We investigated the temporal expression profiles of long noncoding RNA (lncRNA) and mRNA in the peripheral blood of pigs during development and identified the lncRNAs that are related to the blood-based immune system.Peripheral blood samples were obtained from the pigs at 0, 7, 28, and 180 days and 2 years of age. RNA sequencing was performed to survey the lncRNA and mRNA transcriptomes in the samples. Short time-series expression miner (STEM) was used to show temporal expression patterns in the mRNAs and lncRNAs. Gene ontology and Kyoto encyclopedia of genes and genomes analyses were performed to assess the genes’ biological relevance. To predict the functions of the identified lncRNAs, we extracted mRNAs that were nearby loci and highly correlated with the lncRNAs.TCONS-00086451 may promote blood-based immune system development by upregulating nuclear factor of activated T-cells cytoplasmic 2 expression.In total of 5,946 lncRNA and 12,354 mRNA transcripts were identified among the samples. STEM showed that most lncRNAs and mRNAs had similar temporal expression patterns during development, indicating the expressional correlation and functional relatedness between them. The five stages were divided into two classes: the suckling period and the late developmental stage. Most genes were expressed at low level during the suckling period, but at higher level during the late stages. Expression of several T-cell-related genes increased continuously during the suckling period, indicating that these genes are crucial for establishing the adaptive immune system in piglets at this stage. Notably, lncRNA This study provides a catalog of porcine peripheral blood-related lncRNAs and mRNAs and reveals the characteristics and temporal expression profiles of these lncRNAs and mRNAs during peripheral blood development from the newborn to adult stages in pigs. A better understanding of porcine immune system development is needed because of the agricultural importance of pigs, the role of pigs as potential reservoir hosts for zoonotic diseases, and increasing awareness of the benefits of using pigs as preclinical models for human disease and therapy . Previoulnc-DC is essential for differentiating monocytes into dendritic cells |≥1.5, p<0.05) between adjacent time points (D0 vs D7 and D7 vs D28) in the suckling period . To better understand the changes in biological processes between adjacent time points within each stage, we first identified the DE genes . NFATC2 is a transcription factor that plays a critical role in regulating lymphocyte effector differentiation. Moreover, NFATC2 is an important factor controlling mast cell accessory function at the interface of innate and adaptive immunity [TCONS-00086451 may be associated with NFATC2 and appears to play a role in adaptive immune system development.Our research also identified 980 DE lncRNAs in the three comparison groups  . Among tf NFATC2 . This lnf NFATC2 , with a immunity . ConsequCD20, NFATC2, and TCONS-00086451), and verified their expression patterns from our RNA-seq results via qRT-PCR. The results confirmed that the five DE genes were expressed at all five development stages  were divided into two developmental stages: the suckling period  and the late development phase (D180 and Y2). Time points within the same stage had similar snapshots of gene expression. Similarly, using a STEM platform to investigate how gene expression profiles change continuously over time during blood development in pigs showed that most mRNAs in the blood were expressed at low level during the suckling period but at higher levels later in development. Thus, we suggest that genes expressed during each time point within a stage should have similar underlying regulatory mechanisms. We also found that both the number of expressed genes and the gene expression levels in the blood peaked at 180 days among the five time points, indicating that most genes may be activated at 180 days. This can be caused by two factors: i) regulation of gene transcriptional activity and ii) relative changes in the abundances of cell populations expressing transcripts at constant levels . Time-seCD4) and toll-like receptors (TLR3 and TLR7) displayed age-related changes. CD4 was significantly upregulated at D0 to D28. TLR3 and TLR7 were significantly upregulated at D0 to D7. T lymphocytes and TLRs are associated with resistance to pathogenic challenges and play important roles in the mammalian immune system [Changes in the biological processes between adjacent time points during lactation revealed that the DE genes associated with inflammatory response and viral and bacterial infections were significantly upregulated in the peripheral blood after birth. This is consistent with previous reports describing that neonates have little immunological memory, which increases their vulnerability to infectious agents . Notablye system . Age-rele system . In contlnc-IL7R [cis- or trans-acting mechanisms. Here, our results also indicate that DE lncRNA might co-regulate the normal development of blood-based immunity with immune-responsible mRNAs as the lncRNA-mRNA pairs (TCON_S00086451-NFATC2) show. The mRNAs overlap of the corresponding lncRNAs. However, the detailed regulatory mechanisms of the mRNA and lncRNA interaction remains unclear and further research is needed to elucidate this relationship.Recent reports have revealed that many immune-related lncRNAs are located near or overlap with immune-responsible protein-coding genes such as interleukin 1b-regions of bidirectional transcription 46  and lnc-lnc-IL7R . These lHere, we produced an extensive resource for lncRNAs and mRNAs expressed in porcine peripheral blood at five developmental stages and predicted that several lncRNAs and mRNAs have functional effects. Our results, from a combined analysis of mRNA functional enrichment and lncRNA functional prediction, suggest that lncRNAs might regulate blood-based immune system development by co-regulation with mRNAs.


Using genomic and structural information from SARS-CoV-2, we created a biomass function capturing its amino and nucleic acid requirements and incorporated this into a metabolic model of the human lung cell to predict metabolic perturbations that inhibit virus reproduction. Viruses rely on their host for reproduction. Here, we made use of genomic and structural information to create a biomass function capturing the amino and nucleic acid requirements of SARS-CoV-2. Incorporating this biomass function into a stoichiometric metabolic model of the human lung cell and applying metabolic flux balance analysis, we identified host-based metabolic perturbations inhibiting SARS-CoV-2 reproduction. Our results highlight reactions in the central metabolism, as well as amino acid and nucleotide biosynthesis pathways. By incorporating host cellular maintenance into the model based on available protein expression data from human lung cells, we find that only few of these metabolic perturbations are able to selectively inhibit virus reproduction. Some of the catalysing enzymes of such reactions have demonstrated interactions with existing drugs, which can be used for experimental testing of the presented predictions using gene knockouts and RNA interference techniques. In summary, the developed computational approach offers a platform for rapid, experimentally testable generation of drug predictions against existing and emerging viruses based on their biomass requirements. One of the crucial steps in the virus life cycle is the synthesis of the virus particles within the host cell. This includes the synthesis of viral structural proteins and new genomic material. For these processes, all viruses are fully dependent on their host for the required energy  and builThe observation of virus synthesis dominating the metabolism and physiology of infected cells suggests that it might be possible to manipulate cell metabolism to control the viral infection . Indeed,These experimental findings show that viral biomass synthesis causes significant metabolic flux changes in host cells and that metabolic perturbations can directly alter virus reproduction. Thus, system-level metabolic models could be used to predict what types of metabolic alterations can cause what kinds of impact on virus reproduction. Although modelling of virus reproduction in host cells has mostly taken a kinetic approach, focusing on translation and transcription processes , it has Motivated by the qualitative predictive power of stoichiometric metabolic models and flux analysis, we apply it here to simulate the production of SARS-CoV-2 virus particles as part of the host metabolism and predict metabolic inhibitions against this virus. Given the emerging literature on SARS-CoV-2 infection primarily targeting lung and intestinal cells, we focus here on human lung cells as the host system. We adapt the available human genome-scale metabolic model with a biomass maintenance function based on human lung tissue–derived expression data, and include in this model also a viral biomass reaction, estimated using structural information available from SARS-CoV-2 and related viruses. By optimising flux distributions in this “infected lung cell model” for viral biomass reaction, we were able to predict reactions whose suppression or constraint can theoretically inhibit viral reproduction. We explored the possible impact of these predicted inhibitions on the host metabolism itself, as well as the experimental feasibility of implementing the predicted metabolic perturbations using available drug and inhibitor information on metabolic enzymes. Our results indicate that individual and double perturbation of several metabolic reactions from central metabolic pathways can inhibit SARS-CoV-2 reproduction in general and some of these can do so selectively without affecting normal metabolic functions of the host. We highlight these reactions as experimentally testable drug targets for inhibiting SARS-CoV-2 reproduction in human lung cells and provide details of the implemented computational approach for further development.To simulate the production of SARS-CoV-2 virus particles in a human cell, we used an existing, community-developed human genome scale model known as RECON2.2  see the. Within By incorporating the SARS-CoV-2 viral biomass function into the human metabolic model and assuming a minimal media composition , we predict a metabolic flux distribution for optimal virus production in a human cell . We thenResults of the flux variability analysis, where flux range for each reaction is calculated while maintaining an optimal biomass flux to SARS-CoV-2 production.Table S1 To identify reaction perturbations which, when inhibited, can halt or reduce virus production, we systematically simulated a knockout of each flux-carrying reaction. Excluding reactions involved in uptake from media, this analysis highlighted 35 reaction knockouts that can stop virus production and an additional eight reactions that can reduce it below 80% of the original . The forResults of the single and combined reaction perturbations (gene knockout simulations) on SARS-CoV-2 production, as performed on all reactions identified as flux-carrying in the flux variability analysis.Table S2 Considering that it is possible for the effects of single perturbations to be circumvented by re-directing of fluxes, we also explored combined perturbations. We created all possible pairs of flux-carrying reactions according to the flux variability analysis  and simulated the effect of setting their reaction fluxes to zero. This has identified more than 400 reaction pairs, co-inhibition of which results in the reduction of virus optima to 80% or less of the original  and S2. In the case of simulating the minimal media with higher uptake fluxes, we have also identified pairs of completely new enzyme inhibitions, which were not causing any effect on their own (Table S2). Some of these pairs exert their effects by blocking multiple pathways from a given compound and thereby causing disruption in steady state balances in the model. For example, co-inhibition of citrate synthase (CSm) and several other enzymes such as histidase (HISD) totally prevents flux in SARS-CoV-2 biosynthesis reaction by making impossible the mass balance of protons in the cytosol see .In the above discussed analyses, we considered host metabolism as optimised for viral production and evaluated impact of perturbations only on this process. Such metabolic perturbations should also be evaluated for their impact on the normal metabolic functions of uninfected host cells. In previous studies, normal state of metabolism in human cell lines are either represented through a pseudo reaction for cellular maintenance  or throuHere, we expanded from this approach to focus on human lung cells, which are shown to be the primary target of SARS-CoV-2 infections along with intestinal cells The composition and stoichiometries of building block metabolites in the virus biomass and human lung cell–specific maintenance pseudo reactions.Table S3 Results of the flux variability analysis, where flux range for each reaction is calculated while maintaining an optimal flux on the SARS-CoV-2 biomass or human lung cell–specific maintenance pseudo reactions.Table S4 Given the above finding that the same reactions carrying flux for SARS-CoV-2 production also carry flux for host metabolic maintenance, we re-analysed the effects of enzyme perturbations on both virus and the host. We found that many of the previously identified single perturbations limiting virus production also limited significantly the host metabolic maintenance, with only one single perturbation—that involving CYOR—showing more than 5% difference in its impact on virus versus the host (Table S5). The same finding prevailed for double perturbations. The only pairs that displayed 5% or more difference in their effects on virus versus host are those involving CYOR paired with other enzymes (Table S5).Results of the single and combined reaction perturbations (gene knockout simulations) on SARS-CoV-2 biomass and human lung cell–specific maintenance pseudo reactions.Table S5 The limited differential impact of full inhibition of enzymes made us postulate that more refined perturbations could provide a better strategy to just impact SARS-CoV-2 production without affecting the host. In particular, given the differences in optimal metabolic fluxes between virus production and host maintenance states, we argued that there might be flux values for some reaction that are compatible with only one of these states. To explore this possibility, we systematically analysed the flux variability of each reaction given either the optimisation of host maintenance or virus production. This has allowed us to see if any of the reactions would have flux regimes that are only compatible with the optimal host maintenance but not with optimal virus production and then “enforce” such flux regimes on the model. This approach allowed us to identify few single and double reaction perturbations that are fully selective on their effect and solely reduce virus production without causing any impact on the host (see Table S6). They involved a small number of metabolic reactions, namely, aspartate carbamoyltransferase (ASPCTr), carbamoyl-phosphate synthase (CBPS), dihydroorotic acid dehydrogenase (DHORD9), dihydroorotase (DHRTS), orotidine-5-phosphate decarboxylase (OMPDC), orotate phosphoribosyltransferase (ORPT), asparagine synthase (ASNS1), glutamine synthase (GLNS), and threonine deaminase (THRD) and caused up to 27% reduction in SARS-CoV-2 production (Table S6).Results of single and double flux enforcement perturbations on SARS-CoV-2 biomass and human lung cell–specific maintenance pseudo reactions.Table S6 The flux enforcement approach creates further constraints on how the metabolic fluxes in the system can be balanced at steady state. For example, in the optimal flux distribution for SARS-CoV-2 production in the unmodified model, threonine is obtained from the medium through a threonine:leucine shuttle, with both of these amino acids being in relatively similar demand between host and virus requirements. In the case of THRD fluxes enforced to specific ranges, threonine is instead obtained through a threonine:isoleucine shuttle and is now further intertwined with the arginine:isoleucine shuttle, thereby creating a trade-off among these amino acids . CrucialMotivated by this finding, we also simulated the effect of the decreased availability of specific metabolites in the culture medium on the host and virus biomass fluxes. The maximum import rate of the different molecules of the minimal medium we defined was set to values ranging between 0.1 and 10 mmol/gDW/h (where DW stands for grams of dry weight) and host and viral biomass optima compared (Table S7). As the maximum import rate of any molecule of the minimal medium decreases, so does the value of the optimal biomass flux of both host and virus. In some cases, namely, for threonine, glycylphenylalanine, and 3-methyl-2-oxovalerate, this decrease occurs more readily for the virus’ optimal biomass flux than for the host. This could be interpreted that a situation of low availability of the aforementioned molecules in the medium is more detrimental to the virus replication than to the host’s maintenance.The composition of the minimal media used in this study and results of simulations with limitations on different media component fluxes.Table S7 The metabolic approach used here allowed prediction of several reactions and reaction combinations that could limit SARS-CoV-2 production in human cells in general and differentially in human lung cells. The significant ones of these are re-summarised in Here, we have created a stoichiometric biomass function for the COVID-19–causing SARS-CoV-2 virus and incorporated this into a human lung cell genome scale metabolic model. The viral biomass function highlights the key building blocks required to synthesize a SARS-CoV-2 virus and its simulation within the human metabolic model enables predicting optimal flux distributions in the host for sustaining either SARS-CoV-2 reproduction or host maintenance. We used this capability to identify reaction perturbations that can inhibit SARS-CoV-2 reproduction in general or selectively, without inhibiting the host metabolic maintenance. The identified reactions primarily fall onto glycolysis and oxidative phosphorylation pathways and their connections to amino acid biosynthesis pathways. The latter finding is in line with the additional observation we made here that the relative stoichiometries of specific amino acids differ in SARS-CoV-2 biomass versus host cell maintenance estimated using human lung cell protein expression data. Together, these results highlight the possibility of targeting host metabolism for inhibition of SARS-CoV-2 reproduction in human cells in general and in human lung cells specifically.The predictions presented here are based crucially on the structure of the metabolic model as well as the two key assumptions of the flux balance analysis (FBA), namely, the assumptions of metabolic steady state and the optimality of metabolic fluxes towards a specific metabolic function. In the case of the key assumptions of FBA , these aThere is unfortunately not much available literature yet on SARS-CoV-2–infected cells and their metabolism, to allow us detailed comparison between model predictions and experiments. This said, we have identified a pre-print under review, which used a colon epithelial carcinoma cell line as a model system to study impact of SARS-CoV-2 infection on cell physiology . In brieIn terms of model result dependence on the model structure, we used the RECON2.2 model . This cuIn our view, this approach should be more informative than using existing cell-specific models that are not experimentally shown the be SARS-CoV-2 targets, such as the macrophage model, which has been used recently in a pre-print to study SARS-CoV-2 infection . BesidesThe virus life cycle consists of environmental circulation, infection, and subsequent host cell attachment and entry, reproduction within the host cell, and exit for a new round of infection. The presented approach focuses solely on the reproduction within the host cell and the metabolic aspects of that. Although this is a limited focus, reproduction in the host cell is a crucial and essential aspect of the virus life cycle. The importance of this stage is highlighted in several studies, which demonstrate that viruses tend to re-program host metabolism for increased viral production  or encodWe argue that differences in metabolic requirements of a virus versus its host could be a “physiological mismatch” that contributes to this evolutionary dynamic. Before metabolic adaptations happen, however, inhibition of the host metabolism might be a possible strategy to selectively inhibit reproduction of emergent viruses in new hosts. Specific host-based metabolic perturbations have already been shown experimentally to be effective against viruses , whereasIn the development of host-based metabolic strategies to inhibit viruses, metabolic modelling, as presented here, can play a useful role. In particular, our approach can be applied relatively rapidly to any host–virus pair both for existing and emerging viruses, and allow generating experimentally testable hypotheses for virus inhibition. This approach can be applied as long as structural and genomic information can be converted into an estimation of biomass composition for the virus and a suitably detailed metabolic model for the host can be constructed. The former process can be enhanced by further databases of viral structural and genomic information, whereas the latter process would benefit from extending human-focussed efforts such as the human metabolic atlas database  to coverTo identify specific host metabolic reactions that can alter viral production, we make use of a generic human cell genome-scale metabolic model that has been previously developed  and that2O in the scaling) (see the Data Availability section). The remaining elements of the maintenance function were retained as in the RECON2.2 model. The final, lung cell–specific maintenance function is provided in Table S3.As discussed in the main text, when identifying metabolic perturbations that can selectively affect viral production without much affecting the host, the specifics of the used host maintenance representation must be cell specific. To align this representation to a lung cell, we have made use of the gene expression data available from the Human Protein Atlas project . In partNC_045512). For one virus particle, the number of copies of genome (Cg) is assumed to be 1, whereas the number of copies of structural proteins is assumed to be 1,540 for membrane glycoproteins (M), 270 for surface glycoproteins (S), 490 for nucleocapsid phosphoproteins (N), and 5 for each of the other structural proteins of SARS-CoV-2. These protein copy numbers and stoichiometries are estimated from electron microscopy and mass spectrometry studies on other coronaviruses, including SARS-CoV  to represent a rich media . We have used here both this approach and additionally implemented a minimal media containing only essential metabolites, carbon and nitrogen source, and oxygen. The identification of the minimal media was achieved using a linear optimisation based algorithmic approach .The integrated genome-scale metabolic model was simulated using the FBA approach . FBA assapproach , where ahttps://www.ebi.ac.uk/biomodels/MODEL2010280002). All source code used in this work are available on our research group GitHub web pages at: https://github.com/OSS-Lab/FBAhv.All relevant data are presented in the main and supplementary texts. The SBML model of the SARS-CoV-2–infected lung cell is also available at BioModels  under id


MicroRNA (miRNA) regulation is associated with several diseases, including neurodegenerative diseases. Several approaches can be used for modeling miRNA regulation. However, their precision may be limited for analyzing multidimensional data. Here, we addressed this question by integrating shape analysis and feature selection into miRAMINT, a methodology that we used for analyzing multidimensional RNA-seq and proteomic data from a knock-in mouse model (Hdh mice) of Huntington’s disease (HD), a disease caused by CAG repeat expansion in huntingtin (htt). This dataset covers 6 CAG repeat alleles and 3 age points in the striatum and cortex of Hdh mice.Remarkably, compared to previous analyzes of this multidimensional dataset, the miRAMINT approach retained only 31 explanatory striatal miRNA-mRNA pairs that are precisely associated with the shape of CAG repeat dependence over time, among which 5 pairs with a strong change of target expression levels. Several of these pairs were previously associated with neuronal homeostasis or HD pathogenesis, or both. Such miRNA-mRNA pairs were not detected in cortex.These data suggest that miRNA regulation has a limited global role in HD while providing accurately-selected miRNA-target pairs to study how the brain may compute molecular responses to HD over time. These data also provide a methodological framework for researchers to explore how shape analysis can enhance multidimensional data analytics in biology and disease. Several neurodegenerative diseases (NDs) such as Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis and Huntington’s disease (HD) may evolve through gene deregulation, which has fostered a large number of studies aiming to explore the role of micro-RNA (miRNA) regulation in driving gene deregulation in these diseases –5. MiRNABesides predicting binding sites, another strategy for predicting miRNA targets is to search for negative correlations between miRNA and target expression levels. Such approaches include the use of Bayesian analysis such as GeneMiR++ . HoweverP-value computed for each predictor variable and each predictor variable stable across RF iterations involving different seeds) and shape analysis (surface matching) for building discriminative and accurate ensembles of negatively correlated miRNA-mRNA pairs. We used RF analysis for feature selection as this method does not make any prior hypothesis on the existence of a relationship, whether direct or indirect, between a miRNA and a target. To select the most interesting miRNAs, this analysis was supplemented with evidence for binding sites as instructed from multiple databases and followed by data prioritization using criteria such as CAG-repeat-length dependence and the fold change of target expression. We applied this approach to the analysis of multidimensional data in the allelic series HD knock-in mice (Hdh mice), currently the largest and more comprehensive datasets  to understand how miRNA regulation may work on a system level in neurodegenerative diseases [Although network inference methods such as Bayesian analysis and WGCNA may provide insights into the features of miRNA regulation, they may be prone to aggregation of a large number of hypotheses around strongly deregulated entities , 20, lacTo understand how the dynamics of miRNA regulation may work on a system level in the brain of Hdh mice, we applied miRNA regulation analysis via multimodal integration (miRAMINT), a pipeline in which novelty is to combine shape analysis with random forest analysis Fig. .Fig. 1Mhttp://www.broca.inserm.fr/MiRAMINT/index.php for edge lists). Amongst all possible associations (144) between miRNA modules and target modules, 12 negative correlations between eigen-vectors  were retained in the striatum and in the cortex  is computed for each predictor variable using the Altmann’s approach [We then tested whether the log fold change (LFC) for miRNA expression across the 15 CAG-repeat and age-dependent conditions tested in Hdh mice might explain target expression levels across these conditions. To this end, we applied RF analysis, which allows this question to be addressed in an unbiased manner (i.e. with no a priori hypothesis on the existence of miRNA-target relationships) and which has been successfully used to study miRNA regulation on a binding site level , 29. To approach  and in wapproach , MicroCoapproach  and miRDapproach  databaseGene and protein expression data from the same cells under similar conditions usually do not show a strong positive correlation –35. As sAlthough selective, data analysis in miRAMINT enables a diversity of profiles in terms of CAG-repeat dependence, age dependence and magnitude of effects across conditions to be retained. Several criteria may then be used for prioritizing the most interesting pairs, including (i) the overall shape of the gene deregulation plane  and the maximal amplitude of gene deregulation at any point in the CAG repeat- and age-dependent plane, (ii) the strength of plane matching (i.e. the Spearman’s score for surface-matching), (iii) the number of databases concluding to a binding site between miRNA(s) and predicted target(s) and (iv), if available, positive correlations between changes in the expression of proteins and of genes encoding these proteins.The analysis retained 31 miRNA-mRNA pairs in the striatum, among which 17 top pairs corresponding to either binding sites found in more than one miRNA target database or highest Spearman’s score for surface matching, or both Fig. a, includIn the cortex, miRAMINT retained 9 miRNA-target pairs that tend to show a biphasic  age-dependent profile, including 6 miRNAs and 3 targets annotated for inflammatory pathways (Tnfrs11a) such as NF-kappa B signaling, a pathway involved in neuronal apoptosis , and forp-value is computed for each predictor variable and in which RF analysis is iterated (involving different seeds) until the number of hypotheses is stable across consecutive iterations, (ii) shape analysis for matching the miRNA and mRNA expression profiles across conditions, (iii) evidence for binding sites and (iv) bona fide comparison of the gene targets retained into the model to protein expression profiles.As multi-point data become available for modeling miRNA regulation , comprehSince the coverage and dynamics of proteomic data in the allelic series of Hdh mice are limited compared to those of miRNA and mRNA data, we focused our study on modeling miRNA regulation mediated by mRNA degradation. Depending on the features of input data layers, miRAMINT analysis may be used to analyze gene expression repression mediated by mRNA degradation or inhibition of protein translation, or both.Combining shape analysis and feature selection for negatively correlating miRNA and mRNA data suggests that miRNA regulation via mRNA degradation may have a limited global role in the striatum and cortex of Hdh mice. This conclusion is supported by the small number of miRNA-target relationships that show a consistent pattern (i.e. strong and linear effects) of expression in the surface defined by CAG-repeat lengths and age points in the striatum of these mice. This conclusion is reminiscent of a similar trend detected in the brain of wild-type mice, where miRNA regulation may be poorly correlated to gene expression signatures across cell types . This copost-mortem brain samples of HD patients compared to control individuals [post-mortem brain samples relied on a simple overlap analysis (based on binding sites in TargetScan) between lists of differentially expressed miRNAs and mRNAs [A former bioinformatic analysis of miRNA expression identified 33 possible miRNA-target relationships in ividuals . We founnd mRNAs  and as mThe lack of miRNA-target pairs that may truly function in a CAG-repeat dependent manner in the cortex of Hdh mice is intriguing. Although some of the miRNAs retained in our analysis showed age- and CAG-repeat-dependent profiles, all nine miRNA-target pairs (involving 3 targets) show a bi-phasic response with deregulation at 6 months of age and return to initial (2-month) expression levels at 10 months of age. Since miRNA regulation may be highly dependent on cellular context, we speculate this observation could relate to the large heterogeneity of neuronal populations in cortex, which could preclude a sufficiently sensitive analysis of HD and age-dependent miRNA regulation in whole cortex extracts compared to whole striatum extracts. Alternatively, this observation could relate to a strong level of miRNA-regulation reprogramming and impairment in the HD cortex, as further discussed below.Although we cannot exclude the possibility that the conclusion about a limited global role of miRNA regulation in the brain of Hdh mice might be biased by the current lack of cell-type specific RNA-seq data in HD mice, our data highlight a new set of precisely matched and highly prioritized miRNA-target relationships see Fig.  that areSince miRAMINT finely accounts for the disease- and time-dependent features of miRNA and mRNA data in Hdh mice, miRAMINT miRNA-target pairs are strongly relevant to how cells and tissues may compute responses to HD on a miRNA regulation level. Amongst the 14 miRNAs retained by MiRAMINT analysis in the striatum see Fig. a, it is In summary, we addressed the problem of accurately modeling the dynamics of miRNA regulation from the analysis of multidimensional data. Our study puts forth the added value of combining shape analysis with feature selection for predictive accuracy and biological precision in modeling miRNA regulation from complex datasets, as illustrated by precise self-organised learning from multidimensional data obtained in the striatum and cortex of HD knock-in mice. MiRAMINT provides a convenient framework for researchers to explore how combining shape analysis with feature selection can enhance the analysis of multidimensional data in precisely modeling the interplay between layers of molecular regulation in biology and disease.RNA-seq (mRNA and miRNA) data were obtained from the striatum and cortex of Hdh knock-in mice  as previously reported [https://www.bioconductor.org/). Gene symbols that could be not mapped to a single Entrez ID were kept with the Entrez identifiers.To identify genes, we used Entrez identifiers. To this end, we converted gene symbols to Entrez identifiers by using the Bioconductor package , down-regulated  or unchanged  for each condition. The set age is k, and Q20 was used as reference for each condition at age k and Q > 20.mRNA and miRNA significant read-count data for eight individuals  as available in the RNA-seq data in the allelic series of Hdh mice was fed into Deseq2 implemented in the R package DESeq2  in orderTo build an accurate model of miRNA regulation from the analysis of highly dimensional data such as the one available for the brain of Hdh mice , we devehttps://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/). We applied standart settings as previously described [To reduce data complexity, we used WGCNA analysis. To this end, we used the R package WGCNA  is randomly permutated. Permutation data are then used to grow new random forests and compute the scores for the predictor variables. Permutation were repeated 100 times (default parameter), thus generating 100 scores of importance for each miRNA variable that can be regarded as realizations from the unknown null distribution. These 100 scores were used to compute a p-value for each predictor variable. If the classification error rate for a mRNA was higher than 10%, we rejected the possibility that this mRNA could be under miRNA regulation. When the error rate of classification was lower that 10%, we retained the miRNA(s) associated with mRNA(s) with a p-value < 0.1. Finally, to further ensure the reliability of feature selection, the entire RF analysis, each round recruiting different starting seeds, was repeated until the pool of hypotheses at the intersection of all ensembles of hypotheses generated by all RF iterations is stable. A pool of hypotheses was considered to be stable and RF iterations were stopped when greater than 80% of the hypotheses were conserved across 3 consecutive rounds of analysis. A stable pool of hypotheses was obtained for a range of 3–13 iterations  as possible explanatory variables of the target expression profile across conditions. Then, RF analysis implemented in the R package Ranger was performed by using the Altmann’s approach . This apThe LFCs of a miRNA and a mRNA across multiple conditions  defines a surface that provides a strong basis for associating a miRNA with its putative target(s). To refine feature selection (see above), we computed the slope of each edge between two conditions. We then computed the Spearman’s score between the slopes for each gene and those for explanatory miRNA(s). Finally, we retained the miRNA-target pairs for which the Spearman’s score is negative and such that the false discovery rate is lower than 0.05 using the Benjamini-Hochberg method .https://www.hdinhd.org/). These data cover 6 CAG-repeat lengths across 3 age points, similarly to RNA-seq data. Briefly, the label-free quantification (LFQ) of the proteins was obtained as previously described [p-value < 0.05 on the Spearman’s score was considered significant.Previous studies have shown that RNA-seq may validate proteomic data whereas few proteomic data may validate gene deregulation . Nonetheescribed . We usedAdditional file 1: Table S1. Lists of nodes in miRNA and mRNA WGCNA modules. Module membership is indicated for mRNAs and miRNAs. NA, not applicable.Additional file 2: Table S2. miRNA-target pairs retained by RF analysis. The p-values are the ones provided by the Altmann’s RF algorithm [lgorithm . This taAdditional file 3: Table S3. Surface-matched miRNA-target pairs for which there is evidence for a binding site. This table shows the miR-target pairs for which (i) the Spearman’s score for plane matching is positive and has a p-value < 0.01 upon Bonferroni correction for multiple testing and (ii) there is evidence for a binding site as supported at least by one database including MicroCosm, TargetScan and miRDB. The LFC amplitude of the target is indicated (see Fig.


The Coronavirus Disease 2019 (COVID-19) pandemic may have altered emergency workflows established to optimize the outcome of patients with large-vessel occlusion (LVO) stroke.We here analyzed workflow time intervals and functional outcomes of LVO patients treated with endovascular thrombectomy (ET) during the COVID-19 pandemic in Germany.N = 795).We compared the frequency, pre- and intrahospital workflow time intervals, rates of reperfusion, and functional outcome of patients admitted from March 1st to May 31st 2020 with patients admitted during the same time interval in 2019 to 12 university and municipal hospitals across Germany (p = 0.005). Rates of reperfusion were not significantly different between 2020 and 2019. Functional outcome at discharge of LVO patients treated in 2020 was not significantly different compared to patients treated in 2019.The number of LVO patients treated with ET between March to May 2020 was similar when compared to the same interval in 2019. Direct-to-center patients and patients admitted through interhospital transfer in 2020 showed similar pre- and intrahospital workflow time intervals compared to patients admitted in 2019, except for a longer door-to-groin time in patients admitted through interhospital transfer in 2020 (47 min vs 38 min, Pre- and intrahospital workflows, ET efficacy, and functional outcome of LVO patients treated with ET were not affected during the COVID-19 pandemic in our large cohort from centers across Germany. The outcome of patients with ischemic stroke and myocardial infarction depends on optimized pre- and intrahospital emergency workflows to minimize the time to reperfusion . The rapThe data that support our findings are available from the corresponding author upon reasonable request.Total numbers of patients treated with ET, patients with ischemic stroke, and patients with transient ischemic attacks were retrieved from hospital databases using the OPS code 8–836.80 and the ICD-10 codes I63 and G45, respectively. Detailed clinical data of this database were available from 795 patients treated with ET (87%) from the German Stroke Registry Endovascular Treatment (NCT03356392) ; Fig. In this study, based on data from almost 800 patients, we found no changes in workflow time intervals, ET efficacy, and functional outcomes in LVO patients treated with ET during the COVID-19 pandemic in Germany when compared to 2019.Our study needs to be put into perspective and compared to recent reports on stroke admission numbers and workflow time intervals from other countries and regions. While we found a slight reduction of patients admitted with ischemic stroke and transient ischemic attack but no change in patients treated with ET, reductions in stroke patient numbers during the COVID-19 pandemic were also found in China and Spain when all stroke admissions were analyzed , 7, and The strengths of our study include the large sample size and multi-center nature of our study, as well the report of functional outcome measures in addition to procedural times. Importantly, our data were collected from both university and municipal hospitals and from regions differently affected by the COVID-19 pandemic. Our study is limited in its observational character, and we thus cannot rule out residual confounding. Moreover, our data were obtained from stroke centers with highly standardized prehospital and intrahospital algorithms, and may thus not be generalizable to lower-volume or non-specialized hospitals.We conclude that pre- and intrahospital ET workflows, ET efficacy, and functional outcome of LVO patients were not affected during the COVID-19 pandemic in our large German cohort. Close monitoring of workflow intervals remains important to secure optimized care of hyperacute emergencies during the pandemic.


Multiple studies have demonstrated the importance of adequate catheter–tissue contact in the creation of effective lesions during radiofrequency catheter ablation. The development of contact force (CF)-sensing catheters has contributed significantly to improve clinical outcomes in atrial fibrillation. However, CF-sensing technology is not used in the ablation of paroxysmal supraventricular tachycardia (PSVT). The possible reason for this is that PSVT ablation with the conventional approach  is considered a relatively low-risk procedure with fairly high success rates (short and long term). The aim of this study is to determine whether CF sensing can further improve the outcomes of PSVT ablation.The COBRA-PATH study is a single-center, two-armed, randomized controlled trial. Patients without structural heart disease being referred for electrophysiology study, because of PSVT and potential treatment with radiofrequency (RF) catheter ablation, will be randomly assigned to either manual ablation with standard nonirrigated ablation catheters or manual ablation with an open-irrigated ablation catheter equipped with CF sensing . The primary study endpoint is the difference in the number of RF applications during the ablation of atrioventricular nodal re-entry tachycardia, and that of Wolff–Parkinson–White syndrome and atrioventricular re-entrant tachycardia. Secondary outcome parameters include acute and long-term procedural success rates, overall duration of RF applications, procedure/fluoroscopy durations and safety parameters.We expect to see a reduced number/duration of RF applications required to achieve effective lesion creation, and consequently a decrease in total procedure/fluoroscopy times. Although a significant improvement in procedural success rates (acute/long term) might not be feasible to demonstrate (given the relatively high success rate of the standard ablation method), the possible decrease in procedure duration and the consequential reduction of radiation exposure has important clinical implications for both operators and patients undergoing the procedure.NCT04078685. Retrospectively registered on 2 September 2019.ClinicalTrials,  Contact force (CF) is a relatively new technology that provides real-time feedback on catheter–tissue contact during radiofrequency (RF) ablation. There is compelling evidence that CF between the tip of the catheter and the target tissue within the cardiac chamber is a key factor for safe and effective lesion creation. Insufficient CF leads to ineffective lesion formation whereas excessive CF may result in procedural complications. Before the development of CF-sensing ablation catheters, operators could only rely on surrogate indicators of tissue contact, such as tactile feedback, intracardiac electrograms, baseline impedance, impedance change, electrode temperature and catheter location by fluoroscopy or electroanatomic mapping. These measures were less accurate and did not provide instant feedback on contact –3.Since the introduction of CF-sensing catheter ablation therapy, many studies have demonstrated the importance of this novel technology in the field of atrial fibrillation ablation, as presented in Table Despite compelling evidence in atrial fibrillation ablation, no randomized controlled clinical trial has yet assessed the feasibility of CF sensing in the ablation of  supraventricular tachycardia ((P)SVT). The relatively high success rate of SVT ablations using the conventional (non-CF-based) methodology might have contributed to this virtual ignorance of CF sensing for these procedures. The acute success rates of catheter ablation of atrioventricular nodal re-entry tachycardia (AVNRT) and of accessory pathway ablations  with the conventional (non-CF-based) approach has been reported to be 96–97% and 93% respectively, whereas the recurrence rate of AVNRT ablation is approximately 5% and that of WPW-AVRT ablation is around 8% –25. FurtThe aim of this randomized controlled trial (RCT) is to demonstrate the superiority of the CF-sensing-based approach compared to the conventional (non-CF-sensing) approach in the catheter ablation of PSVTs by assessing the improvement in the number of RF applications, and to prove the safety of the open-irrigated, CF-sensing ablation catheters.The COBRA-PATH study is a prospective, single-center, randomized controlled trial. Patients will be randomized to ablation using a standard (noncontact force-sensing) catheter or ablation using a contact force-sensing catheter Fig. . The folReferral for electrophysiology study because of pre-excitation on 12-lead surface ECG and/or documented (or suspected) symptomatic PSVT: palpitations associated with narrow-complex tachycardia (≥ 1 episode) documented by a 12-lead electrogram, Holter monitoring, trans-telephonic event recorder, telemetry strip or implanted device  within 12 months prior to referral to EPS; or frequent symptoms of palpitation  associated with clinical signs highly suggestive of PSVT—sudden onset and termination of rapid (and regular) palpitations usually accompanied with neck pulsation and/or dizziness, termination with Valsalva maneuver or carotid sinus massage, and no evidence for an underlying structural heart diseaseIdentification of AVNRT or WPW-AVRT  during standard EP studyVerbal consent to continue with ablation therapy after the diagnostic steps of the EPS identified the aforementioned arrhythmia mechanisms, and after the patient received sufficient information about the benefits and potential risks of the ablative treatment of the individual arrhythmia substrateInformed written consent to being included in the studyAll patients without structural heart disease being referred to participating centers for standard electrophysiological study either because of pre-excitation detected on 12-lead surface ECG or because of palpitations associated with documented narrow-complex tachycardia  will be eligible. In addition, in order to be eligible for participating in the trial, an electrophysiology study must identify AVNRT or WPW-AVRT (see clarification in the following) as the underlying arrhythmia mechanism –29. In oEvidence of structural heart disease and/or myocardial ischemiaPregnancy Age below 18 yearsA potential subject who meets any of the following criteria will be excluded from participating in this study:Patients with evidence of a manifest accessory pathway (i.e. pre-excitation on surface ECG and/or findings supporting anterograde conduction over an accessory pathway during standard EP study) who also present with documented narrow (or wide)-complex tachycardia, which proves to be orthodromic (or antidromic) AVRT by the EP study. (Note: antidromic AVRT is a quite rare entity.)Patients presenting with documented orthodromic AVRT using a concealed bypass tract (accessory pathway conducting only in the retrograde direction).Patients who possess a manifest accessory pathway  but have never experienced palpitations and have no prior documentation of tachycardia. In these patients, RF ablation of the accessory pathway will be offered for those individuals who possess a manifest accessory pathway with an anterograde ERP less than 240 ms, and/or for whom EP study identifies a reproducibly inducible AVRT. These patients can be enrolled in our study, even if no tachycardia had been experienced or documented in the past.In this study protocol, we use the term WPW-AVRT to describe the following disease entities:The study protocol was approved in September 2017 by the Medical Research Ethics Committee (2017-394) of Erasmus Medical Center in Rotterdam, the Netherlands. Patient inclusion began in June 2018 and is still ongoing. Written informed consent has and will be obtained from each participant.The primary outcome of this study is the number of radiofrequency ablations during the ablation procedures of AVNRT and WPW-AVRT. The secondary outcome parameters are overall duration of radiofrequency applications, acute procedural success/failure, fluoroscopy time, total procedural duration, (serious) adverse events and long-term procedural success. The following additional characteristics and parameters are collected and investigated during the study: baseline demographic characteristics of patients, previous antiarrhythmic drug treatment, history of cardiac arrhythmias -sustained VT) and prior ablation history.Consecutive patients on the waiting/referral list for PSVT ablations  will be screened for eligibility by dedicated study coordinators. When they meet the inclusion criteria, the patients will be enrolled in the study . Standard pacing maneuvers  are performed to confirm the diagnosis of AVNRT or WPW-AVRT. After confirming the diagnosis of AVNRT/WPW-AVRT, the operator informs the patients about the specific diagnosis and briefly describes the expected success rates and safety concerns regarding the ablation of the particular subtype of arrhythmia substrate. Next, in order to continue with ablation, verbal consent is obtained from the patient  is left to the discretion of the operator. Transseptal punctures are guided by intracardiac echocardiography (ICE).Depending on the results of the randomization, the subsequent steps of mapping/ablation are performed using either a conventional  manual RF ablation catheter  or the TactiCath Quartz open-irrigated contact force-sensing catheter (CF-sensing arm). Stable catheter–tissue contact (minimum of 10 s in duration) with a minimum CF exceeding 5–10 g  is targeted in the CF-sensing group. The TactiCath catheter is used in a virtual “nonirrigated mode”  to achieve a similar degree of temperature control as in the case of conventional nonirrigated catheters. In both groups, the power  and the duration  settings of individual RF applications will follow local laboratory standards and will be left at the operator’s discretion. The ablation procedures are continued until the clinical tachycardia is no longer inducible and/or the conduction through an accessory pathway is terminated, provided that these clinical endpoints can be achieved with acceptable clinical risk–benefit ratios . Thereafter, the inducibility of the clinical tachycardia and/or conduction over an accessory pathway is reassessed throughout a waiting time of 30 min from the last RF application. No additional applications are implemented until further programmed stimulation shows evidence for recurrence of inducibility and/or re-conduction over an accessory pathway.The ablation catheter is not introduced into the patient due to prior occurrence of a serious AE (note: not during the ablation process itself), to the withdrawal of consent or to any type of technical difficulty with the setup.The ablation catheter is not introduced into the patient or ablation is discontinued before the achievement of acute success due to a clinical decision to utilize a different type of catheter that is not compatible with the study design . Such clinical scenarios include, for example, parahisian accessory pathways (switching to cryoablation to prevent AV node injury), ablation of epicardial accessory pathways from a coronary sinus branch (magnetic navigation guided catheter for better access) or any other situation where the specific anatomy of the underlying arrhythmia-substrate involves an increased potential risk thereby necessitating the utilization of special approaches.Patients who were originally enrolled into the study (registered in the database and randomized to one of the two treatment arms) are excluded from further follow-up in the case of the following scenarios:. non-CF sensing during catheter ablation.However, those individuals for whom ablation was implemented with a “study-compatible” ablation catheter yet the applications were prematurely discontinued due to the occurrence of (serious) AEs will indeed be taken into consideration for further analyses (i.e. these patients will not be excluded from further follow-up) in order to be able to assess the safety aspects of CF sensing vsPatients are discharged home within 2 days after the index procedure. Antiarrhythmic medication is discontinued after the ablation procedure. Follow-up of study patients is implemented as outlined in Table Adverse events are registered and reported according to regulations of the medical ethics committee. The occurrence of the following complications are considered a major (serious) adverse event: death, acute myocardial infarction/coronary artery damage, major bleeding, abdominal bleeding, cardiac tamponade or pericardial effusion requiring intervention , ischemic cerebral event and other procedure-related embolic events, high-degree AV-block requiring PM implantation, atrial esophageal fistula, phrenic nerve palsy and vascular access complications . Minor adverse events are as follows: mild pericardial effusion, minor bleeding, and minor vascular access complications that do not require interventions and do not prolong the duration of hospitalization; in addition, nonsustained high-degree AV-block or sustained first-degree AV-block not requiring pacemaker implantation.Since the conduct of this trial is considered low risk for participating subjects , the ethical committee has agreed to omit the establishment of a safety committee.t test or the Mann–Whitney test, again depending on the underlying distribution of the data.The number of radiofrequency applications during the ablation procedures of AVNRT and WPW-AVRT (primary study endpoints), as well as other continuous procedural parameters from the group of secondary study endpoints , will be expressed as mean ± SD or median (interquartile range) depending on their distributions, and statistical analysis of these variables will be conducted using either the two-sample p value of 0.05 will be considered significant for all statistical determinations, and all reported p-values will be based on two-sided tests.The recurrence rate of PSVT at 1 year (as an indicator of long-term success rate of the index procedure), as well as the acute success rate and the major/minor complication rates of the index procedures (secondary study endpoints), will be presented as absolute and relative frequencies. The associations between treatment and these secondary endpoints will be analyzed with the chi-squared test or with Fischer’s exact test, as appropriate. Moreover, logistic regression will be performed to examine these associations and will, if needed, be adjusted for unbalanced baseline characteristics. Freedom from PSVT recurrence will be determined with Kaplan–Meier analysis, and differences in recurrence-free survival rates will be evaluated with the log-rank test. A A retrospective analysis of AVNRT/WPW-AVRT ablations performed in our institute within the last 6 months revealed that the mean number of radiofrequency applications required for successful ablations was 4.81 ± 3.21, which is in line with previous results found in the literature .According to previous studies, the ablation efficiency for patients with atrial fibrillation has been increased with the use of CF-sensing technology by approximately 15–20% –19, as rThe sample size calculations were based on the following:Evaluation of the distribution of RF applications in our database of AVNRT/WPW-AVRT ablations (mentioned under the first point) revealed a positive skewness, and therefore logarithm transformation  was performed in order to approximate a normal distribution of the variables. The mean and standard deviation of the natural logs of application numbers were computed and amounted to 1.39 ± 0.59; these values were subsequently used in our sample size calculations.http://powerandsamplesize.com/Calculators/Compare-2-Means/2-Sample-Equality) and G*power (http://www.gpower.hhu.de/en.html). In both methods, we applied the appropriate test comparing the difference of two independent means of two groups . The mean application number of the group with the study catheter was assumed to be reduced by 25%  compared to the control group (thus amounting to 1.39 × 0.75 = 1.043) and the standard deviation of this group was assumed to be identical to the control group. The power was set to 80% and the type 1 error (α) to 5% (two-tailed). Using these parameters, both methods/calculators determined the same sample size of patients (2 × 46 = 92), which would be required to show a statistical significance in the case of a clinically significant reduction  of RF application number.For the sample size calculations, we used two different calculator tools: an online sample-size calculator tool SVTs. Although the TOCCATA study enrolled patients with right-sided SVTs as well, it was conducted in a nonrandomized, uncontrolled fashion, comparing the results of CF-sensing-based catheter ablations (performed in this study with the TactiCath catheter) with the results of conventional (non-CF-sensing-based) ablation techniques reported in the literature (for right-sided SVT) prior to the study. In addition, the focus of this trial was the safety profile of the CF-sensing catheter, and parameters such as the recurrence rate, total procedure/ablation time and fluoroscopy time have not been evaluated for right-sided SVTs , 6.Since catheters equipped with CF-sensing technology already have an established role in atrial fibrillation ablation, and are also demonstrated to represent a safe methodology, we raise no potential concerns regarding the usage of the same types of catheters in the population of patients with PSVT. On the contrary, we strongly believe that the aforementioned results of the CF-sensing technology in AF ablations are also highly suggestive for the potential of this technology to be beneficial for PSVT patients. Since clear evidence  on the superiority of this technology in PSVT patients is currently lacking, we believe that our study can give an answer to the question of whether CF sensing is beneficial to use in ablation procedures targeting less complex substrates, like the slow-pathway of the AV node and the accessory pathways in different anatomic locations.Commercially available CF-sensing catheters utilize irrigated-tip technology as a standard feature . However, the standard catheter type used for PSVT ablations is a nonirrigated catheter. In order to create a comparable ablation approach, we intend to use the CF-sensing catheter in a “virtual nonirrigated modus” (irrigation rate set at 2 ml/min), thereby achieving similar temperature-controlled ablation characteristics to those with the nonirrigated catheters. The small amount of residual irrigation could, however, lead to potential bias and inconsistencies as far as the homogeneity of the two ablation approaches are concerned, thereby adding another possible non-negligible factor to the equation, which can have a potential effect on outcome and efficacy. However, based on our experience , the efficacy and safety profiles of an irrigated catheter utilized in a “nonirrigated modus” are comparable to the characteristics of nonirrigated ablation.Recruitment of participants started on 25 August 2017 and is expected to be completed on 30 June 2020. This manuscript is based on protocol version 02 of 25 August 2017.Additional file 1. SPIRIT 2013 Checklist.Additional file 2. SPIRIT figure.


The increasing understanding of breast cancer biology has provided the basis for the development of multigene signatures aimed to improve the capability of clinicians to assess patients’ prognostication and risk stratification. Incorporating these tools in clinical practice has profoundly impacted on the decision-making process for the adjuvant therapy of patients with ER+/HER2- early breast cancer and the results from prospective adjuvant trials have strengthened the clinical utility of multigene signatures in this setting. In July 2019, Lombardy was the first Region in Italy to reimburse genomic testing for patients with ER+/HER2- early breast cancer. Three years later, a group of investigators from six referral Cancer Centers in Lombardy convened to debate the use of multigene signatures in clinical practice and share their own experience with the tests after reimbursement. Here, we reviewed relevant data on the role of multigene signatures in tailoring adjuvant chemotherapy for patients with ER+/HER2- early breast cancer and discussed about the optimal use of these assays in current clinical practice. As the treatment landscape of early breast cancer evolves and novel questions about the possible additional applications of multigene assays arise, we also provide our viewpoint on the potential implementation of the assays in the evolving scenario ER+/HER2- early breast cancer treatment. The decision-making process on adjuvant treatment for patients with estrogen receptor positive and human epidermal growth factor receptor 2-negative (ER+/HER2-) early breast cancer may prove challenging. Clinicians should estimate the individual risk of recurrence and weigh the predicted benefit of adjuvant therapies against their short- and long-term toxicities in order to avoid both over- and undertreatment. The careful assessment of the classical clinicopathologic variables is essential to estimate the risk of disease recurrence but is little informative for prediction of chemotherapy benefit for individual patients .The increasing understanding of breast cancer biology has led to the development of multigene signatures that provide prognostic information independent of that provided by standard clinicopathologic features and may help clinicians to better identify those patients with low-risk disease who can be safely spared the toxic effects of chemotherapy . All theBased on this striking evidence, in July 2019 Lombardy was the first Region in Italy to reimburse genomic testing for patients with ER+/HER2- early breast cancer . The indSeveral multigene assays have been developed over the past two decades to guide adjuvant therapy decisions. Among them, Oncotype DX, MammaPrint, EndoPredict and Prosigna are the tests currently available in Italy Table 1.Oncotype DX  was among the earliest clinically validated genomic assay for use in patients with early breast cancer. This assay measures the expression of 16 tumor-associated genes and 5 control genes to compute a Recurrence Score (RS) from 0 to 100, resulting in assignment of patients into low-risk RS <18), intermediate-risk (RS 18 to 30) or high-risk (RS ≥31) groups Table 1 , intermeMammaPrint (Agendia) is a microarray-based 70-gene assay performed by a central laboratory in Amsterdam. This assay stratifies patients into low-risk or high-risk prognostic groups and was first validated in 295 breast cancer patients both ER-positive and ER-negative Table 1 EndoPredict (Myriad Genetics) is an assay marketed in Europe as a diagnostic kit that can be performed by local laboratories. The test measures the expression of 8 cancer-related and 3 reference genes using RT-PCR, in order to calculate a risk score and stratify patients into a low- or high-risk of recurrence group, if treated with adjuvant endocrine therapy alone Table 1. (PAM50 (Prosigna) is a diagnostic kit that uses Nanostring technology to quantify mRNA expression of 50 genes used in the molecular subtypes’ classification of breast cancer. When integrated with a proliferation score and tumor size, the test leads to the risk-of-recurrence (ROR) score, that reflects the 10-year risk of distant recurrence  . The proThe ongoing OPTIMA trial (ISRCTN42400492), which randomized patients with ER+/HER2- early breast cancer (pN1-2 or pN1mi with pT ≥20mm or pN0 with pT ≥30mm) to receive chemo-endocrine therapy or to undergo Prosigna testing and omit chemotherapy in case of low- or intermediate-Prosigna Score (≤ 60), hopefully will provide information about the clinical utility of this assay as a predictive biomarker.We agreed that all signatures are valid prognostic tools useful to identify low-risk patients with good outcome with adjuvant endocrine therapy alone. However, according to the abovementioned data, only Oncotype DX has been shown to provide convincing predictive information for chemotherapy benefit in prospective trials and is the only one recommended for both post- and premenopausal patients with a level of evidence and a grade of recommendation of I, A .For these reasons, Oncotype DX resulted the preferred test chosen by the Panelists in almost all cases (1098 of 1132 patients who received an indication for genomic testing in the last two years).Modern breast cancer management has become increasingly complex and requires dedicated breast cancer specialists working together to deliver high-quality care throughout the patient journey. The multidisciplinary team (MDT) -defined by the UK Department of Health as a ‘‘group of people of different health care disciplines, which meets together at a given time to discuss a given patient and who are each able to contribute independently to the diagnostic and treatment decisions about the patient’’ - is fundHowever, this process sometimes might lead to different indications for genomic testing by different MDTs on one side, and to a delay in the initiation of the adjuvant systemic therapy beyond the 3-6 weeks recommended by Guidelines  one the In this view, we advocated alternative paths for genomic test prescription in an effort to standardize practice and reduce delays to adjuvant chemotherapy initiation. We endorsed the “Oncotype DX reflex testing”, that is the automated preparation and shipping of tumor sample by the pathologist at the time of the histological examination  in cases that meet pre-defined criteria. The implementation of Oncotype DX reflex testing in clinical practice has been shown to improve the timeliness to chemotherapy initiation , with poWe endorse the reflex testing for postmenopausal patients < 70 years with ER+/HER2- breast cancers, both node-negative and 1-3 node positive and the following tumor characteristics: T1c-2, Grade 2-3 and at least one of Ki67 21-40% or PgR < 20%. We acknowledge that advanced age, by itself, should not be a criterion to preclude a patient from receiving the most appropriate anticancer treatment and that ‘functional’ rather than chronological age should be considered in the decision making about adjuvant therapy. However, older patients require a more careful evaluation of the risk-benefit ratio of any medical intervention, including a geriatric assessment and a careful consideration of life expectancy and competing risks of mortality that should be performed by the various members of the MDT .> 30% has a low RS and therefore a recommendation of adjuvant chemotherapy for these patients based solely on high Ki67 values would have been questionable. Recent analyses from the TransATAC study showed that Oncotype DX is substantially an estrogen-driven signature, since the correlation between RS and its proliferation module is much weaker than the strong negative correlation between RS and its estrogen module  or ‘luminal B-like’  . Howevern module . Therefon module , 37. Indn module . Accordin module  and a lan module . We acknAge at diagnosis is an important prognostic factor in ER+/HER2- early breast cancer  and studConcerning Oncotype DX, both TAILORx and RxPONDER have found significant interaction for outcome between chemotherapy effect and age or menopausal status , 21. In Although Guidelines recommend that genomic testing in premenopausal women with 1-3 positive lymph-nodes should not be offered to guide decisions for adjuvant therapy  and someWe reviewed relevant data about the role of multigene assays in tailoring the decision-making for adjuvant chemotherapy for patients with ER+/HER2- early breast cancer and provided our viewpoint on specific topics regarding the optimal use of these assays in clinical practice. Nevertheless, the treatment landscape of ER+/HER2- early breast cancer is evolving and novel questions about the potential additional applications of multigene assays are emerging.Neoadjuvant therapy is increasingly used in the management of early breast cancer and the feasibility of performing gene expression profiling using tumor tissue from core biopsies has been largely demonstrated –48. SeveProbably yes. As noted above, while other signatures are dominated by proliferative features, Oncotype DX RS has a strong inverse correlation with its estrogen module  and this> 20%) (One attempt of treatment escalation in ER+/HER2- early breast cancer has been to combine adjuvant endocrine therapy with CDK4/6 inhibitors. The role of these agents in the early setting has been investigated in 4 large clinical trials –58. WhilThe increasing understanding of breast cancer biology has provided the basis for the development of multigene assays aimed to improve our capability of patients’ prognostication and risk stratification. Incorporating these tools in clinical practice has profoundly changed the decision-making for adjuvant chemotherapy for patients with ER+/HER2- early breast cancer and the results from large prospective adjuvant trials have strengthened the clinical utility of multigene assays in this setting. As the treatment landscape of early breast cancer broadens, novel questions arise and new areas of research emerge. In this evolving scenario, the contribution of multigene assays to personalized medicine may become even more important.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Pulmonary arterial hypertension (PAH) is a rare heterogeneous disorder in the paediatric population which is mostly associated with congenital heart disease. The management of paediatric idiopathic PAH (IPAH) is difficult due to insufficient comparative data and depends on the results of evidence-based adult studies with several pulmonary vasodilators, as well as the clinical experiences of paediatric experts. Our aim was to present the case of a 9-year-old girl who underwent several methods of treatment, including pharmacotherapy with a significant reaction to treprostinil, as well as bilateral lung transplantation. The patient’s treatment was distinguished by the fact that the dose escalation was as rapid as that observed in the adult population. Due to the limited current evidence and knowledge, the initiation of treatment for these patients remains an individual choice. On the grounds of the number of non-specific symptoms, the diagnosis of this patient was a long process and based mainly on the differential diagnosis. The purpose of this paper is to study this example in order to highlight the importance of early symptoms and the quick implementation of intensive treatment. The applied methods may be useful in doubtful diagnosis processes and treatment procedures in the paediatric population. A 9-year-old girl  was admitted to the Cardiology Department with recurring heart palpitations and exertional dyspnoea. Upon admission, she was categorised in the II World Health Organization functional class (WHO-FC) and reached 413 m on the 6 min walk test (6MWT). Her N-terminal pro-B-type natriuretic peptide (NT-proBNP) serum concentration was increased to 1303 pg/mL (reference range: <391 pg/mL). An electrocardiogram (ECG) indicated sinus rhythm with a frequency of 110 beats per minute (bpm). Moreover, signs of right ventricle hypertrophy and overload were present in ECG.2, assessed by means of a thermodilution method. The acute vasoreactivity test with nitric oxide (dosage 20 ppm) was negative. The differential diagnosis to search for clear causes of pulmonary hypertension was performed according to the current guidelines [Echocardiography  showed didelines . The patFour months after treatment initiation, the patient’s condition started to deteriorate. Moreover, the patient demonstrated central cyanosis associated with low arterial saturation (78%). A physical examination revealed bilateral, subscapular crackles and dyspnoea on minimal exertion (WHO-FC III/IV). Echocardiography showed the progression of the patient’s RV dysfunction with a further diameter enlargement to 3.3 cm (z-score + 2.36), dilatation of the PA to 3.1 cm (z-score + 4.3), and an increase in the assessed RVSP to 92 mmHg A. The quAfter the introduction of triple therapy, the patient underwent successful BLTx from a cytomegalovirus-positive donor in a referral transplant centre in Vienna. A cytological analysis of the postoperative material after bilateral lung transplantation finally excluded PVOD. Cardiac remodelling occurs early after BLTx as a result of a decreased RV afterload and elevated left ventricular volume preload due to the patient’s healthy lung vasculature and parenchyma of the graft . The twoThe scans were carried with a GE Vivid E90 ultrasound machine before transplantation and with a Phillips Epiq CVx ultrasound machine at the two-year follow-up after transplantation, in accordance with the international recommendations .


Objectives: As a stressor in the context of COVID-19 pandemic fatigue is associated with well-being. However, how pandemic fatigue is associated with well-being and what protective factors buffer this negative effect are under investigated. Based on the stress process model and emotion regulation theory, the study examined the indirect effect of pandemic fatigue on subjective well-being through emotional distress and the buffering effect of self-compassion.Methods: Data were collected from 1,162 university students  through an online survey. Indirect effect analysis and conditional process analysis were conducted by the SPSS macro PROCESS.Results: Indirect effect of pandemic fatigue on subjective well-being through emotional distress was identified and self-compassion moderated the association between pandemic fatigue and emotional distress. The indirect effect of pandemic fatigue was weaker among participants with high levels of self-compassion than among those with low levels of self-compassion.Conclusion: Pandemic fatigue was negatively associated with subjective well-being through emotional distress at all levels of self-compassion. The findings deepen our understanding of the link between pandemic fatigue and well-being while considering the indirect role of emotional distress and protective function of self-compassion. Since the end of 2019, the Corona virus disease (COVID-19) pandemic has persisted worldwide and largely affected people’s lives globally. Because of the long-term impacts of the COVID-19 pandemic, the World Health Organization (WHO) . NegativHowever, the underlying mechanism between pandemic fatigue and well-being, and whether this negative impact could be buffered by a protective factor, have not been investigated. To address this gap, we propose that emotional distress could work a pathway linking pandemic fatigue and subjective well-being, and self-compassion could function as a protective factor in alleviating the adverse indirect effect of pandemic fatigue on the basis of the stress process model and emotion regulation theory.The stress process model  emphasizEmotional distress has been found to be one adverse consequence of pandemic fatigue in the populations of teachers, university students, and a community sample –16. IndiEmotion regulation theory highlights the role of personal resources and the approach of emotion regulation strategy in daily life. The importance of self-compassion is emphasized because of its nature as an effective emotional self-regulating strategy and its protective function in buffering the adverse effect of stress . Self-coSelf-compassion is helpful in lessening the negative impacts of external stress  on emotional distress among different populations  , 25, 26.Supported by the stress process model and emotion regulation theory, the present study examined the indirect effect of pandemic fatigue on subjective well-being through emotional distress and the moderating role self-compassion in attenuating the association between pandemic fatigue and emotional distress.Data were from a project on the effect of COVID-19 pandemic on psychological adjustment  among Chinese university students. University students  aged 18 years or above from Beijing Normal University were recruited to participate the study. Data were collected in April 2022. Participants were informed of the research purpose, confidentiality, and their rights to voluntary participation. Valid participants received a small monetary reward . The project was approved by the research ethics committee of the School of Social Development and Public Policy at Beijing Normal University before data collection.n = 37) and those that failed the attention check (n = 118), the final sample size was 1,162 . The average age of the participants was 21.61 (SD = 2.81). The percentages of undergraduates and postgraduates were 65.2% and 34.8% respectively. The educational attainment of 22% of the participants was high school or below. The household monthly income was 19,811.49 RMB .A total of 1,317 qualified participants completed the online survey. After removing cases with duplicated responses (strongly disagree) to 7 (strongly agree). Sample items are “I am tired of all the COVID-19 discussions in TV shows, newspapers, and radio programs, etc.” and “I am tired of restraining myself to save those who are most vulnerable to COVID-19.” A total mean score was calculated by dividing the total score by the number of items. A high mean value represented a higher level of pandemic fatigue. The Cronbach’s α of the scale in the present study was 0.91.Pandemic fatigue is the independent variable in the present study. It was measured by the pandemic fatigue scale developed by Lilleholt . This scdid not apply to me at all) to 4 (applied to me very much). Sample items are “I felt that life was meaningless.” and “I was unable to become enthusiastic about anything.” A total mean score was calculated by dividing the total score by the number of items. A high mean value represented a higher level of emotional distress. The Cronbach’s α of this scale in the present study was 0.89.Emotional distress is the mediator in the hypothesized model. The Depression subscale of the Depression-Anxiety-Stress Scale developed by Lovibond  was usedalmost never) to 5 . Sample items are “When I’m going through a very hard time, I give myself the caring and tenderness I need.” and “When something upsets me I try to keep my emotions in balance.” A total mean score was calculated by dividing the total score by the number of items. A high mean value represented a higher level of self-compassion. The Cronbach’s α of SCS-SF in the present study was 0.80.Self-compassion is the moderator in the hypothesized model. Self-compassion was assessed by the Self-Compassion Scale-Short Form constructed by Raes (SCS-SF) . All 12 strongly disagree) to 7 (strongly agree). Sample items are “I am satisfied with my life.” and “The conditions of my life are excellent.” A total mean score was calculated by dividing the total score by the number of items. A high mean value indicated a higher level of subjective well-being. Cronbach’s α of the SWLS in the present study was 0.87.Subjective well-being is the dependent variable in the present study. Subjective well-being was assessed by the Satisfaction With Life Scale (SWLS) developed by Diener . This scDescriptive statistics  were conducted by SPSS (version 24). Indirect effect and conditional indirect effect (moderating effect of self-compassion) were found by conducting indirect effect analysis and conditional process analysis . Pandemic fatigue was positively associated with emotional distress  and emotional distress was negatively associated with subjective well-being . The direct effect of pandemic fatigue on subjective well-being was not significant .Results of indirect effect analysis showed that pandemic fatigue was indirectly associated with subjective well-being through emotional distress . This result suggests that self-compassion moderated the association between pandemic fatigue and emotional distress. N = 158) and low  levels of self-compassion. The positive association between pandemic fatigue and emotional distress and the negative indirect effect of pandemic fatigue on subjective well-being through emotional distress were identified at all self-compassion levels. The association between pandemic fatigue and emotional distress was positively significant . With the increase of self-compassion, the predictive effect of pandemic fatigue on emotional distress decreased . The indirect effect of pandemic fatigue became weaker as the level of self-compassion increased.Simple slope analysis was employed to investigate the relationship between pandemic fatigue and emotional distress at high , which occupies a lot of attention, energy, and psychological resources. Individuals who spend a lot of energy and resources to cope with COVID-19 related stress are more likely to be fatigued. Exhaustion caused by exposure to COVID-19 pandemic information from social media and behavioral restrictions usually leaves individuals tired , which mOne noteworthy finding is that self-compassion buffered the indirect effect of pandemic fatigue on subjective well-being through emotional distress. Specifically, self-compassion alleviated the association between pandemic fatigue and emotional distress. This association was weaker among the individuals with high levels of self-compassion than among those people with low levels of self-compassion. This finding is in line with previous findings that self-compassion functions as an effective emotion regulation strategy in attenuating the adverse effect of different stressors on emotional distress , 27, 34.The present study has insightful implications. To our knowledge, the present study is the first to investigate the emotional pathway between pandemic fatigue and subjective well-being in association with the buffering effect of a protective factor. Exposure to COVID-19 pandemic information from social media and behavioral restrictions make individuals physically and mentally exhausted. These people are more vulnerable to the emotional problems and distress, which further damages their satisfaction with life. This finding extends the implications of the stress process model in the context of COVID-19. Pandemic fatigue was the initial stressor that was associated with subjective well-being through the secondary stressor of emotional distress. In addition, our findings provide new understanding and supplement the roles of protective factors  in weakening the link between stress and outcomes –40 durinThe present study has several limitations. First, the cross-sectional design of the current study limits the investigation of the causal relationships among pandemic fatigue, emotional distress, and subjective well-being. The association between pandemic fatigue and subjective well-being, the mediating effect of emotional distress, and the buffering role of self-compassion should be examined in the future research with a longitudinal design. Second, the self-reported and retrospective nature of the data may lead to response and recall biases. Other approaches of data collection such as assessment of physical fatigue   could beIn the present study, pandemic fatigue was found to be associated with subjective well-being through emotional distress among university students. In addition, self-compassion alleviated the negative effect of pandemic fatigue on well-being by weakening the association between pandemic fatigue and emotional distress. Future studies and interventions aimed at reducing the adverse effect of COVID-19 pandemic could focus on the emotional pathway in the link between pandemic fatigue and outcomes, and the protective function of emotion regulation strategy.This manuscript is not under review elsewhere and the results have not been published previously or accepted for publication. This manuscript has been seen and approved by all authors.


Respiratory syncytial virus (RSV) is a leading cause of childhood mortality in infants below 6 months of age. In low-income and middle-income countries (LMICs), the public health burden is substantial and resources are limited. It is critical to inform decision makers about effectiveness of new interventions.We developed a mathematical model where individual RSV subtype A (RSV-A) and B (RSV-B) maternally derived neutralizing titers were predicted at time of birth after maternal vaccination with the RSV prefusion F protein-based vaccine. We estimated the subsequent duration of vaccine-induced immunity and compared this to the age at time of death distribution in the RSV GOLD Mortality Database to predict the potential impact of maternal vaccination on RSV-related childhood mortality. We used country-specific timing of antenatal care visits distributions and mortality estimates to make country-specific predictions for number of cases averted.The model predicts that on average a neonate born at 40 weeks gestational age will be protected between 6 and 7 months from RSV-A and approximately 5 months from RSV-B related mortality. We estimated the potential impact of RSV-related mortality for in-hospital and out-of-hospital cases in LMICs and predicted that in 51 GAVI-eligible countries maternal vaccination could avert between 55% and 63% of the RSV-related in-hospital mortality cases below 6 months of age.We show that maternal vaccination could substantially decrease RSV-A and RSV-B related in-hospital and out-of-hospital mortality in LMICs in the first 6 months of life. Respiratory syncytial virus (RSV) is a leading cause of childhood morbidity and mortality in infants below 5 years of age Multiple RSV interventions are currently in development In LMICs, the public health burden is substantial and resources are limited. Thus, it is critical that governments and large funding organizations are informed about effectiveness of new interventions so that interventions that have the highest impact and best value for money can be prioritized The maternal vaccine known as RSVpreF, marketed under the brand name Abrysvo, has recently received approval from the European Medicines Agency (EMA) and could soon be available Gavi considered some of the RSV interventions under development, including both maternal vaccination and monoclonal antibodies in its 2018 vaccine investment strategy for the 2021–2025 funding period. Health impact defined by the number of cases and deaths averted was used as one of the main criteria for prioritizing interventions The present study predicts country-specific impact of maternal vaccination in LMICs on both in-hospital and out-of-hospital mortality. We show that our mathematical model provides a useful tool to make subgroup-specific predictions for future vaccine impact and to explore the impact of timing of vaccination and gestational age at time of birth on vaccine efficacy.As described previously We hence assumed that maternal RSV-A and RSV-B neutralizing antibody titers increase exponentially from 7 days after vaccination, until they reach a peak at 14 days. After 14 days, maternal antibody levels decline exponentially. The maternal antibody titers are described in Equation The foetus-to-mother antibody transfer function was modelled as described previously Using the individual antibody titers of mother-fetus pairs during pregnancy, we predicted the number of days protected (which we refer to as duration of vaccine-induced immunity) from RSV-related death  as the fraction of the number of deaths (N) in the dataset that could have been averted, had the mothers been vaccinated. The simplest approach would be to compare the observed time of death (Tdeath) with the estimated duration of vaccine-induced immunity  visits could be an easy access to administer vaccines that require strict gestational age windows of administration To estimate the probability mass distribution for time of vaccination we summed the proportion of ANC attenders for visit 1, 2 and 3 together per week. The weekly total was divided by the summed total of the weekly proportions to create a probability mass distribution for time of vaccination  . The preFor preterms , the duration of vaccine-induced immunity depends strongly on gestational age at time of vaccination and gestational age at time of birth . The larWe applied our model in a situation without vaccination to estimate the duration of natural immunity. The model predicts no protection from RSV-A and RSV-B related mortality for preterm neonates born 35 weeks’ gestational age or younger . For fulWe applied our model to a subset of the RSV GOLD database By considering the distribution of gestational age at the time of birth from the mortality cases in the RSV GOLD database, we made predictions on the proportion of infants who possess vaccine-induced immunity (i.e. their antibody concentration being above the protection threshold) at different ages b. The frTaking both the age at time of death and gestational age at time of birth distributions into account, we can predict vaccine impact in children younger than 6 months in LMICs. For RSV-A, the predicted vaccine-induced duration of protection exceeds 6 months for almost all combinations of gestational age at time of birth and gestational age at time of vaccination , hence pThe optimal timing of vaccination depends on the relation between gestational age and antibody transfer over the placenta, the gestational age at time of birth distribution in the population of interest, and the half-life of vaccine induced antibodies in the mother.For RSV-A, the model predicts that vaccination at 25 weeks’  and 24 weeks’  gestational age would be optimal . For RSVThe predicted optimal timing of vaccination is largely driven by the slow decay of vaccine induced antibodies in the mother for the RSVpreF vaccine . When we parameterized the model to the Novavax vaccine characteristics We assumed that antenatal care (ANC) visits could be used as opportunities to administer a maternal RSV vaccine. We used the country-specific distribution of time of ANC visits within the administration window as a proxy for the likelihood distribution for timing of maternal vaccination. The country-specific estimates for vaccine efficacy  were mulLastly, we estimated for 51 GAVI-eligible countries the number of RSV-related mortality cases averted yearly, using the mortality estimates from Li et al. We have refined and extended a previously published mathematical model The model predicted that given a probable vaccination coverage, the RSVpreF Vaccine, that recently received approval from the EMA, could decrease the yearly number of RSV-related cases in 51 GAVI-eligible countries in the first half year of life by 15,000–17,000. This corresponds to a weighted average of 55%–63% of all RSV-related mortality cases in the first year. Future research should translate these findings into cost-effectiveness analyses to guide policy makers with prioritizing interventions that have the highest impact and best value for money.In our previous work In our model we fitted the antibody transfer function on observed cord blood to maternal ratio IgG levels as described previously g/ml could avert (or at least postpone) RSV-related mortality. Although this threshold has been the best established correlation with protection to date Another important assumption in our model is the choice for the protection threshold. We assumed that palivizumab serum titers, or RSV neutralizing titers corresponding to palivizumab serum titers, equal or above 100 µA strength of our study is the use of characteristics of a maternal RSV vaccine currently in development One limitation of our study is the reporting bias, quality, and completeness of the RSV GOLD database, which have been described elsewhere We predicted that maternal vaccination against RSV could substantially decrease the number of RSV related in-hospital and out-of-hospital deaths before 6 months of age in LMICs. Our model can be used to estimate duration of protection provided by maternal vaccination on case-level and to help inform policy makers on optimal timing of maternal vaccination during pregnancy. It can give an indication of the population-level effectiveness of new interventions prior to introducing a preventive strategy, which is critical for governments and large funding organizations.10.13039/100000865Bill & Melinda Gates Foundation [OPP1148988.8].The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: LB has regular interaction with pharmaceutical and other industrial partners. He has not received personal fees or other personal benefits. UMCU has received major funding  for investigator initiated studies from AbbVie, MedImmune, AstraZeneca, Sanofi, Janssen, Pfizer, MSD, and MeMed Diagnostics. UMCU has received major funding for the RSV GOLD study from the Bill and Melinda Gates Foundation. UMCU has received major funding as part of the public private partnership IMI-funded RESCEU and PROMISE projects with partners GSK, Novavax, Janssen, AstraZeneca, Pfizer, and Sanofi. UMCU has received major funding by Julius Clinical for participating in clinical studies sponsored by MedImmune and Pfizer. UMCU received minor funding  for consultation and invited lectures by AbbVie, MedImmune, Ablynx, Bavaria Nordic, MabXience, GSK, Novavax, Pfizer, Moderna, Astrazeneca, MSD, Sanofi, Genzyme, Janssen. Dr. Bont is the founding chairman of the ReSViNET Foundation.Since April 1st 2021, LB has been given a new position as medical scientific division manager of the Children's Division of the Wilhelmina Children's Hospital in Utrecht.