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Introduction Setting research priorities together with children and young people (CYP) in James Lind Alliance (JLA) Priority Setting Partnerships (PSPs) is an example of involving CYP from the beginning of the research process. In these PSPs, CYP can be involved in steering groups, surveys, focus groups and the final priority‐setting workshop. The success of JLA PSPs is evident, but it is important to note that specific guidance has not been available on how to involve CYP aged under 18 years. We aimed to collect experiences and tips from CYP who have taken part in a priority‐setting exercise and to work with them to develop guidance for the JLA on involving CYP in PSPs. Methods This project was conducted in the United Kingdom and the Netherlands and was coordinated by the JLA. The project was overseen by a project group consisting of eight people, this group invited CYP who had participated in previous JLA PSPs. A workshop in the Netherlands and in the United Kingdom was organised. Throughout the workshops, the primary focus was exploring the first‐hand experiences of CYP. We developed ‘tips’ on how best to involve CYP. The project was conducted between May 2023 and February 2024. Results Four CYP were involved in the UK workshop and three in the Dutch workshop. One young person had to cancel and was interviewed separately. Tips are divided into three categories, to reflect the different elements of the JLA method: steering group, surveys and the final priority‐setting workshop including preparations and evaluations. Tips centred on three themes: making participants feel comfortable, making the process accessible and making the involvement of CYP meaningful. Discussion This project provides a list of 29 tips for involving CYP in JLA PSPs and guides future partnerships seeking to involve CYP. It guides teams during the planning, execution and completion of the project. Patient and Public Contribution Children and young people with experience in JLA PSPs shared their tips with us for involving CYP in future PSPs. We examined their perceptions of what worked well and their constructive insights into areas for refinement in involving CYP in developing a research agenda.

Introduction There is a growing consensus that children and young people (CYP) should be involved in matters that concern them. Progress is made in involving CYP in developing pediatric research agendas (PRAs), although the impact of their involvement remains unknown. We aimed to evaluate the impact of involving CYP in developing PRAs and assess the extent to which postpatient and public involvement (post‐PPI) activities were planned. Methods We conducted a qualitative study using in‐depth interviews to identify and gain an in‐depth understanding of the impact of involving CYP in developing PRAs. The transcripts were uploaded to Atlas.ti to be coded and organised. Dutch‐language interviews were analysed and interpreted together with vocational education and training (VET) students. These students were aged between 14 and 18 years and were training to become nurses. Results Three CYP and 15 researchers decided to participate. We focused on three categories of impact: agenda‐setting impact, individual impact and academic impact. Involving CYP creates a more enriched and clarified agenda. It ensured that both CYP and researchers underwent personal or professional growth and development, it created a connection between the people involved, awareness about the importance of involving CYP and it ensured that the people involved had a positive experience. The participants were unable to indicate the academic impact of their PRAs, but they did understand the key factors for creating it. In addition, the need to measure impact was highlighted, with a particular focus on assessing individual impact. Discussion Our study outlines the diverse subthemes of impact that arise from involving CYP in developing PRAs. Despite the potential of research agendas to amplify CYP voices, only a minority of researchers strategized post‐PPI activities ensuring impactful outcomes, prompting the need for thorough evaluation of various impact forms and consistent alignment with the overarching goal of transforming the research field. Patient or Public Contribution We involved VET students in the data analysis and interpretation phase by forming a young person advisory group. The data analysis of the interviews analysed by the VET students revealed four distinct themes: 1. Learnt new knowledge. 2. Learnt to collaborate. 3. Learnt to listen. 4. Assessment of the individual impact.

ObjectiveThe objective of this study is twofold: first, to describe the methods used when involving children and young people (CYP) in developing a paediatric research agenda and, second, to evaluate how the existing literature describes the impact of involving CYP. We distinguish three forms of impact: impact on the research agenda (focused impact), impact on researchers and CYP (diffuse impact) and impact on future research (research impact).DesignA narrative review of MEDLINE, PsycINFO, Web of Science and Google Scholar was conducted from October 2016 to January 2022. The included studies involved at least one CYP in developing a research agenda and were published in English.Results22 studies were included; the CYP involved were aged between 6 years and 25 years. Little variation was found in the methods used to involve them. The methods used were James Lind Alliance (JLA) approach (n=16), focus groups (n=2), workshop (n=2), research prioritisation by affected communities (n=1) and combined methods (n=1). Impact was rarely described: focused impact in nine studies, diffuse impact in zero studies and research impact in three studies.ConclusionThis study concludes that the JLA approach is most frequently used to involve CYP and that all methods used to involve them are rarely evaluated. It also concludes that the reported impact of involving CYPs is incomplete. This study implies that to convince sceptical researchers of the benefits of involving CYPs and to justify the costs, more attention should be paid to reporting these impacts.

Background A growing trend in research is to involve co-researchers. It is referred to as Patient and Public Involvement (PPI) and comprises three groups: the patients, the public, and the researchers. Like in adult public involvement, healthy children can also be considered as ‘the public’. Paediatric patients and researchers experienced in conducting child-inclusive research are often asked about their attitudes towards the challenges they encounter. This is not the case for healthy children and researchers without such experience. Our aim was to investigate the attitudes of these children and researchers towards the challenges encountered during child-inclusive research. Methods This was an exploratory study. We interviewed healthy children and adult researchers without prior experience in child-inclusive research. We recruited the children through a foundation for young researchers and the adult researchers from two hospitals, both in Groningen, the Netherlands. We audio recorded the interviews, and they were transcribed verbatim. We analysed the data using qualitative content analysis. Results We interviewed five adult researchers and seven healthy children, aged 9 to 14 years. Both groups thought that it was best to involve children in paediatric research from as early a stage as possible. The children assumed that no prior training would be needed because they had already been trained at school. The researchers’ attitudes varied regarding training children beforehand. Both groups thought that researchers did not need prior training on how to involve children if they worked with children on a daily basis. The children felt that recognition and a modest financial reward was appropriate. Adult researchers were cautious about rewarding the children. They feared it might render the children less intrinsically motivated. Conclusion Our study indicated that young and adult researchers have clear attitudes towards the challenges encountered during child-inclusive research. Young researchers could help adult researchers to find solutions to these challenges, even if they have no prior experience in child-inclusive research. Adult researchers who acknowledge the importance of child-inclusive research represent a significant step towards meaningful involvement of children. Our results imply that children could be involved in the decision-making process concerning the challenges encountered in child-inclusive research. Plain English summary A growing trend in research is to involve co-researchers. It is called Patient and Public Involvement (PPI) and includes three groups: the patients, the public, and the researchers. In child-inclusive research the patients are children with a medical condition while the public is represented by healthy children. Generally, the attitudes of patients and researchers are heard, while the public is often unheard. We aimed to give a voice to healthy children and researchers without prior experience in child-inclusive research to determine their attitudes towards the challenges encountered. We interviewed seven healthy children and five researchers without prior experience in child-inclusive research. Both groups thought that it is best to involve children in research from as early a stage as possible. The children saw no added value in training children to be involved in child-inclusive research. The attitudes of researchers varied on this point. Both groups thought that researchers did not require prior training on how to involve children provided they already worked with children on a day-to-day basis. Children thought a modest financial reward would be appropriate, while researchers were cautious about giving a reward. Our study indicated that both groups had clear attitudes about the challenges encountered during child-inclusive research. Young researchers could help adult researchers to find solutions to these challenges, even if they have no prior experience in child-inclusive research. Our results imply that children could be involved in the decision-making process concerning the challenges encountered in child-inclusive research.

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An extremely bright, truly monomeric RFP, mScarlet, is described that outperforms existing RFPs in diverse labeling applications, especially in FRET with ratiometric imaging. We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.

Genetically encoded fluorescent proteins (FPs) are highly utilized in cell biology research to study proteins of interest or signal processes using biosensors. To perform well in specific applications, these FPs require a multitude of tailored properties. It is for this reason that they need to be optimized by using mutagenesis. The optimization process through screening is often based solely on bacterial colony brightness, but multiple parameters ultimately determine the performance of an optimal FP. Instead of characterizing other properties after selection, we developed a multiparameter screening method based on four critical parametersscreened simultaneously: fluorescence lifetime, cellular brightness, maturation efficiency, and photostability. First, a high-throughput primary screen (based on fluorescence lifetime and cellular brightness using a mutated FP library) is performed in bacterial colonies. A secondary multiparameter screen based on all four parameters, using a novel bacterial–mammalian dual-expression vector enables expression of the best FP variants in mammalian cell lines. A newly developed automated multiparameter acquisition and cell-based analysis approach for 96-well plates further increased workflow efficiency. We used this protocol to yield the record-bright mScarlet, a fast-maturating mScarlet-I, and a photostable mScarlet-H. This protocol can also be applied to other FP classes or Förster resonance energy transfer (FRET)-based biosensors with minor adaptations. With an available mutant library of a template FP and a complete and tested laboratory setup, a single round of multiparameter screening (including the primary bacterial screen, secondary mammalian cell screen, sequencing, and data processing) can be performed within 2 weeks. This protocol describes a screening pipeline to select red fluorescent proteins (FPs) with optimized parameters from a mutant FP library. FPs are screened for fluorescence lifetime, cellular brightness, maturation efficiency, and photostability.

The 15 short chapters that form this 2023 ammonia-for-energy roadmap provide a comprehensive assessment of the current worldwide ammonia landscape and the future opportunities and associated challenges facing the use of ammonia, not only in the part that it can play in terms of the future displacement of fossil-fuel reserves towards massive, long-term, carbon-free energy storage and heat and power provision, but also in its broader holistic impacts that touch all three components of the future global food-water-energy nexus.

Bivalent mRNA-based COVID-19 vaccines encoding the ancestral and omicron spike (S) protein were developed as a countermeasure against antigenically distinct SARS-CoV-2 variants. We aimed to assess the (variant-specific) immunogenicity and reactogenicity of mRNA-based bivalent omicron (BA.1) vaccines in individuals who were primed with adenovirus-based or mRNA-based vaccines encoding the ancestral spike protein. We analysed results of the direct boost group of the SWITCH ON study, an open-label, multicentre, randomised controlled trial. Health-care workers from four academic hospitals in the Netherlands aged 18–65 years who had completed a primary COVID-19 vaccination regimen and received one booster of an mRNA-based vaccine, given no later than 3 months previously, were eligible. Participants were randomly assigned (1:1) using computer software in block sizes of 16 and 24 to receive an omicron BA.1 bivalent booster straight away (direct boost group) or a bivalent omicron BA.5 booster, postponed for 90 days (postponed boost group), stratified by priming regimen. The BNT162b2 OMI BA.1 boost was given to participants younger than 45 years, and the mRNA-1273.214 boost was given to participants 45 years or older, as per Dutch guidelines. The direct boost group, whose results are presented here, were divided into four subgroups for analysis: (1) Ad26.COV2.S (Johnson & Johnson) prime and BNT162b2 OMI BA.1 (BioNTech–Pfizer) boost (Ad/P), (2) mRNA-based prime and BNT162b2 OMI BA.1 boost (mRNA/P), (3) Ad26.COV2.S prime and mRNA-1273.214 (Moderna) boost (Ad/M), and (4) mRNA-based prime and mRNA-1273.214 boost (mRNA/M). The primary outcome was fold change in S protein S1 subunit-specific IgG antibodies before and 28 days after booster vaccination. The primary outcome and safety were assessed in all participants except those who withdrew, had a SARS-CoV-2 breakthrough infection, or had a missing blood sample at day 0 or day 28. This trial is registered with ClinicalTrials.gov, NCT05471440. Between Sept 2 and Oct 4, 2022, 219 (50%) of 434 eligible participants were randomly assigned to the direct boost group; 187 participants were included in the primary analyses; exclusions were mainly due to SARS-CoV-2 infection between days 0 and 28. From the 187 included participants, 138 (74%) were female and 49 (26%) were male. 42 (22%) of 187 participants received Ad/P and 44 (24%) mRNA/P (those aged <45 years), and 45 (24%) had received Ad/M and 56 (30%) mRNA/M (those aged ≥45 years). S1-specific binding antibody concentrations increased 7 days after bivalent booster vaccination and remained stable over 28 days in all four subgroups (geometric mean ratio [GMR] between day 0 and day 28 was 1·15 [95% CI 1·12–1·19] for the Ad/P group, 1·17 [1·14–1·20] for the mRNA/P group, 1·20 [1·17–1·23] for the Ad/M group, and 1·16 [1·13–1·19] for the mRNA/M group). We observed no significant difference in the GMR between the Ad/P and mRNA/P groups (p=0·51). The GMR appeared to be higher in the Ad/M group than in the mRNA/M group, but was not significant (p=0·073). Most side-effects were mild to moderate in severity and resolved within 48 h in most individuals. Booster vaccination with mRNA-1273.214 or BNT162b2 OMI BA.1 in adult healthcare workers resulted in a rapid recall of humoral and cellular immune responses independent of the priming regimen. Monitoring of SARS-CoV-2 immunity at the population level, and simultaneously antigenic drift at the virus level, remains crucial to assess the necessity and timing of COVID-19 variant-specific booster vaccinations. The Netherlands Organization for Health Research and Development (ZonMw).

Copper deficiency is an important disease of cattle that produces several clinical signs and lesions, due to alterations in copper-dependent enzymes. One of the organs affected by this deficiency is the heart (falling disease), but nevertheless, these cardiac lesions have not been extensively studied in bovines. The aim of this work was to propose a possible pathogenic mechanism for cardiac lesions in cattle affected by copper deficiency. Because of the possible existence of oxidative distress caused by low levels of copper-zinc-superoxide dismutase and cytochrome oxidase, ultrastructural and histological lesions have been evaluated in the heart of bovines in which a Cu deficiency had been induced using high molybdenum and sulfur levels in the diet. Our results indicated that copper deficiency produces significant damage in myocardium with high levels of lipid oxidation and a significant reduction in copper-zinc-superoxide dismutase activity leading to an oxidative distress situation. However, cytochrome oxidase activity was not significantly reduced. Histological observation revealed a significant increase in the amount of connective tissue, enlarged basement membranes of myocytes, and numerous Anichkov cells, in the hearts of deficient animals. Ultrastructural observation showed a significant enhancement in the mitochondrial volume density, with presence of lesions such as swelling and cristae disruption. We conclude that copper deficiency in bovines causes morphological lesions in the heart due to an oxidative damage produced by copper-dependent enzyme alterations.