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Abstract Background Traditional medical research infrastructures relying on the Centers of Excellence (CoE) model (an infrastructure or shared facility providing high standards of research excellence and resources to advance scientific knowledge) are often limited by geographic reach regarding patient accessibility, presenting challenges for study recruitment and accrual. Thus, the development of novel, patient-centered (PC) strategies (e.g., the use of online technologies) to support recruitment and streamline study procedures are necessary. This research focused on an implementation evaluation of a design innovation with implementation outcomes as communicated by study staff and patients for CoE and PC approaches for a randomized controlled trial (RCT) for patients with vasculitis. Methods In-depth qualitative interviews were conducted with 32 individuals (17 study team members, 15 patients). Transcripts were coded using the Consolidated Framework for Implementation Research (CFIR). Results The following CFIR elements emerged: characteristics of the intervention, inner setting, characteristics of individuals, and process. From the staff perspective, the communication of the PC approach was a major challenge, but should have been used as an opportunity to identify one “point person” in charge of all communicative elements among the study team. Study staff from both arms were highly supportive of the PC approach and saw its promise, particularly regarding online consent procedures. Patients reported high self-efficacy in reference to the PC approach and utilization of online technologies. Local physicians were integral for making patients feel comfortable about participation in research studies. Conclusions The complexity of replicating the interpersonal nature of the CoE model in the virtual setting is substantial, meaning the PC approach should be viewed as a hybrid strategy that integrates online and face-to-face practices. Trial registrations 1) Name: The Assessment of Prednisone In Remission Trial – Centers of Excellence Approach (TAPIR). Trial registration number: ClinicalTrials.gov NCT01940094 . Date of registration: September 10, 2013. 2) Name: The Assessment of Prednisone In Remission Trial – Patient Centric Approach (TAPIR). Trial registration number: Clinical Trials.gov NCT01933724 . Date of registration: September 2, 2013.

Abstract To gain a deeper understanding of skeletal muscle function in younger age and aging in elderly, identification of molecular signatures regulating these functions under physiological conditions is needed. Although molecular studies of healthy muscle have been conducted on adults and older subjects, there is a lack of research on infant muscle in terms of combined morphological, transcriptomic and proteomic profiles. To address this gap of knowledge, we performed RNA sequencing (RNA-seq), tandem mass spectrometry (LC–MS/MS), morphometric analysis and assays for mitochondrial maintenance in skeletal muscle biopsies from both, infants aged 4–28 months and adults aged 19–65 years. We identified differently expressed genes (DEGs) and differentially expressed proteins (DEPs) in adults compared to infants. The down-regulated genes in adults were associated with functional terms primarily related to sarcomeres, cellular maintenance, and metabolic, immunological and developmental processes. Thus, our study indicates age-related differences in the molecular signatures and associated functions of healthy skeletal muscle. Moreover, the findings assert that processes previously associated solely with aging are indeed part of development and healthy aging. Hence, combined findings of this study also indicate that age-dependent controls are crucial in muscle disease studies, as otherwise the comparative results may not be reliable.

Abstract Regulatory T cells (Tregs) are crucial immune cells for tissue repair and regeneration. However, their potential as a cell-based regenerative therapy is not yet fully understood. Here, we show that local delivery of exogenous Tregs into injured mouse bone, muscle, and skin greatly enhances tissue healing. Mechanistically, exogenous Tregs rapidly adopt an injury-specific phenotype in response to the damaged tissue microenvironment, upregulating genes involved in immunomodulation and tissue healing. We demonstrate that exogenous Tregs exert their regenerative effect by directly and indirectly modulating monocytes/macrophages (Mo/MΦ) in injured tissues, promoting their switch to an anti-inflammatory and pro-healing state via factors such as interleukin (IL)-10. Validating the key role of IL-10 in exogenous Treg-mediated repair and regeneration, the pro-healing capacity of these cells is lost when Il10 is knocked out. Additionally, exogenous Tregs reduce neutrophil and cytotoxic T cell accumulation and IFN-γ production in damaged tissues, further dampening the pro-inflammatory Mo/MΦ phenotype. Highlighting the potential of this approach, we demonstrate that allogeneic and human Tregs also promote tissue healing. Together, this study establishes exogenous Tregs as a possible universal cell-based therapy for regenerative medicine and provides key mechanistic insights that could be harnessed to develop immune cell-based therapies to enhance tissue healing.

Abstract: Understanding how adolescents integrate multiple cultures into their identity is a growing field of research in modern multicultural societies and requires psychometrically sound assessments tools. The Bicultural Identity Integration framework together with the Bicultural Identity Integration Scale 2 (BIIS-2; Huynh et al., 2018) offers an instrument that allows assessing BII with two factors (Blendedness vs. Compartmentalization and Harmony vs. Conflict). However, the psychometric properties of the BIIS-2 remain inconclusive for multicultural youth in Germany. To address this gap, we examined the psychometric properties of the BIIS-2 in a sample comprising 595 diverse ethnic minority adolescents in Germany (Mage = 13.26, SD = 1.3; 45.6% female). Contrary to the proposed two-factor structure (Huynh et al., 2018), our findings provide evidence for a four-factor structure (Blendedness, Compartmentalization, Harmony, and Conflict) which is also supported through unique associations in validation results.

Aminoglycosides (AGs) are a class of antibiotics with a broad spectrum of activity. However, their use is limited by safety concerns associated with nephrotoxicity and ototoxicity, as well as drug resistance. To address these issues, semi-synthetic approaches for modifying natural AGs have generated new generations of AGs, however, with limited types of modification due to significant challenges in synthesis. This study explores a novel approach that harness the bacterial biosynthetic machinery of gentamicins and kanamycins to create hybrid AGs. This was achieved by glycodiversification of gentamicins via swapping the glycosyltransferase (GT) in their producer with the GT from kanamycins biosynthetic pathway and resulted in the creation of a series of novel AGs, therefore referred to as genkamicins (GKs). The manipulation of the hybrid biosynthetic pathway enabled the targeted accumulation of different GK species and the isolation and characterization of six GK components. These compounds display retained antimicrobial activity against a panel of World Health Organization (WHO) critical priority pathogens, and GK-C2a, in particular, demonstrates low ototoxicity compared to clinical drugs in zebrafish embryos. This study provides a new strategy for diversifying the structure of AGs and a potential avenue for developing less toxic AG drugs to combat infectious diseases.

ABSTRACTThe accurate classification of non-coding RNA (ncRNA) sequences is pivotal for advanced non-coding genome annotation and analysis, a fundamental aspect of genomics that facilitates understanding of ncRNA functions and regulatory mechanisms in various biological processes. While traditional machine learning approaches have been employed for distinguishing ncRNA, these often necessitate extensive feature engineering. Recently, deep learning algorithms have provided advancements in ncRNA classification. This study presents BioDeepFuse, a hybrid deep learning framework integrating convolutional neural networks (CNN) or bidirectional long short-term memory (BiLSTM) networks with handcrafted features for enhanced accuracy. This framework employs a combination of k-mer one-hot, k-mer dictionary, and feature extraction techniques for input representation. Extracted features, when embedded into the deep network, enable optimal utilization of spatial and sequential nuances of ncRNA sequences. Using benchmark datasets and real-world RNA samples from bacterial organisms, we evaluated the performance of BioDeepFuse. Results exhibited high accuracy in ncRNA classification, underscoring the robustness of our tool in addressing complex ncRNA sequence data challenges. The effective melding of CNN or BiLSTM with external features heralds promising directions for future research, particularly in refining ncRNA classifiers and deepening insights into ncRNAs in cellular processes and disease manifestations. In addition to its original application in the context of bacterial organisms, the methodologies and techniques integrated into our framework can potentially render BioDeepFuse effective in various and broader domains.

Abstract Background Esophageal carcinoma is a growing concern in regions that have a high incidence of human papillomavirus (HPV) infection such as East Africa. HPV, particularly the high-risk genotypes, is increasingly recognized as a risk factor for esophageal carcinoma. We set out to investigate the prevalence and associated factors of high-risk HPV in formalin-fixed paraffin-embedded (FFPE) tissue blocks with esophageal carcinoma at Bugando Medical Center, a tertiary referral hospital in Mwanza, Tanzania, East Africa. Methods A total of 118 esophageal carcinoma FFPE tissue blocks, collected from January 2021 to December 2022, were analyzed. Genomic DNA was extracted from these tissues, and multiplex polymerase chain reaction (PCR) was performed to detect HPV using degenerate primers for the L1 region and type-specific primers for detecting HPV16, HPV18, and other high-risk HPV genotypes. Data were collected using questionnaires and factors associated with high-risk HPV genotypes were analyzed using STATA version 15 software. Results Of the 118 patients’ samples investigated, the mean age was 58.3 ± 13.4 years with a range of 29–88 years. The majority of the tissue blocks were from male patients 81/118 (68.7%), and most of them were from patients residing in Mwanza region 44/118 (37.3%). Esophageal Squamous Cell Carcinoma (ESCC) was the predominant histological type 107/118 (91.0%). Almost half of the tissue blocks 63/118 (53.3%) tested positive for high-risk HPV. Among these, HPV genotype 16 (HPV16) was the most common 41/63 (65.1%), followed by HPV genotype 18 (HPV18) 15/63 (23.8%), and the rest were other high-risk HPV genotypes detected by the degenerate primers 7/63 (11.1%). The factors associated with high-risk HPV genotypes were cigarette smoking (p-value

Abstract Regulatory T cells (Tregs) are key immune regulators that have shown promise in enhancing cardiac repair post-MI, although the mechanisms remain elusive. Here, we show that rapidly increasing Treg number in the circulation post-MI via systemic administration of exogenous Tregs improves cardiac function in male mice, by limiting cardiomyocyte death and reducing fibrosis. Mechanistically, exogenous Tregs quickly home to the infarcted heart and adopt an injury-specific transcriptome that mediates repair by modulating monocytes/macrophages. Specially, Tregs lead to a reduction in pro-inflammatory Ly6CHi CCR2+ monocytes/macrophages accompanied by a rapid shift of macrophages towards a pro-repair phenotype. Additionally, exogenous Treg-derived factors, including nidogen-1 and IL-10, along with a decrease in cardiac CD8+ T cell number, mediate the reduction of the pro-inflammatory monocyte/macrophage subset in the heart. Supporting the pivotal role of IL-10, exogenous Tregs knocked out for IL-10 lose their pro-repair capabilities. Together, this study highlights the beneficial use of a Treg-based therapeutic approach for cardiac repair with important mechanistic insights that could facilitate the development of novel immunotherapies for MI.

Abstract Purpose Reaction databases are a key resource for a wide variety of applications in computational chemistry and biochemistry, including Computer-aided Synthesis Planning (CASP) and the large-scale analysis of metabolic networks. The full potential of these resources can only be realized if datasets are accurate and complete. Missing co-reactants and co-products, i.e., unbalanced reactions, however, are the rule rather than the exception. The curation and correction of such incomplete entries is thus an urgent need. Methods The SynRBL framework addresses this issue with a dual-strategy: a rule-based method for non-carbon compounds, using atomic symbols and counts for prediction, alongside a Maximum Common Subgraph (MCS)-based technique for carbon compounds, aimed at aligning reactants and products to infer missing entities. Results The rule-based method exceeded 99% accuracy, while MCS-based accuracy varied from 81.19 to 99.33%, depending on reaction properties. Furthermore, an applicability domain and a machine learning scoring function were devised to quantify prediction confidence. The overall efficacy of this framework was delineated through its success rate and accuracy metrics, which spanned from 89.83 to 99.75% and 90.85 to 99.05%, respectively. Conclusion The SynRBL framework offers a novel solution for recalibrating chemical reactions, significantly enhancing reaction completeness. With rigorous validation, it achieved groundbreaking accuracy in reaction rebalancing. This sets the stage for future improvement in particular of atom-atom mapping techniques as well as of downstream tasks such as automated synthesis planning. Scientific Contribution SynRBL features a novel computational approach to correcting unbalanced entries in chemical reaction databases. By combining heuristic rules for inferring non-carbon compounds and common subgraph searches to address carbon unbalance, SynRBL successfully addresses most instances of this problem, which affects the majority of data in most large-scale resources. Compared to alternative solutions, SynRBL achieves a dramatic increase in both success rate and accurary, and provides the first freely available open source solution for this problem.

Abstract Blast wave exposure, a leading cause of hearing loss and balance dysfunction among military personnel, arises primarily from direct mechanical damage to the mechanosensory hair cells and supporting structures or indirectly through excessive oxidative stress. We previously reported that HK-2, an orally active, multifunctional redox modulator (MFRM), was highly effective in reducing both hearing loss and hair cells loss in rats exposed to a moderate intensity workday noise that likely damages the cochlea primarily from oxidative stress versus direct mechanical trauma. To determine if HK-2 could also protect cochlear and vestibular cells from damage caused primarily from direct blast-induced mechanical trauma versus oxidative stress, we exposed rats to six blasts of 186 dB peak SPL. The rats were divided into four groups: (B) blast alone, (BEP) blast plus earplugs, (BHK-2) blast plus HK-2 and (BEPHK-2) blast plus earplugs plus HK-2. HK-2 was orally administered at 50 mg/kg/d from 7-days before to 30-day after the blast exposure. Cochlear and vestibular tissues were harvested 60-d post-exposure and evaluated for loss of outer hair cells (OHC), inner hair cells (IHC), auditory nerve fibers (ANF), spiral ganglion neurons (SGN) and vestibular hair cells in the saccule, utricle and semicircular canals. In the untreated blast-exposed group (B), massive losses occurred to OHC, IHC, ANF, SGN and only the vestibular hair cells in the striola region of the saccule. In contrast, rats treated with HK-2 (BHK-2) sustained significantly less OHC (67%) and IHC (57%) loss compared to the B group. OHC and IHC losses were smallest in the BEPHK-2 group, but not significantly different from the BEP group indicating lack of protective synergy between EP and HK-2. There was no loss of ANF, SGN or saccular hair cells in the BHK-2, BEP and BEPHK-2 groups. Thus, HK-2 not only significantly reduced OHC and IHC damage, but completely prevented loss of ANF, SGN and saccule hair cells. The powerful protective effects of this oral MFRM make HK-2 an extremely promising candidate for human clinical trials.

Abstract Hepatitis C virus (HCV) is a plus-stranded RNA virus that often chronically infects liver hepatocytes and causes liver cirrhosis and cancer. These viruses replicate their genomes employing error-prone replicases. Thereby, they routinely generate a large ‘cloud’ of RNA genomes (quasispecies) which—by trial and error—comprehensively explore the sequence space available for functional RNA genomes that maintain the ability for efficient replication and immune escape. In this context, it is important to identify which RNA secondary structures in the sequence space of the HCV genome are conserved, likely due to functional requirements. Here, we provide the first genome-wide multiple sequence alignment (MSA) with the prediction of RNA secondary structures throughout all representative full-length HCV genomes. We selected 57 representative genomes by clustering all complete HCV genomes from the BV-BRC database based on k-mer distributions and dimension reduction and adding RefSeq sequences. We include annotations of previously recognized features for easy comparison to other studies. Our results indicate that mainly the core coding region, the C-terminal NS5A region, and the NS5B region contain secondary structure elements that are conserved beyond coding sequence requirements, indicating functionality on the RNA level. In contrast, the genome regions in between contain less highly conserved structures. The results provide a complete description of all conserved RNA secondary structures and make clear that functionally important RNA secondary structures are present in certain HCV genome regions but are largely absent from other regions. Full-genome alignments of all branches of Hepacivirus C are provided in the supplement.

Abstract Resistance to clinical malaria takes years to develop even in hyperendemic regions and sterilizing immunity has rarely been observed. To evaluate the maturation of the host response against controlled repeat exposures to P. falciparum (Pf) NF54 strain-infected mosquitoes, we systematically monitored malaria-naïve participants through an initial exposure to uninfected mosquitoes and 4 subsequent homologous exposures to Pf-infected mosquitoes over 21 months (n = 8 males) (ClinicalTrials.gov# NCT03014258). The primary outcome was to determine whether protective immunity against parasite infection develops following repeat CHMI and the secondary outcomes were to track the clinical signs and symptoms of malaria and anti-Pf antibody development following repeat CHMI. After two exposures, time to blood stage patency increases significantly and the number of reported symptoms decreases indicating the development of clinical tolerance. The time to patency correlates positively with both anti-Pf circumsporozoite protein (CSP) IgG and CD8 + CD69+ effector memory T cell levels consistent with partial pre-erythrocytic immunity. IFNγ levels decrease significantly during the participants’ second exposure to high blood stage parasitemia and could contribute to the decrease in symptoms. In contrast, CD4-CD8 + T cells expressing CXCR5 and the inhibitory receptor, PD-1, increase significantly after subsequent Pf exposures, possibly dampening the memory response and interfering with the generation of robust sterilizing immunity.

We describe a new optical diagnostic for determining the composition of gases by measuring the motion of atoms and molecules trapped within very deep optical lattices. This non-resonant method is analogous to conventional Raman scattering, except that the observed spectral features relate to the oscillatory center-of-mass motion of each species within the lattice, determined uniquely by their respective polarizability-to-mass ratio. Depending on the density of the probed sample, detection occurs either via optical scattering at the high end or via non-resonant ionization at the lower end. We show that such a technique is complementary to conventional mass spectrometry techniques and envision its implementation in existing instruments.

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing.MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5’ reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs.ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster.DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

BackgroundWithin the city of Bradford in West Yorkshire, South Asian and Eastern European communities have an increased risk of childhood tooth decay, especially among families with Limited English Proficiency. Tooth decay is preventable, with national guidelines advocating home-based behaviours (toothbrushing with fluoride toothpaste and reducing sugar intake). In England, Health Visitors have opportunities to undertake oral health conversations during universal visits for children aged 0–24 months. The HABIT (Health visitors delivering Advice in Britain on Infant Toothbrushing) intervention provides structured oral health conversations, underpinned by complex intervention methodology. A feasibility study found HABIT acceptable to parents, feasible to deliver and led to improvements in home-based behaviours. However, the reach of this original study was limited to those proficient in English. This new study focused on exploring and enhancing the accessibility of the HABIT intervention to parents with Limited English Proficiency.MethodTwenty-four parents participated in interviews and focus groups, with 21 requesting support from interpreters. Community centres and WhatsApp were used to maximise inclusivity. Interviews and focus groups, followed a topic guide and the “Think Aloud” technique, were professionally transcribed, managed in NVivo, and thematically analysed. Team discussions facilitated analytical rigour. Recruitment continued until data saturation.ResultsThree themes were developed: (1) Navigating linguistic barriers; (2) Engagement through visuals; and (3) Addressing oral health challenges. Parents employed diverse strategies to interpret resources, including Google Translate, as well as family and wider community members. Consequently, the HABIT resources were modified to include simple text, subtitles, and translation tools. Parents highlighted the benefits of shorter oral health messages with clear visuals to help understanding, and this strategy was applied across all resources. Challenges surrounding children's resistance to toothbrushing, high sugar intake within their wider families and communities, and limited dental access were all raised. The HABIT resources were updated to address these challenges.ConclusionCollaborative community engagement has enhanced the HABIT resources, enabling access for high-risk communities to preventive oral-health programmes thereby promoting health equity.

C3 glomerulopathy (C3G) is an ultra-rare complement-mediated kidney disease caused by to the deregulation of the alternative pathway (AP) of proximal complement. Consequently, all effector loops of the complement are active and can lead to pathologies, such as C3a- and C5a-mediated inflammation, C3b opsonization, surface C3b-mediated AP C3 convertase assembly, C3 cleavage product deposition in the glomerulus, and lytic C5b-9/MAC cell damage. The most common pathologic mechanisms are defective chronic alternative pathway deregulation, mostly occurring in the plasma, often causing C3 consumption, and chronic complement-mediated glomerular damage. C3G develops over several years, and loss of renal function occurs in more than 50% of patients. C3G is triggered by both genetic and autoimmune alterations. Genetic causes include mutations in individual complement genes and chromosomal variations in the form of deletions and duplications affecting genes encoding complement modulators. Many genetic aberrations result in increased AP C3 convertase activity, either due to decreased activity of regulators, increased activity of modulators, or gain-of-function mutations in genes encoding components of the convertase. Autoimmune forms of C3G do also exist. Autoantibodies target individual complement components and regulators or bind to neoepitopes exposed in the central alternative pathway C3 convertase, thereby increasing enzyme activity. Overactive AP C3 convertase is common in C3G patients. Given that C3G is a complement disease mediated by defective alternative pathway action, complement blockade is an emerging concept for therapy. Here, we summarize both the causes of C3G and the rationale for complement inhibition and list the inhibitors that are being used in the most advanced clinical trials for C3G. With several inhibitors in phase II and III trials, it is expected that effectice treatment for C3G will become availabe in the near future.

IntroductionStudying plant-microbe interactions is one of the key elements in understanding the path to sustainable agricultural practices. These interactions play a crucial role in ensuring survival of healthy plants, soil and microbial communities. Many platforms have been developed over the years to isolate these highly complex interactions however, these are designed for small model plants. This creates a need for complementary devices for larger plants, such as sorghum.MethodsThis work introduces a novel platform, EcoFAB 3.0, which is designed to enable studying bioenergy plants such as sorghum for up to 4 weeks in a controlled sterile environment. Several other advantages of this platform such as dark root chambers and user-friendly assembly are also discussed in this work.Results and discussionEcoFAB 3.0 was found to replicate previous greenhouse and field observations when comparing an engineered sorghum line overproducing 4-hydroxybenzoic acid (4-HBA) and wildtype (variety BTx430). Consistent with greenhouse and field observations, it was found that the engineered line of sorghum grown in EcoFAB 3.0 had a higher 4-HBA content and a lower dry biomass.

Summary: Background: PfSPZ Vaccine, a promising pre-erythrocytic stage malaria vaccine candidate based on whole, radiation-attenuated Plasmodium falciparum (Pf) sporozoites (SPZ), has proven safe and effective in mediating sterile protection from malaria in malaria-naïve and exposed healthy adults. Vaccine-induced protection presumably depends on cellular responses to early parasite liver stages, but humoral immunity contributes. Methods: On custom-made Pf protein microarrays, we profiled IgG and IgM responses to PfSPZ Vaccine and subsequent homologous controlled human malaria infection (CHMI) in 21 Tanzanian adults with (n = 12) or without (n = 9) HIV infection. Expression of the main identified immunogens in the pre-erythrocytic parasite stage was verified by immunofluorescence detection using freshly purified PfSPZ and an in vitro model of primary human hepatocytes. Findings: Independent of HIV infection status, immunisation induced focused IgG and IgM responses to circumsporozoite surface protein (PfCSP) and merozoite surface protein 5 (PfMSP5). We show that PfMSP5 is detectable on the surface and in the apical complex of PfSPZ. Interpretation: Our data demonstrate that HIV infection does not affect the quantity of the total IgG and IgM antibody responses to PfCSP and PfMSP5 after immunization with PfSPZ Vaccine. PfMSP5 represents a highly immunogenic, so far underexplored, target for vaccine-induced antibodies in malaria pre-exposed volunteers. Funding: This work was supported by the Equatorial Guinea Malaria Vaccine Initiative (EGMVI), the Clinical Trial Platform of the German Center for Infection Research (TTU 03.702), the Swiss Government Excellence Scholarships for Foreign Scholars and Artists (grant 2016.0056) and the Interdisciplinary Center for Clinical Research doctoral program of the Tübingen University Hospital. The funders had no role in design, analysis, or reporting of this study.

Background Pregnant patients with single ventricle (SV) physiology carry a high risk of spontaneous pregnancy loss (SPL), yet the clinical factors contributing to this risk are not well defined.Methods Single-centre retrospective study of pregnant patients with SV physiology seen in cardio-obstetrics clinic over the past 20 years with chart review of their obstetric history. Patients without a known pregnancy outcome were excluded. Univariable Bayesian panel-data random effects logit was used to model the risk of SPL.Results The study included 20 patients with 44 pregnancies, 20 live births, 21 SPL and 3 elective abortions. All had Fontan palliation except for two with Waterston and Glenn shunts. 10 (50%) had a single right ventricle (RV). 14 (70%) had moderate or severe atrioventricular valve regurgitation (AVVR). Atrial arrhythmias were present in 16 (80%), Fontan-associated liver disease (FALD) in 15 (75%) and FALD stage 4 in 9 (45%). 12 (60%) were on anticoagulation. Average first-trimester oxygen saturation was 93.8% for live births and 90.8% for SPL. The following factors were associated with higher odds of SPL: RV morphology (OR 1.72 (95% credible interval (CrI) 1.0008–2.70)), moderate or severe AVVR (OR 1.64 (95% CrI 1.003–2.71)) and reduced first-trimester oxygen saturation (OR 1.83 (95% CrI 1.03–2.71) for each per cent decrease in O2 saturation.Conclusion Pregnant patients with SV physiology, particularly those with RV morphology, moderate or severe AVVR, and lower first-trimester oxygen saturations, have a higher risk of SPL. Identifying these clinical risk factors can guide preconception counselling by the cardio-obstetrics team.

Background The presence of dental caries impacts on children’s daily lives, particularly among those living in deprived areas. There are successful interventions across the United Kingdom for young children based on toothbrushing with fluoride toothpaste. However, evidence is lacking for oral health improvement programmes in secondary-school pupils to reduce dental caries and its sequelae. Objectives To determine the clinical and cost effectiveness of a behaviour change intervention promoting toothbrushing for preventing dental caries in secondary-school pupils. Design A multicentre, school-based, assessor-blinded, two-arm cluster randomised controlled trial with an internal pilot and embedded health economic and process evaluations. Setting Secondary schools in Scotland, England and Wales with above-average proportion of pupils eligible for free school meals. Randomisation occurred within schools (year-group level), using block randomisation stratified by school. Participants Pupils aged 11–13 years at recruitment, who have their own mobile telephone. Interventions Two-component intervention based on behaviour change theory: (1) 50-minute lesson delivered by teachers, and (2) twice-daily text messages to pupils’ mobile phones about toothbrushing, compared with routine education. Main outcome measures Primary outcome: presence of at least one treated or untreated carious lesion using DICDAS4–6MFT (Decayed, Missing and Filled Teeth) in any permanent tooth, measured at pupil level at 2.5 years. Secondary outcomes included: number of DICDAS4–6MFT; presence and number of DICDAS1–6MFT; plaque; bleeding; twice-daily toothbrushing; health-related quality of life (Child Health Utility 9D); and oral health-related quality of life (Caries Impacts and Experiences Questionnaire for Children). Results Four thousand six hundred and eighty pupils (intervention, n = 2262; control, n = 2418) from 42 schools were randomised. The primary analysis on 2383 pupils (50.9%; intervention 1153, 51.0%; control 1230, 50.9%) with valid data at baseline and 2.5 years found 44.6% in the intervention group and 43.0% in control had obvious decay experience in at least one permanent tooth. There was no evidence of a difference (odds ratio 1.04, 95% confidence interval 0.85 to 1.26, p = 0.72) and no statistically significant differences in secondary outcomes except for twice-daily toothbrushing at 6 months (odds ratio 1.30, 95% confidence interval 1.03 to 1.63, p = 0.03) and gingival bleeding score (borderline) at 2.5 years (geometric mean difference 0.92, 95% confidence interval 0.85 to 1.00, p = 0.05). The intervention had higher incremental mean costs (£1.02, 95% confidence interval −1.29 to 3.23) and lower incremental mean quality-adjusted life-years (−0.003, 95% confidence interval −0.009 to 0.002). The probability of the intervention being cost-effective was 7% at 2.5 years. However, in two subgroups, pilot trial schools and schools with higher proportions of pupils eligible for free school meals, there was an 84% and 60% chance of cost effectiveness, respectively, although their incremental costs and quality-adjusted life-years remained small and not statistically significant. The process evaluation revealed that the intervention was generally acceptable, although the implementation of text messages proved challenging. The COVID-19 pandemic hampered data collection. High rates of missing economic data mean findings should be interpreted with caution. Conclusions Engagement with the intervention and evidence of 6-month change in toothbrushing behaviour was positive but did not translate into a reduction of caries. Future work should include work with secondary-school pupils to develop an understanding of the determinants of oral health behaviours, including toothbrushing and sugar consumption, particularly according to free school meal eligibility. Trial registration This trial is registered as ISRCTN12139369. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 15/166/08) and is published in full in Health Technology Assessment; Vol. 28, No. 52. See the NIHR Funding and Awards website for further award information. Plain language summary What was the problem? Tooth decay has an impact on children and young people’s daily lives, particularly those living in deprived areas. For young children, programmes to improve toothbrushing with fluoride toothpaste help prevent tooth decay. The Brushing RemInder 4 Good oral HealTh trial (BRIGHT) investigated whether a secondary-school-based toothbrushing programme would work. What did we do? We developed a new programme which included a lesson and twice-daily text messages sent to pupils’ phones. In total, 4680 pupils, aged 11–13 years, from 42 secondary schools in the United Kingdom took part in the trial. At each school, one year group was randomly selected to receive the programme, while the other year group did not receive it. All pupils were followed up for 2.5 years to see whether there were any differences in levels of tooth decay, frequency of toothbrushing, plaque or quality of life. We also considered the programme’s value for money and the views of pupils and school staff. What did we find? We followed up 2383 pupils and found no difference in tooth decay, plaque or quality of life. We found those who had the programme were more likely to brush their teeth twice daily after 6 months than those who did not. The programme was not good value for money overall. However, the programme appeared to be of more benefit at preventing tooth decay in pupils eligible for free school meals compared to those not eligible. In the schools with more pupils eligible for free school meals, the chance of the programme representing good value for money increased. The programme was generally liked by the pupils and school staff. Some pupils found the text messages useful, although others said they were annoying. What does this mean? The programme helped pupils brush their teeth more frequently in the short term, but this did not lead to less tooth decay. Further research is needed to understand how to prevent tooth decay in secondary-school pupils. Scientific summary Background Reducing the high prevalence and severity of dental caries in the UK is a public health priority. Dental caries is largely preventable with several evidence-based child oral health promotion interventions including toothbrushing with fluoride toothpaste. However, evidence is lacking for interventions targeting toothbrushing practices to reduce dental caries and its sequelae (pain, infection and tooth loss) in secondary-school pupils. The Brushing RemInder 4 Good oral HealTh (BRIGHT) trial investigated a two-component behaviour change intervention (lesson and text messages) to prevent dental caries in secondary-school pupils. Objectives Conduct an internal pilot phase with feasibility components to: Tailor the intervention to young people. Test trial processes. Assess feasibility of within-school cluster randomisation (by year group). Investigate the intervention’s effect on caries prevalence, twice-daily toothbrushing, oral health-related quality of life and oral health behaviours. Investigate the intervention’s cost effectiveness. Explore implementation, mechanisms of impact and context through process evaluation. Methods Design A multicentre, school-based, assessor-blinded, two-arm cluster randomised controlled trial with an internal pilot phase, and embedded health economic and process evaluations. Criteria determining progression to the main phase were pre-specified and reviewed within the internal pilot phase. Setting Secondary schools in Scotland, England and Wales with above national average percentage of pupils eligible for free school meals (FSM) were recruited. Schools had to have pupils aged 11–16 years and at least 60 pupils per year group. Participant recruitment Pupils aged 11–13 years (Years 7 and 8 in England and Wales; S1 and S2 in Scotland) in participating schools were eligible to take part and received an information session about BRIGHT. Parents/carers had 2 weeks to decline their child’s participation. Pupils who had not been opted out were invited to participate by completing a consent form and providing their mobile telephone number. If they did not own a mobile telephone or could not provide their number, they were ineligible. Sample size We proposed to recruit 5040 pupils from 42 schools (84 year groups) to give 90% power to detect a reduction in the proportion of pupils with obvious decay experience from 34% to 26%, assuming: within-school (year-group level) randomisation, partial contamination effects (i.e. those contaminated gain half the treatment benefits) for 27% of the control group, recruiting an average of 60 pupils per year group, an intracluster correlation coefficient of 0.02% and 20% attrition at follow-up. A sample size of 10 schools was considered sufficient to test internal pilot objectives. Randomisation Year groups within schools were randomised 1 : 1 to the intervention or control arm. An allocation sequence, stratified by school using blocks of size two, was generated by an independent statistician. Once each school’s baseline assessments were completed, the year groups were randomised by allocation to the next available block. Trial interventions The intervention consisted of two components: (1) a 50-minute classroom-based session (CBS) about dental health delivered by teachers using a lesson plan and pupil-facing materials followed by (2) a series of twice-daily text messages to participants’ mobile phones about toothbrushing. Year groups allocated to the control arm received routine education only. Follow-up Assessment time points were baseline, after the lesson (internal pilot only), 12 weeks (internal pilot only), 6 months, 1, 2 (internal pilot only) and 2.5 years following lesson delivery. Pupils completed self-report questionnaires at all these time points. Dental examinations at baseline, 2 and 2.5 years included assessment of caries, plaque and gingival bleeding. Parents/carers were sent a resource use questionnaire at baseline, and 1, 2 and 2.5 years. Note, the descriptors of the time points for the assessments (e.g. 2.5 years) reflect the planned follow-up schedule and are used throughout the report; however, some of the actual average time intervals of follow-up varied from that planned (e.g. the actual average length of follow-up at the 2.5-year assessment was closer to 3 years as this was delayed due to disruptions caused by the COVID-19 pandemic). Primary outcome The primary outcome was the presence of at least one treated or untreated carious lesion in any permanent tooth, measured at the pupil level during the 2.5-year dental assessment using DICDAS4–6MFT (Decayed, Missing and Filled Teeth). Secondary outcomes Secondary outcomes assessed during the dental assessments were DICDAS4–6MFT at 2 years, and the following at 2 and 2.5 years: the presence of at least one treated or untreated carious lesion in any permanent tooth measured using DICDAS1–6MFT, the number of permanent DICDAS4–6MFT and DICDAS1–6MFT, plaque score (modified gingival index of Löe), bleeding score (gingival index) and number of teeth with bleeding gingivae. Participant-reported secondary outcome measures were: twice-daily toothbrushing, health-related quality of life measured by the Child Health Utility 9D (CHU9D) and oral health-related quality of life assessed using Caries Impacts and Experiences Questionnaire for Children (CARIES-QC). Statistical methods Both the clinical effectiveness and economic analyses were conducted to pre-specified and externally endorsed analysis plans. Analyses were conducted in STATA v17 (StataCorp LP, College Station, TX, USA) following the principles of intention to treat using two-sided statistical tests assessed at the 5% significance level. Baseline data were summarised descriptively overall and by randomised arm. The primary outcome was analysed using mixed-effect logistic regression, adjusting for the number of DICDAS4–6MFT and school year at baseline as fixed effects, and school as a random effect. Sensitivity analyses were conducted in which year group, nested within school, was additionally included as a random effect, and including additional covariates that were significantly associated with missing primary outcome data. A further sensitivity analysis excluded pupils whose dental assessments were completed outside of 3 months either side of the average length of follow-up for the 2.5-year time point. A complier-average causal effect (CACE) analysis, using a two-stage instrumental variable regression approach with the randomised group as the instrumental variable, was implemented to assess the impact of: attending the lesson; attending the lesson and receiving at least seven text messages a week for the first 12 weeks; and number of text messages sent. Subgroup analyses considered whether or not the intervention effect differed by FSM status, number of carious teeth at baseline and whether the school was recruited during the pilot or main trial phase. The secondary outcome of presence of at least one DICDAS1–6MFT was analysed as described for the primary outcome. The number of permanent DICDAS4–6MFT and DICDAS1–6MFT and number of teeth with bleeding gingivae were analysed using mixed-effect negative binomial regression, self-reported twice-daily toothbrushing via mixed-effect logistic regression and plaque, bleeding and CARIES-QC scores via mixed-effect linear regression. Models were adjusted for school year and associated baseline value of the outcome as fixed effects and school as a random effect. Attendance data, adverse events and safeguarding issues are summarised descriptively. Economic analysis The economic evaluation was undertaken using individual-level trial data, took the NHS perspective and generated quality-adjusted life-years (QALYs) using the CHU9D. The time horizon of the analysis was set to cover all relevant costs and outcomes. Parental questionnaires were used to collect data on dental treatments and child health-related quality of life. Other sources of cost data were used in relation to the text messages and the lesson. Results Clinical effectiveness results Of the 14,083 pupils approached in the 42 recruited schools, 4699 (33.4%) were eligible and consented and were asked to complete baseline data collection; however, 19 withdrew pre randomisation leaving 4680 pupils (92.9% of our target of 5040) included in the randomised sample (intervention, n = 2262; control, n = 2418). The average number of pupils recruited per school was 111.4 [standard deviation (SD) 35.9, median 107, range 46–189] and per year group was 55.7 (SD 21.6, median 53, range 13–119). The average age of pupils at recruitment was 12.7 years (SD 0.6), 54.2% were female and 21.9% were eligible for FSM. Over three-quarters (77.6%) reported brushing their teeth at least twice a day. There was a valid baseline dental assessment for 4625 pupils; 1603 (34.7%) had evidence of obvious decay experience (presence of DICDAS4–6MFT in at least one permanent tooth) and 2929 (63.3%) had at least one treated or untreated carious lesion in any permanent tooth (DICDAS1–6MFT). Baseline data were similar between the intervention and control groups. Confirmation of lesson delivery was received from 39 of the 42 schools, with an estimated 2016 (89.1%) of 2262 pupils randomised to the intervention group attending. Text messages were sent to 2258 (99.8%) intervention pupils. The other four withdrew shortly after receiving the lesson and so their messages were not commenced. Participants were sent text messages until they requested them to stop or until 12 July 2020 (when a technical error occurred with the text provider that meant texts stopped being sent). A total of 962 intervention participants (42.5%) withdrew from receiving the text messages, a median of 2.8 months after they commenced (range 1 day to 30 months). Participants were sent messages for between 0 and 127 weeks (approximately 30 months, mean 53.4 weeks, SD 35.4, median 62). This equated to between 1 and 1708 text messages (mean 694.5, SD 468.9, median 789). On average, 71.4% of the text messages sent to a participant were successfully delivered. At 2.5 years, 1043 out of 2383 pupils (43.8%; intervention 514, 44.6%; control 529, 43.0%) had obvious decay experience in at least one permanent tooth. There was no evidence of a difference between the intervention and control groups [odds ratio (OR) 1.04, 95% confidence interval (CI) 0.85 to 1.26, p = 0.72]. The sensitivity analyses produced similar results to the primary analysis. The CACE estimates of the treatment effect based on attending the CBS session and on attending the CBS session and receiving at least 50% of their messages per week for the first 12 weeks were similar to the intention to treat estimate (OR 1.05, 95% CI 0.85 to 1.31, p = 0.64, and 1.07, 95% CI 0.72 to 1.59, p = 0.74, respectively). The CACE estimate associated with the number of texts sent was OR 1.00 (95% CI 0.999 to 1.001, p = 0.93), which indicates that for every additional text message sent, there was no evidence of a decrease in likelihood of having a carious lesion. There was no significant interaction between treatment allocation and either number of carious teeth at baseline or pilot/main trial schools, but there was evidence of a qualitative interaction for FSM status, with a benefit of the intervention seen among FSM pupils (OR 0.69, 95% CI 0.44 to 1.08, p = 0.10) but not among non-FSM pupils (OR 1.17, 95% CI 0.93 to 1.46, p = 0.18). There was evidence of a statistically significant difference for twice-daily toothbrushing at 6 months (OR 1.30, 95% CI 1.03 to 1.63, p = 0.03) and borderline evidence of a difference in gingival index score between the two groups (geometric mean difference 0.92, 95% CI 0.85 to 1.00, p = 0.05) at 2.5 years. One non-serious adverse event was recorded during the trial, which was deemed possibly related and unexpected, for a pupil in the control group. No suspected serious pathologies were identified during dental assessments. Fifteen safeguarding issues arose during the course of the trial. All were dealt with according to the trial safeguarding procedure. Cost-effectiveness results Clinical results suggested that there was unlikely to be effects beyond 2.5 years, which was therefore set as the time horizon. Due to high rates of missing data, treatment costs were estimated from dental assessment data using multiple imputation. Text messages cost £32.53 per pupil on average, while mean dental costs over the 2.5-year follow-up were £20.73 and £21.02 for control and intervention groups, respectively. QALYs were similar in the control and intervention groups (2.196 and 2.193, respectively). Regression analysis estimated incremental costs and QALYs of the intervention, relative to the control group, to be £1.02 (95% CI −1.29 to 3.23) and −0.003 (95% CI −0.009 to 0.002), respectively, with a 7% chance that the intervention is cost-effective using a £20,000 per QALY gained threshold. The results were robust to the changes explored in the sensitivity analyses, except for an alternative approach to estimating QALYs using the CARIES-QC. While this only had a very small impact on incremental costs and QALYs, it was enough to generate an incremental QALY gain, which, in combination with a very small incremental cost, generates an incremental cost-effectiveness ratio (ICER) of £79 per QALY gained and an associated probability of the intervention being cost-effective of 96%. The subgroup analyses suggest that there is no evidence of a difference in the probability of the intervention being cost-effective within schools in Scotland versus England/Wales (6% in both cases). A positive QALY gain is observed in those schools with higher levels of pupils eligible for FSM than schools with lower levels of FSM eligibility. This produces an ICER of £2254 per QALY gained and a probability of the intervention being cost-effective of 60%. We also observed a QALY gain for pilot schools which leads to the intervention having an ICER of £3049 per QALY gained (and an 84% chance of it being cost-effective). Process evaluation The process evaluation showed that the BRIGHT intervention was generally implemented as intended, although there were challenges establishing the dose delivered and received for both components and technical difficulties delivering the text messages. Overall, pupils, staff members and stakeholders felt the intervention was acceptable. Conclusions At the 2.5-year follow-up, there was no evidence of a difference between the control and intervention groups in the prevalence of caries extending to dentine (primary outcome) or including enamel and dentine lesions. The proportion of participants with caries into dentine was high (intervention group 34.4% at baseline and 44.7% at the 2.5-year follow-up, and control group 34.9% at baseline and 43.1% at follow-up). There was an indication of a positive effect on short-term toothbrushing behaviour at 6 months. The subgroup analysis of participants eligible for FSM suggests a significant, qualitative interaction effect whereby the intervention appeared to be beneficial in terms of caries prevalence within pupils eligible for FSM but not for those not eligible for FSM. The process evaluation found the intervention to be broadly acceptable with some technical issues of text message delivery. The primary economic analysis shows that the intervention is not likely to be cost-effective. Further research is needed to understand how to prevent dental caries in secondary-school pupils. Trial registration This trial is registered as ISRCTN12139369. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 15/166/08) and is published in full in Health Technology Assessment; Vol. 28, No. 52. See the NIHR Funding and Awards website for further award information.

Summary: Osteosarcoma is a highly aggressive primary bone tumor that has seen little improvement in survival rates in the past three decades. Preclinical studies are conducted on a small pool of commercial cell lines which may not fully reflect the genetic heterogeneity of this complex cancer, potentially hindering translatability of in vitro results. Developing a single-site laboratory protocol to rapidly establish patient-derived primary cancer cell lines (PCCL) within a clinically actionable time frame of a few weeks will have significant scientific and clinical ramifications. These PCCL can widen the pool of available cell lines for study while patient-specific data could derive therapeutic correlation. This endeavor is exceedingly challenging considering the proposed time constraints. By proposing key definitions and a clear theoretical framework, this evaluation of osteosarcoma cell line establishment methodology over the past three decades assesses feasibility by identifying barriers and suggesting solutions, thereby facilitating systematic experimentation and optimization.

Abstract Aerobic methanotrophic bacteria are considered strict aerobes but are often highly abundant in hypoxic and even anoxic environments. Despite possessing denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we show that acidophilic methanotrophs can respire nitrous oxide (N2O) and grow anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. We study two strains that possess N2O reductase genes: Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6. We show that N2O respiration supports growth of Methylacidiphilum caldifontis at an extremely acidic pH of 2.0, exceeding the known physiological pH limits for microbial N2O consumption. Methylocella tundrae simultaneously consumes N2O and CH4 in suboxic conditions, indicating robustness of its N2O reductase activity in the presence of O2. Furthermore, in O2-limiting conditions, the amount of CH4 oxidized per O2 reduced increases when N2O is added, indicating that Methylocella tundrae can direct more O2 towards methane monooxygenase. Thus, our results demonstrate that some methanotrophs can respire N2O independently or simultaneously with O2, which may facilitate their growth and survival in dynamic environments. Such metabolic capability enables these bacteria to simultaneously reduce the release of the key greenhouse gases CO2, CH4, and N2O.