies between the predicted patterns and those observed in biology, the existence of weak stereochemical interactions means that random RNA sequences could template non-random peptides. This offers a compelling explanation for the emergence of genetic information in biology.

What Makes a Quality Health App-Developing a Global Research-Based Health App Quality Assessment Framework for CEN-ISO/TS 82304-2: Delphi Study.

BACKGROUND: The lack of an international standard for assessing and communicating health app quality and the lack of consensus about what makes a high-quality health app negatively affect the uptake of such apps. At the request of the European Commission, the international Standard Development Organizations (SDOs), European Committee for Standardization, International Organization for Standardization, and International Electrotechnical Commission have joined forces to develop a technical specification (TS) for assessing the quality and reliability of health and wellness apps. OBJECTIVE: This study aimed to create a useful, globally applicable, trustworthy, and usable framework to assess health app quality. METHODS: A 2-round Delphi technique with 83 experts from 6 continents (predominantly Europe) participating in one (n=42, 51%) or both (n=41, 49%) rounds was used to achieve consensus on a framework for assessing health app quality. Aims included identifying the maximum 100 requirement questions for the uptake of apps that do or do not qualify as medical devices. The draft assessment framework was built on 26 existing frameworks, the principles of stringent legislation, and input from 20 core experts. A follow-up survey with 28 respondents informed a scoring mechanism for the questions. After subsequent alignment with related standards, the quality assessment framework was tested and fine-tuned with manufacturers of 11 COVID-19 symptom apps. National mirror committees from the 52 countries that participated in the SDO technical committees were invited to comment on 4 working drafts and subsequently vote on the TS. RESULTS: The final quality assessment framework includes 81 questions, 67 (83%) of which impact the scores of 4 overarching quality aspects. After testing with people with low health literacy, these aspects were phrased as "Healthy and safe," "Easy to use," "Secure data," and "Robust build." The scoring mechanism enables communication of the quality assessment results in a health app quality score and label, alongside a detailed report. Unstructured interviews with stakeholders revealed that evidence and third-party assessment are needed for health app uptake. The manufacturers considered the time needed to complete the assessment and gather evidence (2-4 days) acceptable. Publication of CEN-ISO/TS 82304-2:2021 Health software - Part 2: Health and wellness apps - Quality and reliability was approved in May 2021 in a nearly unanimous vote by 34 national SDOs, including 6 of the 10 most populous countries worldwide. CONCLUSIONS: A useful and usable international standard for health app quality assessment was developed. Its quality, approval rate, and early use provide proof of its potential to become the trusted, commonly used global framework. The framework will help manufacturers enhance and efficiently demonstrate the quality of health apps, consumers, and health care professionals to make informed decisions on health apps. It will also help insurers to make reimbursement decisions on health apps.

A Method for the Classification of Digital Health Architectures as Medical Devices; a Digital Health Research Perspective.

It is typical for many digital health research projects to develop IT architectures that will implement integrated care services that may also deliver interventions. As part of compliance with the requirements of the regulation, the components that are considered as a medical device will need to be classified to a medical device category. This is often seen as task that may increase the business risk and a major barrier of the project, particularly during the earlier stages when not all information is available. The paper offers a method assisting with classification of such architectures in the context of the Medical Devices Rregulation, offering a structured way to identifying how the initial deliverables of a project can be used to provide assurance to the justification of the classification.

Hazards for the Implementation and Use of Artificial Intelligence Enabled Digital Health Interventions, a UK Perspective.

BACKGROUND: Artificial Intelligence (AI) has seen an increased application within digital healthcare interventions (DHIs). DHIs use entails challenges about their safety assurance. Exacerbated by regulatory requirements, in the UK, this places the onus of safety assurance not only on the manufacturer, but also on the operator of a DHI. Clinical Safety claims and evidencing safe implementation and use of AI-based DHIs require expertise, to understand and act to control or mitigate risk. Current health software standards, regulation, and guidance do not provide the insight necessary for safer implementation. OBJECTIVE: To interpret published guidance and policy related to AI and justify clinical safety assurance of DHIs. METHOD: Assessment of UK health regulation policy, standards, and AI institution insights, utilizing a published Hazard Assessment framework, to structure safety justifications, and articulate hazards relating to AI-based DHIs. RESULTS: AI enabled DHI hazard identification, relating to implementation and use within healthcare delivery organizations. CONCLUSION: By application of the method, we postulate that UK research of AI DHIs highlighted issues that may affect safety, in need of consideration to justify safety of a DHI.

A biophysical basis for the emergence of the genetic code in protocells.

The origin of the genetic code is an abiding mystery in biology. Hints of a 'code within the codons' suggest biophysical interactions, but these patterns have resisted interpretation. Here, we present a new framework, grounded in the autotrophic growth of protocells from CO2 and H2. Recent work suggests that the universal core of metabolism recapitulates a thermodynamically favoured protometabolism right up to nucleotide synthesis. Considering the genetic code in relation to an extended protometabolism allows us to predict most codon assignments. We show that the first letter of the codon corresponds to the distance from CO2 fixation, with amino acids encoded by the purines (G followed by A) being closest to CO2 fixation. These associations suggest a purine-rich early metabolism with a restricted pool of amino acids. The second position of the anticodon corresponds to the hydrophobicity of the amino acid encoded. We combine multiple measures of hydrophobicity to show that this correlation holds strongly for early amino acids but is weaker for later species. Finally, we demonstrate that redundancy at the third position is not randomly distributed around the code: non-redundant amino acids can be assigned based on size, specifically length. We attribute this to additional stereochemical interactions at the anticodon. These rules imply an iterative expansion of the genetic code over time with codon assignments depending on both distance from CO2 and biophysical interactions between nucleotide sequences and amino acids. In this way the earliest RNA polymers could produce non-random peptide sequences with selectable functions in autotrophic protocells.

A method for machine learning generation of realistic synthetic datasets for validating healthcare applications.

Digital health applications can improve quality and effectiveness of healthcare, by offering a number of new tools to users, which are often considered a medical device. Assuring their safe operation requires, amongst others, clinical validation, needing large datasets to test them in realistic clinical scenarios. Access to datasets is challenging, due to patient privacy concerns. Development of synthetic datasets is seen as a potential alternative. The objective of the paper is the development of a method for the generation of realistic synthetic datasets, statistically equivalent to real clinical datasets, and demonstrate that the Generative Adversarial Network (GAN) based approach is fit for purpose. A generative adversarial network was implemented and trained, in a series of six experiments, using numerical and categorical variables, including ICD-9 and laboratory codes, from three clinically relevant datasets. A number of contextual steps provided the success criteria for the synthetic dataset. A synthetic dataset that exhibits very similar statistical characteristics with the real dataset was generated. Pairwise association of variables is very similar. A high degree of Jaccard similarity and a successful K-S test further support this. The proof of concept of generating realistic synthetic datasets was successful, with the approach showing promise for further work.

A Method of Justifying Confidence in the Safety of Digital Health Interventions.

Digital health interventions (DHIs) enable improvements in health strategy and address health system challenges. The World Health Organization provides a formal classification for DHIs. However, safety claims, about such interventions, vary in quality and are often vague as to how they are communicated between technical, clinical experts and stakeholders. By combining the classifications with a method of safety analysis and justification, we postulate confidence in the safety of digital technology. Confidence is resulting from the application of the framework to the DHI, using defined health system challenges. The framework and derived safety justifications can be applied to any DHI. It can serve as guideline for health strategy, regulatory and standards based compliance.

Safety Justification of Healthcare Applications Using Synthetic Datasets.

BACKGROUND: Increasing numbers of intelligent healthcare applications are developed by analysing big data, on which they are trained. It is necessary to assure that such applications will be safe for patients; this entails validation against datasets. But datasets cannot be shared easily, due to privacy, and consent issues, resulting in delaying innovation. Realistic Synthetic Datasets (RSDs), equivalent to the real datasets, are seen as a solution to this. OBJECTIVE: To develop the outline for safety justification of an application, validated with an RSD, and identify the safety evidence the RSD developers will need to generate. METHOD: Assurance case argument development approaches were used, including high level data related risk identification. RESULT: An outline of the justification of such applications, focusing on the contribution of the RSD. CONCLUSIONS: Use of RSD will require specific arguments and evidence, which will affect the adopted methods. Mutually supporting arguments can result in a compelling justification.

Life as a guide to prebiotic nucleotide synthesis.

Synthesis of activated nucleotides has been accomplished under 'prebiotically plausible' conditions, but bears little resemblance to the chemistry of life as we know it. Here we argue that life is an indispensable guide to its own origins.

Exploring the Notion of Hazards for Health IT.

Safety analysis is centred on identifying a set of hazards that form the basis of risk assessment. In healthcare, hazards are potential sources of harm to patients and as such the risk of these has to be assessed and managed. With the increased reliance on Health IT systems in health and social care settings, some of these hazards are associated with the development and use of these systems. In this paper we examine current practices in hazard identification, focusing on how clinicians and engineers approach this task within the Health IT safety assurance process. We highlight certain technical and organisational challenges and discuss approaches to improving current practices and promoting learning initiatives.

Sulfate radicals enable a non-enzymatic Krebs cycle precursor.

The evolutionary origins of the tricarboxylic acid cycle (TCA), or Krebs cycle, are so far unclear. Despite a few years ago, the existence of a simple non-enzymatic Krebs-cycle catalyst has been dismissed 'as an appeal to magic', citrate and other intermediates have meanwhile been discovered on a carbonaceous meteorite and do interconvert non-enzymatically. To identify the non-enzymatic Krebs cycle catalyst, we used combinatorial, quantitative high-throughput metabolomics to systematically screen iron and sulfate reaction milieus that orient on Archean sediment constituents. TCA cycle intermediates are found stable in water and in the presence of most iron and sulfate species, including simple iron-sulfate minerals. However, we report that TCA intermediates undergo 24 interconversion reactions in the presence of sulfate radicals that form from peroxydisulfate. The non-enzymatic reactions critically cover a topology as present in the Krebs cycle, the glyoxylate shunt and the succinic semialdehyde pathways. Assembled in a chemical network, the reactions achieve more than ninety percent carbon recovery. Our results show that a non-enzymatic precursor for the Krebs cycle is biologically sensible, efficient, and forms spontaneously in the presence of sulfate radicals.

Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection.

Resistance against the leaf mold fungus Cladosporium fulvum is mediated by the tomato Cf proteins which belong to the class of receptor-like proteins and indirectly recognize extracellular avirulence proteins (Avrs) of the fungus. Apart from triggering disease resistance, Avrs are believed to play a role in pathogenicity or virulence of C. fulvum. Here, we report on the avirulence protein Avr4, which is a chitin-binding lectin containing an invertebrate chitin-binding domain (CBM14). This domain is found in many eukaryotes, but has not yet been described in fungal or plant genomes. We found that interaction of Avr4 with chitin is specific, because it does not interact with other cell wall polysaccharides. Avr4 binds to chitin oligomers with a minimal length of three N-acetyl glucosamine residues. In vitro, Avr4 protects chitin against hydrolysis by plant chitinases. Avr4 also binds to chitin in cell walls of the fungi Trichoderma viride and Fusarium solani f. sp. phaseoli and protects these fungi against normally deleterious concentrations of plant chitinases. In situ fluorescence studies showed that Avr4 also binds to cell walls of C. fulvum during infection of tomato, where it most likely protects the fungus against tomato chitinases, suggesting that Avr4 is a counter-defensive virulence factor.

Clinical safety of England's national programme for IT: a retrospective analysis of all reported safety events 2005 to 2011.

OBJECTIVE: To analyse patient safety events associated with England's national programme for IT (NPfIT). METHODS: Retrospective analysis of all safety events managed by a dedicated IT safety team between September 2005 and November 2011 was undertaken. Events were reviewed against an existing classification for problems associated with IT. The proportion of reported events per problem type, consequences, source of report, resolution within 24h, time of day and day of week were examined. Sub-group analyses were undertaken for events involving patient harm and those that occurred on a large scale. RESULTS: Of the 850 events analysed, 68% (n=574) described potentially hazardous circumstances, 24% (n=205) had an observable impact on care delivery, 4% (n=36) were a near miss, and 3% (n=22) were associated with patient harm, including three deaths (0·35%). Eleven events did not have a noticeable consequence (1%) and two were complaints (<1%). Amongst the events 1606 separate contributing problems were identified. Of these 92% were predominately associated with technical rather than human factors. Problems involving human factors were four times as likely to result in patient harm than technical problems (25% versus 8%; OR 3·98, 95%CI 1·90-8.34). Large-scale events affecting 10 or more individuals or multiple IT systems accounted for 23% (n=191) of the sample and were significantly more likely to result in a near miss (6% versus 4%) or impact the delivery of care (39% versus 20%; p<0·001). CONCLUSION: Events associated with NPfIT reinforce that the use of IT does create hazardous circumstances and can lead to patient harm or death. Large-scale patient safety events have the potential to affect many patients and clinicians, and this suggests that addressing them should be a priority for all major IT implementations.

A comparative review of patient safety initiatives for national health information technology.

OBJECTIVE: To collect and critically review patient safety initiatives for health information technology (HIT). METHOD: Publicly promulgated set of advisories, recommendations, guidelines, or standards potentially addressing safe system design, build, implementation or use were identified by searching the websites of regional and national agencies and programmes in a non-exhaustive set of exemplar countries including England, Denmark, the Netherlands, the USA, Canada and Australia. Initiatives were categorised by type and software systems covered. RESULTS: We found 27 patient safety initiatives for HIT predominantly dealing with software systems for health professionals. Three initiatives addressed consumer systems. Seven of the initiatives specifically dealt with software for diagnosis and treatment, which are regulated as medical devices in England, Denmark and Canada. Four initiatives dealt with blood bank and image management software which is regulated in the USA. Of the 16 initiatives directed at unregulated software, 11 were aimed at increasing standardisation using guidelines and standards for safe system design, build, implementation and use. Three initiatives for unregulated software were aimed at certification in the USA, Canada and Australia. Safety is addressed alongside interoperability in the Australian certification programme but it is not explicitly addressed in the US and Canadian programmes, though conformance with specific functionality, interoperability, security and privacy requirements may lead to safer systems. England appears to have the most comprehensive safety management programme for unregulated software, incorporating safety assurance at a local healthcare organisation level based on standards for risk management and user interface design, with national incident monitoring and a response function. CONCLUSIONS: There are significant gaps in the safety initiatives for HIT systems. Current initiatives are largely focussed on software. With the exception of diagnostic, prognostic, monitoring and treatment software, which are subject to medical device regulations in some countries, the safety of the most common types of HIT systems such as EHRs and CPOE without decision support is not being explicitly addressed in most nations. Appropriate mechanisms for safety assurance are required for the full range of HIT systems for health professionals and consumers including all software and hardware throughout the system lifecycle. In addition to greater standardisation and oversight to ensure safe system design and build, appropriate implementation and use of HIT is critical to ensure patient safety.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs.

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H(+)-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Isolation and characterisation of a class of carbohydrate oxidases from higher plants, with a role in active defence.

In a search for novel plant-derived antimicrobial proteins, we screened extracts from salicylic acid (SA)-treated lettuce and sunflower leaves. These extracts displayed very potent antimicrobial activity against a set of phytopathogens. Characterisation of these extracts revealed that in both extracts, proteins of approximately 60 kDa were responsible for the antimicrobial activity. Further characterisation of these proteins and cloning of the respective cDNAs revealed close homology to a range of (plant) oxidases. Dissection of the enzymatic activity of both proteins revealed them to be carbohydrate oxidases (Helianthus annuus carbohydrate oxidase (Ha-CHOX) and Lactuca sativa carbohydrate oxidase (Ls-CHOX)) with broad substrate specificity and with hydrogen peroxide (H(2)O(2)) as one of the reaction products. The sunflower transcript, in addition to being SA inducible, was also inducible by fungal pathogens but not by ethylene and jasmonate. To determine whether Ha-CHOX plays a role in pathogen defence, it was transformed into tobacco and the effect of resistance to Pectobacterium carotovorum ssp. carotovorum was examined. Transgenic plants overexpressing Ha-CHOX displayed enhanced resistance to infection by this pathogen, and the resistance level was proportional to enzyme expression.