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Introduction: Medical professionals meet many transitions during their careers, and must learn to adjust rapidly to unfamiliar workplaces and teams. This study investigated the use of a digital educational escape room (DEER) in facilitating medical students' learning around managing uncertainty in transitioning from classroom to clinical placement.Methods: We used design-based research to explore the design, build, and test of a DEER, as well as gain insight into how these novel learning environments work, using Community of Inquiry (CoI) as a guiding conceptual framework. This study represented a mixed methods pilot test of a prototype DEER. Twenty-two medical students agreed to participate, and data were collected through qualitative (i.e., focus groups, game-play observations) and quantitative (i.e., questionnaires) methods.Results: Eighty-two per cent of participants agreed or strongly agreed that the DEER supported their learning around uncertainty. Participants offered diverse examples of how the game had facilitated new insights on, and approaches to, uncertainty. With respect to the learning environment, multiple indicators and examples of the three domains of CoI - cognitive, teaching and social presence - were observed.Discussion: Our findings suggested that DEERs offer a valuable online learning environment for students to engage with complex and emotion-provoking challenges, such as those experienced at transitions. The study also suggested that CoI can be applied to the design, implementation, and evaluation of DEER learning environments, and we have proposed a set of design principles that may offer guidance here.

The emergence of bacterial strains resistant to topical antimicrobials in both human and veterinary medicine has raised concerns over retaining the efficacy of these preparations. Yet, little information is available regarding the use of topical antimicrobials in either sector for planning targeted interventions. This study aims to quantify the use of topical antimicrobials in 44 Dutch companion animal clinics before and during their participation in an antimicrobial stewardship programme (ASP), to explore the effect of the intervention on topical antimicrobial use (AMU). Hence, prescription and clinic animal population data, collected from July 2012 until June 2018 were used. Specifically, the period from July 2012 until June 2015 was defined as pre-intervention period, whereas clinics started to participate in the ASP from March 2016 onwards. As quantification metric, the Defined Daily Dose for Animals (DDDA) was used and a mixed effect times series model with auto-regression was applied to monthly topical AMU data. The intervention effect was modelled using a step function with a change in (linear) time trend and clinic characteristics, as potential determinants of topical AMU, were assessed using a multivariable regression model. A seasonal pattern was identified, in the pre-intervention period, where topical AMU was highest in July-August and lowest in February-March. In addition, total topical AMU appeared to significantly decrease over time in the pre-intervention period and the proportion of dogs in the clinic was positively associated with topical AMU. The intervention effect was significant only for second line and for skin product AMU. This study demonstrates that during participation in an ASP, second line and skin product AMU decreased in Dutch companion animal clinics. Additionally, this study demonstrates the existence of a seasonal effect and a decrease in topical AMU over time already before introduction of a targeted intervention.

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales have been increasingly isolated from pigs, highlighting their potential for transmission to humans living and/or working within pig farms. As longitudinal data on the prevalence and the molecular characteristics of such isolates from the high-risk farming population remain scarce, we performed a long-term study on 39 Dutch pig farms. Fecal samples from pigs, farmers, family members, and employees were collected during four sampling occasions with a 6-month period. The presence of ESBL-producing Enterobacterales and their molecular characteristics (ESBL gene, plasmid, and sequence types) were determined by standard methods. Data on personal and farm characteristics were collected using questionnaires. ESBL-producing Escherichia coli was present in pigs at least once for 18 of 39 farms and in 17 of 146 farmers, family members, and/or employees. Among these 417 E. coli isolates, bla CTX-M-1 was the most frequently observed ESBL gene in pigs ( n = 261) and humans ( n = 25). Despite the great variety in plasmid (sub)types and E. coli sequence types (STs), we observed genetic similarity between human- and pig-derived isolates in (i) ESBL gene, plasmid (sub)type, and ST, suggesting potential clonal transmission in seven farms, and (ii) only ESBL gene and plasmid (sub)type, highlighting the possibility of horizontal transfer in four farms. Five pig farmers carried ESBL producers repeatedly, of whom two carried an identical combination of gene, plasmid (sub)type, and ST over time. Human ESBL carriage was associated with both presence of ESBL producers in pigs and average number of hours working on the pig farm per week, while prolonged human carriage was observed only incidentally. IMPORTANCE Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli represents a public health hazard due to reduced therapeutic options for the treatment of infections. Although direct contact with pigs is considered a risk factor for human ESBL-producing E. coli carriage through occupational exposure, nationwide data regarding the occurrence of such isolates among pigs and humans living and/or working on farms remain scarce. Therefore, we determined (i) the longitudinal dynamics in prevalence and molecular characteristics of ESBL-producing E. coli in Dutch pig farmers and their pigs over time and (ii) the potential transmission events between these reservoirs based on genetic relatedness and epidemiological associations in longitudinal data. Our data suggesting the possibility of clonal and horizontal dissemination of ESBL-producing Escherichia coli between pigs and pig farmers can be used to inform targeted intervention strategies to decrease the within-farm human exposure to ESBL-producing E. coli.

In context of an increasing interest in immunotherapeutic treatments, the work described in this thesis expands the current knowledge of several potential antibody-based therapeutic approaches. A new development is the use of bispecific antibodies (BsAb). To introduce BsAb, chapter 2 describes the different platforms currently used to generate IgG-like and non-IgG-like BsAbs, as well as the strategies to gain clinical approval for the different BsAbs. A highly selective binding, as can be achieved with BsAbs, could be beneficial to ‘target’ certain cell populations, such as Tregs, against which current treatments appear to be insufficiently specific. Focusing on BsAb-based therapeutic approaches, in chapter 3 we investigate a novel strategy to optimize a T-cell redirecting bispecific antibody. Two Fab x sdAb-Fc, directed against CD3 and EGFR, with different lengths of the hinge region, were compared. The results show that a shorter hinge design improves BsAb-induced anti-tumor activity, induces more effector cell/target cell clustering and activates T cells more efficiently. In addition, we show (chapter 4) how important it is to select the right isotype antibody. In a mouse tumor model, only IgG2a antibodies were found to exert anti-tumor function, achieving a survival rate of approximately 50%. None of the isotypes tested showed a synergistic effect with adoptive T-cell therapy. An increasing interest in the costimulatory receptor TNFR2 in cancer has prompted us to design, generate and produce novel anti-mTNFR2 antibodies (chapter 5). The characterization and different features of these antibodies are described in this chapter, and they could be used as tools in follow-up studies to further investigate i) the role of TNFR2 in cancer, and ii) how TNFR2 targeting with different antibody candidates that either block or activate this receptor could improve immunotherapy. In chapter 6, two anti-mTNFR2 antibodies were selected for assessment of their application in vitro, ex vivo and in vivo, which revealed their capacity to bind to Tregs but also to CD4+ effector T cells. Taken together, the results presented in this thesis highlight the progress achieved in immunotherapy approaches, with focus on BsAbs and novel strategies to improve cancer treatments. Furthermore, novel anti-mTNFR2 antibodies were developed and featured as useful tools to unravel the role of this receptor in tumor growth.

Bispecific antibodies (BsAbs) are a class of monoclonal antibodies that can target two different antigens/epitopes simultaneously. BsAbs can be roughly divided into two classes: IgG-like BsAbs and fragment-based BsAbs. Fragment-based BsAbs have a much shorter serum half-life due to the absence of the Fc fragment, which has limited their application in the clinic. In contrast, IgG-like BsAbs have relatively long serum half-lives, however, generating pure IgG-like BsAbs and improving their yield is still challenging due to chain association issues. The production of a BsAb in one expression cell line is very difficult and unfavourable due to challenges in extracting the desired BsAb from the lysates and the inherently low yield (Chapter 1, Figure 2). Thus, the aim of the research described in chapter 3 was to design a novel format to improve the purity and yield of BsAb during production. We have combined a conventional antigen-binding fragment with a single-domain antibody and generated BsAbs in a Fab x sdAb-Fc format. Our data show that this format can avoid potential heavy-light chain mis-pairing during the production of BsAbs, and increases the purity of BsAbs to above 95%. Further characterization assays showed that the BsAbs in this configuration nevertheless maintained the ability of binding to two distinct antigens concurrently. During the design of this novel BsAb format, we found that the hinge region can be adapted for different application scenarios. There are already several studies indicating that enhanced T-cell mediated tumor cell elimination can be achieved by decreasing the distance between T cells and tumor cells. In our case, the distance between tumor cells and T cells can be modulated by different hinge designs. Thus, we modulated the hinge region in T-cell redirecting bispecific antibodies (TRBAs) and studied their anti-tumor activity in in vitro assays. Our data show that with less space separating the two arms of the TRBA, tumor cells and effector T cells can bridge more tightly, which strengthens T cell activation and, in turn, increases tumor cell death. Therefore, our data indicate that the modulation of the ‘hinge region length’ parameter can possibly contribute to future design of similar molecules (Chapter 4). Since the antibody Fc-tail activates specific immune effector mechanisms, antibody isotype plays an important role in cancer therapy. Previous studies in a mouse tumor model showed the efficacy of prophylactic application of mIgG2a isotype antibodies. However, human cancer patients usually receive antibodies in a therapeutic setting. Thus, in the last study, we developed a panel of anti-Thy1.1 antibodies with various isotypes (mIgG1, mIgG2a, mIgE and Fc-silenced) and evaluated their effectiveness in a therapeutic setting in mouse tumor models. Our data demonstrated that mIgG2a is the most effective isotype in treating cancer in a therapeutic setting in mice. Therefore, isotype selection is a critical parameter determining the efficacy of tumor-targeting antibody therapy. We believe future research in tumor immunotherapy may benefit from the knowledge we gained in designing tumor antigen targeting antibodies for cancer therapy (Chapter 5).

The binding of a viral pathogen to the host cell is the first, crucial, step of infection. This first step in host-pathogen interactions is dependent on the attachment tools of the viral pathogen and the presentation of attachment factors by the host. Pathogen receptor specificity and host receptor distribution govern which cells and tissues are susceptible, known as host tropism. Using our own developed tissue microarrays to represent the host and the viral attachment proteins to represent the pathogen, we set out to study how receptor-binding interactions can improve our understanding of receptor distribution of both domestic and wild animals and how glycobiology plays a key role in driving these receptor-binding interactions. We used these tools to study and map the receptor binding of influenza A & D attachment proteins in respiratory tissues of various domesticated and wild animals. Receptor binding is the first step in infection and it is therefore of high importance to study it. The use of tissue microarrays is a great in-between system for studying the distribution of these host receptors. The system does have its shortcomings, particularly in terms of glycolipid and mucin detection. To summarize, TMAs are not a replacement for current methods used, such as glycan arrays and animal experiments, but are an excellent addition that complements those systems for studying receptor interactions.

Extracellular vesicles (EVs) are cell-derived multicomponent messengers that participate in diverse intercellular communication processes regulating immune responses, cell survival and proliferation. However, EVs also play a role in disease processes like cancer. Since EVs contain biological information that reflects the status of the cell of origin, they might be used in liquid biopsies as disease biomarkers. The detection and characterization of EVs is a fundamental step towards EV-based biomarker profiling of pathological processes and is complicated due to their small size, heterogeneity and complex composition. Therefore, technological solutions that allow high throughput detection and analysis of single EVs are of utmost importance. Flow cytometry (FCM) is a promising technology for this purpose. However, traditional flow cytometers, designed for cells, lack the sensitivity for the detection of nano-sized EVs, whose signals are typically below or at the detection limit of conventional instruments. In my PhD thesis I explored the limits of flow cytometry of single EVs, provide guidance for reproducible and robust EV FCM and address the challenges of EV analysis in body fluids. First, we developed and characterized a reference material for EV research. Next, we focused on high-sensitivity FCM for the detection of EVs and I present a reporting framework developed by the ISEV-ISAC-ISTH EV Flow cytometry workgroup to increase reproducibility of results. Since fluorescence calibration of EV signals is of importance for inter-instrument and inter-lab comparisons, we provide insights into the utilization of current calibrators and show that intrinsic variations related to these calibrators compromise accurate calibration of EV signals. In addition to the fluorescence, light scatter signals also contain valuable information that it is often lost within the background noise. By using an open-architecture flow cytometer, we optimized the configuration by combining differently sized hardware components to improve light scatter-based EV detection by reducing optical background signals. Lastly, we applied these technological advancements in the field of high-sensitivity FCM to investigate interactions between biological particles, namely EVs and lipoprotein particles (LPPs), which are known to co-isolate. Liquid biopsy EV-based biomarker profiling is complicated because LPPs in blood outnumber EVs by orders of magnitude and have biophysical overlapping properties with EVs, including size and density. We here demonstrate by high-sensitivity FCM, in combination with orthogonal methods; i.e., Rayleigh and Raman scattering and cryo-electron microscopy, that EV-LPP complexes can be formed and can impact EV-analysis in blood. Overall, the findings presented in my thesis can contribute to the development of robust liquid biopsy EV biomarker profiling by FCM and shed light on the complexity of single EV analysis in body fluids, which will ultimately help to a successful clinical translation of EVs as disease biomarkers.

The emergence and spread of infectious diseases, especially zoonoses, is complex and depends on various determinants or drivers, including their interrelations at the human-animal-environment interface (HAEI). This thesis provides novel insights into the potential role of environmental transmission in the epidemiology of selected exemplary zoonotic pathogens (i.e. Campylobacter, Shiga toxin-producing E. coli O157, and non-typhoid Salmonella) that are typically considered as foodborne pathogens. This was done mainly by studying some of their characteristics in the environment and the spatial associations between their incidence among human cases and the level of exposure to local livestock density, using novel methods within an eco-epidemiological framework. In addition, this thesis explored the potential human health effects linked to extreme rainfall events and the spatial association between the urban-rural gradient and the diversity and composition of the human gut microbiome. Chapter 2 showed that direct exposure to pluvial floodwater, and activities leading to such exposure, are associated with an increased risk of developing acute gastroenteritis (AGE) and acute respiratory infection (ARI). Chapter 3 showed that surface water is mainly contaminated with Campylobacter originating from meat-producing poultry (i.e. broilers and turkeys) in areas with high poultry densities and from wild birds in recreational waters and WWTP discharge points. The results of Chapter 4 and 5 revealed that human STEC O157 infections can be caused by environmental exposure to small ruminants besides food, while the route of transmission of Salmonella and Campylobacter to humans is mainly foodborne. Chapter 6 revealed that bacterial diversity, richness and composition of the adult gut microbiome are associated with the urban-rural gradient in the Netherlands. This thesis showed that we cannot ignore the role of the environment in mediating the transmission of (foodborne) zoonotic pathogens from livestock and wildlife, and more holistically in shaping the human gut microbiome. However, evidence about specific routes of transmission (e.g. air-borne, via soil or water) and their relative importance, was not provided. To study all interactions between different drivers of zoonotic pathogens, we recommend future studies to focus on pathogen transmission and their related transmission pathways to be able to look beyond the three drivers of change included in the One Health disease triangle (humans, animals, environment). This can be visualized by including a white triangle based on the Gestalt idea that the sum of visual perception is more than its parts. This triangle illustrates the interactions to include to study zoonotic pathogens and provide a complete picture of how they may influence one another. This visualization can be considered as a first step towards further unraveling the complexity of zoonotic pathogens by showing “the complete picture” or at least an attempt to do so, to, in the end, achieve optimal health outcomes.

Evidence suggests that the prevalence of asthma and allergic symptoms have risen rapidly over the past decades. It has been suggested that occupational exposure to pesticides is associated with an increased risk of asthma and related respiratory symptoms in adults. There is also the suggestion that adults who use cleaning agents professionally or in the home have an increased risk of asthma and related respiratory symptoms. In addition, there is increasing concern that some cleaning agents being used in households might be associated with an increased risk of asthma and allergic symptoms in children. However, the current evidence on the effect of exposure to pesticides and cleaning products among children and adolescents is limited. In Chapter 2, It was found that a small proportion of the study participants lived near (i.e., within 50 or 100 m) agricultural fields with the selected crops relevant for pesticide use (approximately 7% of the population residing within the 100 m). The results indicated further that 40 and 64 percent of the PIAMA participants lived within 500 and 1,000m buffers, respectively. It was hypothesised that participants who lived within 500 and 1,000m buffers tended to have minimal pesticide exposure compared to participants who lived within 50 and 100 m. Chapter 3 examined whether living close to crops that are likely treated with pesticides is associated with asthma and related respiratory symptoms among PIAMA participants at the time of 14 years of follow-up. However, we found no evidence that living close to agricultural fields assumed to have been treated with pesticides is associated with an increased risk of asthma and related respiratory symptoms. In Chapter 4, it was found that the use of cleaning agents in the household was not associated with increased risks of asthma, rhinitis, and eczema in adolescents who participated in the PIAMA study at the time of 14 years of follow-up. Chapter 5 presents the opportunity for the development of studies investigating the effects of environmental exposure to pesticides on the development of asthma and related respiratory symptoms in children. However, this Chapter cannot provide a comprehensive review of the effects of pesticide exposure on human health in Tanzania due to limited data and literature on the subject. This thesis set out to investigate the effects of environmental exposure to pesticides and cleaning agents on the prevalence of asthma and allergic symptoms in adolescents who participated in the PIAMA. We observed no association between these environmental factors (environmental exposure to pesticides and cleaning agents) and the risk of asthma, related respiratory symptoms, and allergic symptoms in adolescents. There is a need to develop a birth cohort study that will investigate the influence of environmental exposure to pesticides and the use of cleaning agents on the development of asthma, allergic symptoms and lung function in children in developing countries.

Quality control of vaccine batches before their release onto the market is mandatory to demonstrate that they are safe and efficacious. For inactivated vaccines, this is often achieved using immunization challenge tests, requiring large numbers of laboratory animals. Ethical concerns about animal use and the questionable relevance of many animal models for the target species urged for implementation of the 3R’s principle (replacement, reduction and refinement of animal use) in vaccine manufacturing and quality control. Increased knowledge of vaccine antigens, implementation of in-process testing, Good Manufacturing Practice (GMP) and Quality Assurance in vaccine manufacturing enabled standardized manufacturing processes. Together, these implementations facilitate production of better defined and less variable vaccine batches. Therefore, demonstration of batch-to-batch consistency has been proposed as a new approach for batch release testing. Once consistency in production has been demonstrated by showing that new batches do not differ from previous batches of sufficient quality by measuring a set of crucial parameters, testing of subsequent batches could rely on assessment of these same parameters, ideally by a panel of in vitro tests. This testing strategy is known as the consistency approach which has the potential to abolish the need for animal tests for batch release onto the market. Potency assessment of inactivated canine Leptospira vaccine batches still relies, to varying degrees among vaccine manufacturers, on the use of in vivo hamster challenge experiments. In view of the 3R principle, there is a clear need for development of novel host specific in vitro tests to study vaccine potency and/or immune activation. Pathogenic Leptospira are the causative agents of leptospirosis, an emerging bacterial zoonosis, potentially affecting all vertebrates, including humans and dogs. Leptospira spp. are Gram-negative spirochetes found in water and soil and comprise of saprophytic and pathogenic species. Numerous Leptospira serovars have been identified based on the carbohydrate structure of the leptospiral LPS. Vaccination of dogs against Leptospira is recommended to protect the animals against potentially fatal infection and to prevent transmission to the environment. Commercially available Leptospira vaccines consist of inactivated whole bacteria of different serovars. Monoclonal antibodies (mAbs) recognising LPS epitopes of specific Leptospira serovars were developed for quantification of Leptospira antigen in vaccines using an enzyme-linked immunosorbent assay (ELISA), for quality control purposes. Although this LPS-based ELISA is a reproducible and affordable alternative to the animal batch release test, it is serovar specific, which means that mAbs need to be developed for each serovar. Furthermore, this method does not provide any information about the protective immune responses induced by inactivated Leptospira vaccines. The purpose of the studies described in this thesis was to develop cell-based assays to determine the immunostimulatory properties of inactivated canine Leptospira vaccines and individual serovars in vitro, and to assess the applicability of these assays for future routine quality control testing. Aspects of innate immunity and its response to inactivated Leptospira vaccines in the dog were investigated and described in Chapters 2, 3 and 4, while Chapter 5 addresses the examination of adaptive immune responses induced by these vaccines.

Andrés Martin,1– 3 Marco A de Carvalho Filho,3,4 Debbie Jaarsma,3,5 Robbert Duvivier3,6 1Child Study Center, Yale School of Medicine, New Haven, CT, USA; 2Simulated Participant Program, Teaching and Learning Center, Yale School of Medicine, New Haven, CT, USA; 3Center for Educational Development and Research in Health Sciences (CEDAR), Lifelong Learning, Education and Assessment Research Network (LEARN), University Medical Center Groningen, Groningen, the Netherlands; 4School of Medical Sciences, University of Minho, Braga, Portugal; 5Faculty of Veterinary Medicine, University of Utrecht, Utrecht, the Netherlands; 6Parnassia Psychiatric Institute, The Hague, the NetherlandsCorrespondence: Andrés MartinYale Child Study Center, 230 South Frontage Road, New Haven, CT, 06520-7900, USATel +1 203-494-2355Fax + 1 203-785-7400Email andres.martin@yale.eduObjective: Innovations in contemporary medical education could inform remedies to address enduring challenges such as the marginalization and stigmatization of psychiatry, of mental illnesses, and of those affected by them.Methods: In blending the works of Bleakley, Bligh, and Brown (2011) and of Kumagai and Naidu (2015), we developed an overarching heuristic with practical relevance and concrete applications to psychiatric education.Results: We identify three areas to enhance psychiatric education embedded into this blended framework: 1) Showing, or the more accurate depiction or imaging of mental illnesses and of psychiatric practice, as exemplified by the incorporation into didactic content of asynchronous video-based clinical materials produced with specific educational objectives in mind; 2) Sharing, or addressing the image problem of mental illnesses, of those living with or affected by them, and of psychiatry as a profession, as exemplified by psychiatrists embracing their role as experts by professional and personal experience when sharing their own journeys with mental illness, treatment, and recovery; and 3) Doing, or reimagining reflective psychiatric practice, as exemplified by the novel methodology of co-constructive patient simulation (CCPS), through which learners can engage in reflective practice and supervision in a participatory and democratic setting that does not privilege participants’ hierarchical standing.Conclusion: The blended model and the sample applications we describe offer a range of teaching, learning, and professional development opportunities, should psychiatric educators choose to pursue them and reap their promise.Keywords: medical education, psychiatry, reflective practice, simulated patients, stigma, video-enhanced didactic

IntroductionThe urgency of preventing the increase of antimicrobial resistance has been emphasized by international authorities such as the World Health Organization, European Medicines Agency, and World Organization for Animal Health. Monitoring systems capable of reporting antimicrobial consumption data are regarded as a crucial pillar of this fight. The Vet-AMNet system was developed to collect and analyze national antimicrobial consumption data in Portuguese dairy farms to support the veterinary authority in stewardship actions and to assist both veterinarians and farmers in daily decisions related to antimicrobials.MethodsTo evaluate the robustness of the system and other identified critical success factors, it was used to analyze antimicrobial consumption data available from the Dutch dairy cow sector over the period from 2012 to 2020. The data previously used for publications by the Netherlands Veterinary Medicines Institute (SDa) were imported and pre-processed by the Vet-AMNet system according to the SDa's standard operating procedure and the Dutch metrics to measure antimicrobial consumption were calculated.ResultsBy comparing the outputs with the figures generated by the system established in the Netherlands, the Portuguese system was validated. Antimicrobial consumption data from the Dutch dairy sector during the 9-year period will be presented in unpublished graphs and tables, where each molecule's pharmaceutical formulation, pharmacotherapeutic group and line of choice will be related and discussed, illustrating the evolution of sectorial antimicrobial consumption against a background of a strong national antimicrobial policy initiated by public-private cooperation and supported by legislation.

Most people, if not all, will be exposed at least once in their life-time to a certain level of pesticide(s). For non-occupationally exposed groups, a great portion of this exposure can be attributed to dietary intake and the remaining to environmental exposure. In the Netherlands, the application of pesticides on agricultural fields has raised concerns from residents living close to these. In order to understand if a certain disease is related to an exposure to any given chemical, we first need to identify the exposed vs non-exposed population and also understand the levels, duration and frequency of exposure. The aim of my thesis is on investigating residents’ environmental exposure to pesticides by studying drivers of this exposure as well as temporal and spatial variability in different pesticides concentration. Chapter 2 details the methodology used to study residential exposure to pesticides and a list of (practical) lessons learned. Chapter 3 described the spatial and temporal variation of 46 different pesticide concentrations in air outside and inside homes. Here, we saw that most pesticide concentrations were 5–10 times higher in outdoor air during the spraying period when comparing with the non-spraying period. Concentrations were overall higher closer to the fields (

To exchange information cells participate in the continuous exchange of small, membranous particles termed “extracellular vesicles” (EVs). These EVs carry status updates and instructions to neighbors, and can be compared to the cellular equivalent of sending an email or posting messages on social media. Although cell-to-cell communication via EVs is typically beneficial, these messages have the potential to turn corrupted during the development of disease. In the last years, a growing number of viruses were found capable of hiding in the EVs shuttled between cells. Similar to an email carrying a computer virus, the packaging of viruses within EVs enables viruses to bypass our body’s defenses by adopting a seemingly harmless exterior, and provides an alternative method for virus spread. In this dissertation, it was investigated how EV production of cells changes in response to virus infection and which proteins produced by the virus and the host cell regulate this process. Our data uncovers a previously unknown level of heterogeneity among the virus-containing EVs that are released during infection. We show that the spread of viruses via EVs is enhanced by drastic changes in the number and type of EVs that a cell produces, along with the activation of new EV-cargo sorting pathways. Finally, we demonstrate that these changes in EV production are the result of the interplay between the activity of dedicated viral proteins and cellular stress responses. These results provide novel insight into the virus-host interactions that determine the amount and phenotype of virus-carrying EVs released during infection.

Highly Pathogenic Avian Influenza (HPAIv) H5N1 virus of the A/Goose/Guandong/1/96 lineage was first reported in Asia in 1996 and has been circulating in Indonesia since 2003. Its spread caused a global impact on poultry health and severe losses to the poultry industry. Furthermore, 200 laboratory-confirmed human cases were reported alone in Indonesia with a case-fatality rate of 84%. Analysis of the contact structure facilitating the transmission of HPAIv(A)H5N1 between different types of poultry farms in West Java showed that visitors of backyard chicken farms had the highest average contact rate, either direct contact with poultry on other farms before the visits or contact during their visits in the farms. These results suggest that backyard chicken farms are most at risk for transmission of HPAIv compared to farms of the other poultry production types and also serve as a vector for transmitting HPAIv H5N1 to commercial poultry. Passive surveillance from outbreak farms in six regions of West Java from November 2015 to November 2016 showed the highest mortality in backyard chickens with dermal apoptosis and lesions and respiratory signs. Neurological signs were most frequently observed in ducks. The rate of visitor contacts onto a farm was positively associated with the odds of HPAI infection. Duck farms had higher odds of being infected than backyard farms. The larger farms had lower odds than small farms. Results indicate that better external biosecurity is needed to reduce transmission of HPAIvA(H5N1) in Indonesia. The phylogenetic analyses of the analysis of 39 full-genome HPAIv(A)H5N1 viruses with additional reference sequences revealed 2 genetic clusters in clade 2.3.2.1c based on the hemagglutinin gene as well as the neuraminidase, nucleoprotein, polymerase, and polymerase basic 1 gene. The polymerase basic 2 gene had a close relation with Eurasian low pathogenic avian influenza virus (LPAIv). Also, several matrix, nonstructural protein and polymerase basic 2 genes were detected in HPAIv with higher identity with clade 2.1.3 than with clade 2.3.2.1c. The phylogenetic analysis of the samples in 2015-2016 thus identified 13 types of reassortment in HPAIv in Indonesia, mostly in native chickens in Indramayu. The BEAST analysis of the Indonesian poultry HPAIvA(H5N1) sequences collected from 2003-2016 were performed with a set of represented global H5Nx that have identities greater than 98%. Temporal dynamic analysis revealed that most likely only two introductions of HPAIvA(H5N1) occurred in Indonesia. The common ancestor (TMRCA) of the first introduction to Indonesian poultry HPAIvA(H5N1) emerged about five to seven years after the global common ancestor of HPAIv A(H5Nx). The divergences and TMRCA of H5N1 clade 2.3.2.1c and clade 2.3.2.1b were identified. The TMRCA of clade 2.3.2.1c and clade 2.3.2.1 indicate that the second introduction of both HPAIvA(H5N1) into Indonesian poultry originated from viruses from the same clade as viruses. detected in China and other Asian countries. Results of studies presented the importance of continued and improved (genomic) surveillance in poultry, live bird markets and wild birds. Adequate control measures such poultry farm biosecurity and poultry reconstruction are required to prevent a new emerging pandemic threat.

There are almost one billion pigs worldwide, which are threatened by pathogenic bacteria. One of those is Streptococcus suis. There is no effective vaccine against this bacterium, leading to many diseased pigs each year, which in turn poses risks to humans. Treatment with antibiotics is possible, but in pigs the infection must be diagnosed in time, and treatment could lead to antibiotic resistance development. One solution for S. suis infections could be the host defence peptides (HDPs). HDPs are antimicrobial without inducing resistance and have immune modulatory functions which make them suitable as vaccine components. However, there is a lack of specific knowledge of HDP activity in porcine cell systems, and particularly in the context of an S. suis infection. The HDP CATH-2 is effective in inhibiting the immune response of porcine macrophages. This could be important in, for example, effective vaccines that have sizable side effects. S. suis has a remarkable resilience to HDPs originating in pigs, but much less against CATH-2. More research is needed to clarify the mechanism of resilience. CATH-2 effectively inhibits porcine dendritic cell activation by both E. coli and S. suis. This could mean that CATH-2 could potentially lower side effects of a vaccine, without damaging the capacity to process the antigen (and thereby the vaccine effectivity). CATH-2 and some derivatives have the potential to protect mice against S. suis induced disease symptoms. These CATH-2 derivatives could be used prophylactically, although this has not yet been tested in pigs.

In this thesis, we study cysteine rich secretory protein 2 (CRISP2) in pig sperm. CRISP2, which is synthesized during spermatogenesis, was localized by confocal immunofluorescent imaging in the tail and in the post-acrosomal region of the sperm head. High resolution localization by immunogold labeling electron microscopy (EM) of ultrathin cryosections revealed that CRISP2 was present in the perinuclear theca (PT) and neck region of the sperm head, as well as in the outer dense fibers and the fibrous sheath of the sperm tail. CRISP2 was involved in different protein complexes and showed different biochemical properties in the sperm head and tail. Later, we investigated the fate of sperm head CRISP2 post-fertilization. In vitro matured porcine oocytes were co-incubated with boar sperm cells for 6-8 h and the zygotes were processed for CRISP2 immunofluorescent staining. Notably, decondensation of CRISP2, and thus of the sperm PT, occurred while the sperm nucleus was still fully condensed. CRISP2 was no longer detectable in fertilized oocytes in which sperm nuclear decondensation and paternal pronucleus formation were apparent. This rapid dispersal of CRISP2 in the PT is likely regulated by redox reactions for which its cysteine rich domain is sensitive. Further, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa. Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. Finally, we elucidate CRISP2 interacting partners using proteomic technology. We identified acrosin and acrosin binding protein (ACRBP) as novel CRISP2 interacting proteins. Additionally, our data also revealed a list of potentially interacting candidates that are functionally categorized into spermadhesins, protease inhibitors, core histones, chaperons, proteins with a mitochondrial function and ubiquitin. Biochemical validation of these interacting protein candidates is required. Nonetheless, our data suggest that CRISP2 might play multiple roles in sperm function and the fertilization process. It is clear that the ejaculated sperm are showing most robust and condensed state of the dense protein structures. This is ergonomically important for creating a minimal volume of well protected chromatin in the sperm head, a concentrated enzyme matrix for sperm-zona penetration in the acrosome. Condensed structures are also present in the sperm flagellum which has unique flexibility and metabolic properties. These properties can be adjusted to proper sperm motility characteristics during sperm maturation in the epididymis as well as during capacitation in the oviduct (or during IVF treatment). Both the formation of the dense protein structures in the testis and in the epididymal environments as well as the decondensation of these dense protein structures during post-fertilization events deserve further attention with special emphasis on the role of CRISP2 in (de)condensation dynamics.

There is an ongoing debate on airborne transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a risk factor for infection. In this study, the level of SARS-CoV-2 in air and on surfaces of SARS-CoV-2 infected nursing home residents was assessed to gain insight in potential transmission routes. During outbreaks, air samples were collected using three different active and one passive air sampling technique in rooms of infected patients. Oropharyngeal swabs (OPS) of the residents and dry surface swabs were collected. Additionally, longitudinal passive air samples were collected during a period of 4 months in common areas of the wards. Presence of SARS-CoV-2 RNA was determined using RT-qPCR, targeting the RdRp- and E-genes. OPS, samples of two active air samplers and surface swabs with Ct-value ≤35 were tested for the presence of infectious virus by cell culture. In total, 360 air and 319 surface samples from patient rooms and common areas were collected. In rooms of 10 residents with detected SARS-CoV-2 RNA in OPS, SARS-CoV-2 RNA was detected in 93 of 184 collected environmental samples (50.5%) (lowest Ct 29.5), substantially more than in the rooms of residents with negative OPS on the day of environmental sampling (n = 2) (3.6%). SARS-CoV-2 RNA was most frequently present in the larger particle size fractions [>4 μm 60% (6/10); 1–4 μm 50% (5/10)

Livestock animals housed in close proximity to humans can act as sources or intermediate hosts facilitating animal-to-human transmission of zoonotic diseases. Understanding virus diversity in livestock is important for identifying potential zoonotic threats and for ensuring animal health and safe livestock production. Here, we report viral metagenomic characterization of chicken feces (N=51) and paired farm dust samples (N=13) using metagenomic deep sequencing. Samples were collected at 4-5 time points in three broiler farms in the Netherlands. Viruses in the Parvoviridae and Picornaviridae families were the most prevalent, detected in all feces and dust samples and in all feces and 85% of dust samples, respectively. Virus composition found in chicken feces and corresponding dust samples were similar. Great genomic diversity was identified in Picornaviridae and 46 sequences from five picornavirus genera (Sicinivirus, Megrivirus, Anatvirus, Gallivirus and Avisvirus) were detected. For calicivirus, Basovirus and an unclassified novel chicken calicivirus were identified in 13 fecal and 1 dust samples. Two distinct types of chicken astroviruses were identified. Phylogenetic analyses of identified virus sequences from Picornaviridae, Astroviridae and Caliciviridae suggested that viral sequences obtained from different farms are often more closely related to each other than global reference sequences, and sequences from feces and paired dust samples also clustered together. Importantly, our sequencing methodology enabled the recover viral genome sequences from farm dusts, allowing the tracking of virus chatter between livestock animals and their farm environment. This study, albeit relative sample size, does expand current knowledge of virus communities in chickens and surrounding dusts. IMPORTANCEChickens may harbor various zoonotic pathogens, some of which can cause severe clinical outcomes in animals and humans. Farm dust can act as vector to facilitate zoonoses transmission. Here, we report the metagenomic characterization of virus communities of chicken feces and paired farm dust samples collected at multiple time points during the production cycle in broiler farms in the Netherlands. Parvoviridae and Picornaviridae were most frequently detected. We also identified novel astrovirus and calicivirus sequences that would inform future virus taxonomy classification. This is the first study to characterize virus communities in farmed chickens and paired farm dust samples. We also describe a dust sequencing strategy that can be adapted for future dust metagenomic characterization. Our study could help setting up a surveillance baseline for tracking virus flow between chickens and their farm environment which could guide zoonotic outbreak preparedness and health risk assessment of farm exposure.

In embryos of most animal species, the zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the sperm aster and mitotic spindle. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the zygotic MTOC. Here, we show that zebrafish (Danio rerio) embryos lacking either maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle. Although Cfap53 is dispensable for sperm aster function, it aids proper formation of the mitotic spindle. During cell division, Cfap53 colocalizes with γ-tubulin and with other centrosomal and centriolar satellite proteins at the MTOC. Furthermore, we find that γ-tubulin localization at the MTOC is impaired in the absence of Cfap53. Based on these results, we propose a model in which Cfap53 deposited in the oocyte and the sperm participates in the organization of the zygotic MTOC to allow mitotic spindle formation.

Resistance to platinum-based chemotherapy represents a major clinical challenge for many tumors, including epithelial ovarian cancer. Patients often experience several response-relapse events, until tumors become resistant and life expectancy drops to 12-15 months. Despite improved knowledge of the molecular determinants of platinum resistance, the lack of clinical applicability limits exploitation of many potential targets, leaving patients with limited options. Serine biosynthesis has been linked to cancer growth and poor prognosis in various cancer types, however its role in platinum-resistant ovarian cancer is not known. Here, we show that a subgroup of resistant tumors decreases phosphoglycerate dehydrogenase (PHGDH) expression at relapse after platinum-based chemotherapy. Mechanistically, we observe that this phenomenon is accompanied by a specific oxidized nicotinamide adenine dinucleotide (NAD+) regenerating phenotype, which helps tumor cells in sustaining Poly (ADP-ribose) polymerase (PARP) activity under platinum treatment. Our findings reveal metabolic vulnerabilities with clinical implications for a subset of platinum resistant ovarian cancers. ispartof: NATURE COMMUNICATIONS vol:13 issue:1 ispartof: location:England status: published

In response to changes in their environment, bacteria need to change not only their protein repertoire, but also their phospholipid composition. A small fraction of the phospholipids in the bacterial membrane are lysophospholipids (LPLs). Although LPLs are minor phospholipid species of the bacterial cell membrane, in eukaryotic cells they are important signaling molecules. In some pathogenic bacteria, LPLs can be strongly increased under certain stress conditions, however, the biological function of LPLs in bacteria has not been investigated. The goal of the research described in this thesis was to determine the phospholipidome of C. jejuni under different environmental conditions and to assess the role of distinct (classes of) phospholipids in bacterial pathogenesis and/or stress adaptation. In Chapter 2, we provide an overview of the different LPLs species present in bacteria and summarize the knowledge about their role in bacterial adaptation, survival, invasion and host-microbe interaction. In Chapter 3, we for the first time investigated the nature of the phospholipids produced by C. jejuni under different growth conditions. In Chapter 4, we investigated the role of the C. jejuni membrane LPLs during bile salt exposure using an isogenic mutant defective in the phospholipase PldA. In Chapter 5, we focused on the potential role of LPLs as a novel virulence factor of C. jejuni. This work has leaded to the identification of the highly dynamic phospholipidome of the food-borne pathogen C. jejuni and that LPLs can be considered as novel virulence factor of this organism.

In our body, hundreds of billions of cell divisions (mitoses) occur every day, and each mitosis must produce two daughter cells with identical genomic information. This is a highly controlled process to maintain tissue homeostasis. However, in cancers cell proliferation takes place in an uncontrolled manner, leading to malignant tumor formation. To discover better ways to treat cancer, it is of critical importance to understand the molecular mechanisms underlying cell division. In this dissertation, we studied the regulation and function of three E2F factors, that is E2F3, E2F7 and E2F8, during cell cycle. Particularly, in chapter 1, we introduce the roles of E2Fs people have found in regulation of cell cycle progression. In chapter 2, we reveal that E2F7 and -8 are subjected to SCFcyclin F-mediated degradation in G2 phase. In chapter 3, we then describe a noncanonical function of E2F7 and -8. We could show that E2F7 and -8 are required for this cohesin removal during prophase. In chapter 4 we switched our focus to the activating arm of the E2F family, and studied the role of E2F3 amplification, which is often seen in bladder cancer patients. We show in our bladder cancer cell lines that this E2F3-induced replication stress may sensitize cells to a class of drugs called intra-S-phase checkpoint inhibitors. Lastly, in chapter 5, we bring all these findings together and discuss the importance of coordinated regulation of E2F factors during cell cycle progression to preserve genome integrity.

During pregnancy, women are exposed to a broad range of environmental contaminants that could reach the placenta and the fetus with maternal blood. These contaminants could influence placental functions, and fetal development and predispose the fetus to diseases later in life. The developing fetus is specifically susceptible to exposure to endocrine disrupting compounds (EDCs), a diverse group of chemicals that can interfere with hormonally-driven developmental processes. Moreover, there are new contaminants of emerging concern, such as micro- and nanoplastics (MNPs) that could reach the placenta and contribute to fetal exposure. Due to the great diversity of EDCs and MNPs in the environment and the obvious ethical limitations, studying these contaminants during pregnancy is extremely challenging and prenatal exposure is still not well characterized. This thesis aimed at addressing these knowledge gaps. Here, we developed, for the first time, a method that applied a combination of cell bioassays, state-of-the-art analytical techniques, and publicly available online databases and chemical screening tools, to identify a broad range of EDCs in human amniotic fluid. Moreover, we reviewed the state of the science of placental MNP research and human-relevant placental models. Furthermore, we investigated the possible uptake, transport and toxicity of pristine and environmentally relevant MNPs in trophoblast cells, key mediators of placental exchange, endocrine, and immune functions. This study reveals the presence of a large variety of EDCs in the direct fetal environment and suggests that diverse MNPs and compounds associated with them, are internalized and translocated in placental cells where they can induce subtle biological effects. Further studies are urgently needed to better understand the possible effects of these contaminants on pregnancy, fetal development and long-term health.

This thesis describes the daily time budgets of dairy cows and how these time budgets are affected by rising environmental temperatures. Further, associations between sensor based behavioral variables during an extended transition period and two major health issues, reduced fertility and lameness, were presented. In chapter 2, two types of complete time budgets are shown: the lactational time budget averaged per month over the course of lactation and the daily time budget with behavioral patterns over 24h. A main finding was the difference in time budgets between first parity cows and older cows before and after entering the milking herd. These findings showed that first parity cows have to adapt from the pre- to post-partum period, similar to a transition period. Introducing cows in the lactational herd led to major changes in daily behavioral patterns, potentially reflecting the effects of accomplishing the hierarchical order. This effect on daily behavioral patterns was largest in primiparous cows, indicating that these animals may be better housed in a separate group. The main finding from the daily time budget indicates that dairy cows are diurnal animals which eat, stand and walk during the day and lie down and ruminate during the night. For both types of time budgets, the behavioral patterns were quite comparable between farms. Thus, this is what high producing cows in free stalls with or without pasture access do in the Netherlands. These behavioral profiles can be used as a benchmark for comparable dairy farming systems. For other types of systems, e.g. seasonal breeding, full grazing, organic farming or tie stall housing, specific benchmarks should be studied. In chapter 3, associations between daily time budgets and the daily Temperature Humidity Index (THI) and daily temperature were analyzed. Strikingly, behavioral adaptation increased with increasing THI and temperature, and was already present at the relatively low THI values and temperatures commonly found in the mild summers in the Netherlands. This adaptation was noticeable from a mean temperature of 12°C or a mean THI of 56 when dairy cows started spending less time lying, eating and walking, and spent more time standing. Chapter 4 describes associations between eating time in the transition period and the interval between calving and first service. Besides differences between primiparous and multiparous cows, lower eating time in week 4 before calving and week 3 after calving was associated with a prolonged interval between calving and first insemination. In chapter 5, associations between body condition score, locomotion score, and sensor-based time budgets during the dry period and early lactation were studied. The percentage of lame cows increased over the course of the scoring moments and was overall very high. There was a clear association between lameness and daily time budgets as well as with the expected loss of body condition in early lactation.

Sepsis is a potentially fatal bacterial infection in neonatal foals. In this thesis we have shown that correct antimicrobial treatment has a positive effect on survival in foals with sepsis. In important groups of bacteria that cause sepsis in foals, we observed emergence of resistance to commonly used antimicrobial drugs over time. Furthermore, hospitalized foals are at risk for development of healthcare-associated infections. The antimicrobial susceptibility patterns of the bacteria that cause these infections are unpredictable, making these infections challenging to treat. Strict hygiene protocols are necessary and repeated bacteriological cultures (including susceptibility testing) are advised in hospitalized foals to identify infections at an early stage. The microorganisms in the gut (the gut microbiota) play an important role in equine health. Location, age, season, horse type and pasture access affect the composition of the gut microbiota in healthy horses. Oral treatment with antimicrobials disturbs the gut microbiota. A gradual recovery was observed within two weeks; however, significant differences could still be detected six months later. Antimicrobial treatment also increased the presence of antimicrobial resistant bacteria in the faeces. Some of these were still excreted in high numbers six months later. Horses might therefore be a potential reservoir of resistant bacteria and could form a risk to animal and human health. Judicious antimicrobial use in veterinary medicine is important to prevent development and spread of antimicrobial resistance.

The prevalence of ophthalmic abnormalities in horses is high, higher even than most people are aware of and this is why equine ophthalmology has increasingly become a focus of attention. Loss or impairment of vision of horses has negative consequences for performance, usability and welfare. This thesis comprises work that arose from research questions related to clinical (eye)problems encountered in daily equine practice and is divided in 3 parts. The first part is focused on unravelling of a genetic ophthalmic disease in the horse, namely distichiasis. In distichiasis, extra eyelashes grow from the tarsal glands on the eyelids. The treatment, long-term follow-up and the identification of the genomic regions associated with distichiasis have been described. Aspects of pharmacokinetics of corticosteroids, one of the most commonly used anti-inflammatories for the equine eye, have been addressed in the second part. The distribution into the different compartments of the equine eye of corticosteroid eye drops (dexamethasone and prednisolone) and oral corticosteroids (prednisolone) have been investigated. The third part focuses on anaesthesia, improving local anaesthetic techniques and adequate analgesia during and after (standing) surgical ophthalmic procedures in horses. This thesis sheds light on the genetic cause of an important ophthalmic disease like distichiasis in the horse, helps to improve evidence-based medical and surgical treatment, and provides a vision for further research and future perspectives for prevention and treatment of diseases of the equine eye.

Staphylococcus pseudintermedius is a bacterium found on the skin and mucous membranes of healthy dogs. Generally, the bacteria is harmless, but the bacteria can also cause infections, such as ear and skin infections. Occasionally, infections with Staphylococcus pseudintermedius occur in humans. We compared Staphylococcus pseudintermedius bacteria originating from infections in dogs with Staphylococcus pseudintermedius bacteria originating from infections in humans. This comparison showed no difference between S. pseudintermedius bacteria from dogs and humans, thereby confirming our suspicion that the Staphylococcus pseudintermedius bacteria pass from the dog to its owner through intensive contact between them. Infections caused by Staphylococcus pseudintermedius can usually be treated effectively with antibiotics, but Staphylococcus pseudintermedius can become resistant to an antibiotic. If so, the Staphylococcus pseudintermedius bacterium is no longer sensitive to the antibiotic and an infection is difficult to treat. In our research, we looked at the best way to measure resistance in Staphylococcus pseudintermedius bacteria in the laboratory. In addition, we investigated how Staphylococcus pseudintermedius becomes resistant to antibiotics. We showed that Staphylococcus pseudintermedius becomes resistant by picking up pieces of DNA from nearby bacteria. Staphylococcus pseudintermedius can even pick up multiple different pieces of DNA and thus become multi-resistant. This means that the bacterium is insensitive to a large number of antibiotics, which makes treatment even more difficult. That is not just unpleasant for the dog with an infection caused by a multi-resistant Staphylococcus pseudintermedius, but may also pose a risk for its owner.

Phosphorus (P) is an essential macro-mineral necessary for many body functions and needs to be supplied in sufficient quantities to optimize animal performance. In practice cows are commonly fed excess P. A high dietary P concentration leads to high P excretion via feces which can result in freshwater eutrophication. Therefore, overfeeding dietary P should stop. In the Netherlands, fecal P excretion of cattle tends to lower over time from 2017 to 2021, however, further P reduction will be needed. Due to P essentialness, there are some limitations and criteria which need to be understood before making the decisions to lower the P concentration in the diets. First study, 60 pregnant multiparous Holstein Friesian (HF) dairy cows were assigned to a RCBD with repeated measurements. The experimental diets contained either 3.6 (Dry-HP) or 2.2 (Dry-LP) g P/kg DM during the dry period, and either 3.8 (Lac-HP) or 2.9 (Lac-LP) g P/kg DM during 56 days of post-calving period. The proportion of cows being hypocalcemic in the first week after calving was lowest with both Dry-LP and Lac-LP groups. Before calving, plasma Carboxy-terminal collagen crosslinks concentrations (CTX) decreased with Dry-HP and increased with Dry-LP. After calving, plasma CTX concentrations increased but this increase was more pronounced when Lac-LP instead of Lac-HP was fed. Overall, the results suggest that when feeding diets containing low P postpartum, cows excreted less P in the feces than at sufficient dietary P without negative impact on feed intake, milk production and fiber digestion. Moreover, feeding postpartum diets containing low P compared with recommended P increased bone resorption, and increased plasma CTX concentrations, without change in plasma concentrations of PTH and 25(OH)D3. Second study, 18 multiparous, mid lactating HF dairy cows were used and fed high dietary P with coarse artificial dried grass (HPCG), low dietary P with coarse artificial dried grass (LPCG) and low dietary P with grass pellet (LPGP). The diets contained 2.79 (HP), 2.06 (LPCH), and 2.23 (LPGP) g P/kg DM. The cows fed with grass pellet (GP) spent less time than the coarse artificial dried grass (CG) group on ruminating, but no effects were observed on saliva P, plasma Pi, PTH, 25(OH)D3, 1,25(OH)2D3, CTX and osteocalcin (OC) concentration, OM and NDF digestibility, and fecal P excretion. Cows fed the LP diets were in negative P balance however, no upregulation of P absorption was observed. Instead, those cows resorbed P from bone to fulfil their requirement. Third study, every week, from week 14 in 2017 up until week 13 in 2018, tank milk samples from 14 dairy plants across the Netherlands were collected. In total, 52 weekly milk samples were analyzed for macronutrients and minerals. Results indicated that the milk P content is changing upon changes in macronutrients and minerals content in the milk. The mean P content of milk was found to be 101.2 mg/100 g which is 1.2% greater than the value commonly used to calculate the P excretion with manure.

The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis.

The importance of fatty acid (FA) metabolism in cancer is well-established, yet the mechanisms underlying metabolic reprogramming remain elusive. Here, we identify tetraspanin CD37, a prognostic marker for aggressive B-cell lymphoma, as essential membrane-localized inhibitor of FA metabolism. Deletion of CD37 on lymphoma cells results in increased FA oxidation shown by functional assays and metabolomics. Furthermore, CD37-negative lymphomas selectively deplete palmitate from serum in mouse studies. Mechanistically, CD37 inhibits the FA transporter FATP1 through molecular interaction. Consequently, deletion of CD37 induces uptake and processing of exogenous palmitate into energy and essential building blocks for proliferation, and inhibition of FATP1 reverses this phenotype. Large lipid deposits and intracellular lipid droplets are observed in CD37-negative lymphoma tissues of patients. Moreover, inhibition of carnitine palmitoyl transferase 1 A significantly compromises viability and proliferation of CD37-deficient lymphomas. Collectively, our results identify CD37 as a direct gatekeeper of the FA metabolic switch in aggressive B-cell lymphoma.Tetraspanin CD37 deficiency has been reported as a prognostic marker for aggressive B-cell lymphoma. Here, the authors show that CD37 interacts with the fatty acid transporter 1 to inhibit palmitate uptake and its deficiency leads to increased fatty acid metabolism which promotes tumorigenesis in B-cell lymphoma.

Within health professions programs programmatic assessment has been proposed to optimize the assessment of competency-based education. Programmatic assessment aims to create a coherent system of assessment. By providing meaningful feedback, together with creating follow-up, the learner has the opportunity to learn and improve its performance. Pass/fail decisions (i.e., high-stakes decisions) do not take place after each assessment. Alternatively, every assessment is low-stakes and only after a longer period of time a high-stakes decision is made. In this thesis we have tried to unravel programmatic assessment by investigating whether validity evidence can be provided for high-stakes decision-making. Can we justify the high-stakes decision about the learner’s progression and performance within a programmatic approach to assessment? To investigate whether it is possible to generate sufficient and reliable information about the learner as input for the high-stakes decision, we performed a quantitative analysis of the scores provided by raters over time. Furthermore, not only the raters are essential in creating the information to inform the high-stakes decision, learners are involved as well by seeking feedback. To learn more about this, we investigated learner’s motivation toward feedback-seeking. Next to that, the concept of saturation of information is important. We did an initial exploration of the concept of saturation of information by investigating the relationship between the number of datapoints and the inter-rater agreement of the examiners involved in high-stakes decision-making. Then, we dived deeper into the concept by investigating the content of the datapoints, i.e., the quality of narrative information. In the final part of this thesis we investigated whether, and if so, what differences exist in designing a high-stakes decision-making procedure between programs within health professions education. As a follow-up on this, in a commentary we proposed an idea to take a different view on the role of the committee consisting of examiners performing the high-stakes decision. As the final chapter, we discussed the results of the studies performed in this thesis within existing literature and propose suggestions for further research.

Recently, a petition was offered to the European Commission calling for an immediate ban on animal testing. Although a Europe-wide moratorium on the use of animals in science is not yet possible, there has been a push by the non-scientific community and politicians for a rapid transition to animal-free innovations. Although there are benefits for both animal welfare and researchers, advances on alternate methods have not progressed enough to be able to replace animal research in the foreseeable future. This trend has led first and foremost to a substantial increase in the administrative burden and hurdles required to make timely advances in research and treatments for human and animal diseases. The current COVID-19 pandemic clearly highlights how much we actually rely on animal research. COVID-19 affects several organs and systems, and various animal-free alternatives currently available do not even come close to this complexity. In this Essay we therefore argue that the use of animals is essential for the advancement of human and veterinary health., In this Essay, Genzel et al. make the case for animal research in light of the COVID-19 pandemic.

Moisture-damaged buildings are associated with respiratory symptoms and underlying diseases among building occupants, but the causative agent(s) remain a mystery. We first identified specific fungal and bacterial taxa in classrooms with moisture damage in Finnish and Dutch primary schools. We then investigated associations of the identified moisture damage indicators with respiratory symptoms in more than 2700 students. Finally, we explored whether exposure to specific taxa within the indoor microbiota may explain the association between moisture damage and respiratory health. Schools were assessed for moisture damage through detailed inspections, and the microbial composition of settled dust in electrostatic dustfall collectors was determined using marker-gene analysis. In Finland, there were several positive associations between particular microbial indicators (diversity, richness, individual taxa) and a respiratory symptom score, while in the Netherlands, the associations tended to be mostly inverse and statistically non-significant. In Finland, abundance of the Sphingomonas bacterial genus and endotoxin levels partially explained the associations between moisture damage and symptom score. A few microbial taxa explained part of the associations with health, but overall, the observed associations between damage-associated individual taxa and respiratory health were limited.

Background: The field of pharmacy will benefit from pharmacy technicians, a higher educated mid-level support workforce. They support pharmacists in providing pharmaceutical patient care through delegated roles and responsibilities. Empirical research on pharmacy technicians within pharmacy practice community and hospital pharmacy practices tends to focus on the practical outcomes of this workforce addition. It mostly addresses the ‘WHAT’ of service delivered by pharmacy technicians. Literature on the ‘HOW’ of their role development in practice is scarce. Furthermore, it seems difficult for most pharmacy technicians to effectively fulfil this professional role.Objective: This qualitative study explored factors influencing role development of pharmacy technicians in community and hospital pharmacies.Methods: On site, individual and small-group interviews were conducted with pharmacy technicians (n = 10), and two colleagues: pharmacists (n = 7) and pharmacy assistants (n = 6). Interviews were based on a semi-structured interview guide. Participants were asked to describe specific incidents and organisational, relational and pharmaceutical care perspectives, illustrative of the process of developing and implementing the pharmacy technician role. Template analysis was used to develop a list of codes representing themes identified in the data.Results: Five interrelated themes influenced development and implementation of the pharmacy technician role. Two of them were at a more contextual level: (a) experiencing a lack of vision on added value of the new role within the field of pharmacy and (b) learning climate. The other three were related to personal interactions between staff members: (c) role expectations and organisational fit, (d) personal traits of pharmacy technicians and (e) support of pharmacy technicians through task delegation and role enhancement.Conclusions: The data showed that development and implementation of pharmacy technician roles is a complicated process. A detailed plan for addressing and remediating the five identified themes is important to promote role development of pharmacy technicians.

Northern Ireland has a comprehensive bTB programme which faces many challenges. The main subject of this dissertation, chronic bTB herds, is one such example, bringing together many factors which interact in a complex manner providing a difficult backdrop in which to apply control policies. The challenges faced include insufficiencies in diagnostic techniques, programme fatigue among farmers who feel a lack of empowerment about their circumstances and an agricultural industry evolving in ways which potentially make disease control more difficult. However, even though the challenges are great it will only be through logical and well thought out science, including social science, that bTB eradication can be achieved.

Activation of oncogenes in cancer cells causes endogenous DNA replication stress (RS). RS is defined as errors during DNA-replication and is present in the vast majority of cancer cells. As a result, cancer cells are sensitive to additional DNA damage and heavily rely on the intra S-phase checkpoint for survival. Therefore, RS-inducing chemotherapeutics have been the mainstay of anti-cancer treatment for decades. Moreover, inhibitors against key players of the intra S-phase checkpoint, ATR and CHK1, are currently evaluated in clinical trials. Despite these developments, resistance to RS-inducing drugs is a major problem. This suggests that some cancer cells can resist potential severe levels of RS. However, the underlying mechanisms are not well understood. To identify the strategies employed by cancer cells to resist therapy, we studied the response of individual cancer cells to RS-inducing drugs. We found that cancer cells exhibit elevated levels of E2F regulated genes. This allows cell proliferation despite RS. Furthermore, we discovered that the frequently occurring oncogenic version of RAS transcriptionally dampens an important control pathway. As a result, cancer cells with oncogenic RAS are sensitive to RS-inducing drugs. Last, using a novel strategy, we uncovered that high expression of a subset of genes potentially protects cancer cells from drugs that induce RS. Overall, the data presented in this thesis imply a key role for transcription in resistance and sensitivity to drug-induced RS.

There has been a slight shift in animal disease surveillance from input-based standards towards output-based standards, meaning that it is not prescribed what needs to be done, but rather what must be achieved. An objective and standardized assessment of the outputs of differently designed control programmes (CPs) is needed and therefore the aim of this thesis was to develop and test a generic output-based framework to determine the probability of freedom from Bovine Viral Diarrhea virus (BVDV) infection in cattle herds. The in this project developed STOC free framework (Surveillance analysis Tool for Outcome-based Comparison of the confidence of FREEdom), consists of a data collection tool (STOC free DATA) and a model to estimate herd-level freedom from infection (STOC free MODEL). Elements of BVDV CPs in six European countries that contribute to confidence of freedom from BVDV were described and qualitatively compared. Many differences in the context and design of BVDV CPs were found. CPs were either mandatory or voluntary, resulting in variation in risks from (in)direct animal contacts. Risk factors such as cattle density and the number of imported cattle varied greatly between countries. Differences were also found in testing protocols and definitions of freedom from infection. A systematic search and meta-analysis of risk factors for the presence of BVDV in cattle herds in Europe was performed to obtain generic estimates that could be used as default input data for STOC free MODEL. Data from 18 observational studies were analyzed by a random effects meta-analysis and significant higher odds were found for dairy herds compared to beef herds, for larger herds, for herds that participated in shows or markets, for herds that introduced cattle and for herds that had contact with cattle of other herds at pasture. STOC free DATA was developed to evaluate data availability and quality and to collect actual input data required for STOC free MODEL. Initially, the tool was developed for assessment of freedom from BVDV in six Western European countries and was then further generalized to enable inclusion of data for other cattle diseases and for use throughout Europe. STOC free DATA includes a wide range of variables that could reasonably influence confidence of freedom from infection, including those relating to cattle demographics, risk factors for introduction and characteristics of CPs. STOC free MODEL, a Bayesian Hidden Markov model, was applied to BVDV field data from CPs based on ear notch samples from newborn calves in four study regions to estimate the probability of herd level freedom from BVDV. The probability of freedom from BVDV for dairy herds that are free according to each study region’s CP was predicted to be very high ranging from 0.98 to 1.00, regardless of the use of default or country-specific priors. The STOC free model can be used to evaluate and improve BVDV CPs and to determine whether they comply with output-based regulations of the EU. The tool is currently evaluated by other research groups for application to other infectious cattle diseases such as Johne’s disease and salmonella.

Plasmid-mediated dissemination of antibiotic resistance among fecal Enterobacteriaceae in natural ecosystems may contribute to the persistence of antibiotic resistance genes in anthropogenically impacted environments. Plasmid transfer frequencies measured under laboratory conditions might lead to overestimation of plasmid transfer potential in natural ecosystems. This study assessed differences in the conjugative transfer of an IncP-1 (pKJK5) plasmid to three natural Escherichia coli strains carrying extended-spectrum beta-lactamases, by filter mating. Matings were performed under optimal laboratory conditions (rich LB medium and 37°C) and environmentally relevant temperatures (25, 15 and 9°C) or nutrient regimes mimicking environmental conditions and limitations (synthetic wastewater and soil extract). Under optimal nutrient conditions and temperature, two recipients yielded high transfer frequencies (5 × 10–1) while the conjugation frequency of the third strain was 1000-fold lower. Decreasing mating temperatures to psychrophilic ranges led to lower transfer frequencies, albeit all three strains conjugated under all the tested temperatures. Low nutritive media caused significant decreases in transconjugants (−3 logs for synthetic wastewater; −6 logs for soil extract), where only one of the strains was able to produce detectable transconjugants. Collectively, this study highlights that despite less-than-optimal conditions, fecal organisms may transfer plasmids in the environment, but the transfer of pKJK5 between microorganisms is limited mainly by low nutrient conditions.

Precise regulation of gene expression ensures oocytes and embryos accomplish the dynamic events required for successful oocyte maturation and early embryo development. However, most studies of gene expression regulation have focused on the mouse. In this respect, we used the cow as an alternative mammalian model to study gene expression regulation during oocyte maturation and early embryo development. We described dynamic changes in gene expression and poly(A) tail length during bovine oocyte maturation and early embryo development. As a result of these changes, the reference genes most suitable for gene expression normalization are different when different reverse transcription priming methods are employed. On the other hand, epigenetic alterations and differential regulation of transcription factors affect gene expression at the transcriptional level in embryos. One of the major epigenetic events-XCI, is initiated at morula stage in female bovine embryos. However, the initiation of XCI does not lead to complete down-regulation of all X-linked genes. The expression of the transcription factor CDX2 in bovine embryos is up regulated after exposure to LPA. But LPA stimulation does not change embryo constitution in terms of percentages of cells committed to one or another lineage. Overall, a better understanding of gene expression regulation during ocoyte maturation and early embryo development broadens our fundamental knowledge of mammalian embryonic development and will improve our understanding of the requirements for cell lineage determination and pluripotency, and the short and longer term consequences of epigenetic disturbances resulting from sub-optimal maternal health and nutrition or in vitro fertilization and embryo culture conditions.

In preclinical experimental animal research, inter-individual variability in phenotypic response is a major source of within-group variability that may negatively affect the power of animal experiments and the reproducibility of their outcomes. Individual differences however are often not actively accounted for in the design of animal experiments. The importance of accounting for this variability has become especially acknowledged in animal models that study the underlying mechanisms and/or treatment of psychiatric diseases. In humans, the susceptibility to develop psychopathologies and the response to treatment is known to vary greatly between patients. Incorporating this variation in animal models may not only make these models more representative, but could also improve our understanding of the underlying mechanisms that are involved in this differential susceptibility. In the present thesis we focused on inter-individual variability in habituation of anxiety responses in mice. The overarching aim of the studies was twofold: To improve our understanding of this variability by mapping its expression on a behavioral and physiological level in three commonly used mouse inbred strains. And: to explore whether systematic incorporation of such inter-individual variability in the analysis and design of our experiments would affect the quality of our results. By applying a multidimensional, individual-based analysis approach we identified two behavioral response types which were displayed by individuals of all three inbred strains. We subsequently demonstrated how controlling for this type of variability may affect the quality of experimental results, and may facilitate a more comprehensive understanding of inter-individuality in anxiety responses in mice.

Livestock feces with antimicrobial resistant bacteria reaches the farm floor, manure pit, farm land and wider environment by run off and aerosolization. Little research has been done on the role of dust in the spread of antimicrobial resistance (AMR) in farms. Concentrations and potential determinants of antimicrobial resistance genes (ARGs) in farm dust are at present not known. Therefore in this study absolute ARG levels, representing the levels people and animals might be exposed to, and relative abundances of ARGs, representing the levels in the bacterial population, were quantified in airborne farm dust using qPCR. Four ARGs were determined in 947 freshly settled farm dust samples, captured with electrostatic dustfall collectors (EDCs), from 174 poultry (broiler) and 159 pig farms across nine European countries. By using linear mixed modeling, associations with fecal ARG levels, antimicrobial use (AMU) and farm and animal related parameters were determined. Results show similar relative abundances in farm dust as in feces and a significant positive association (ranging between 0.21 and 0.82) between the two reservoirs. AMU in pigs was positively associated with ARG abundances in dust from the same stable. Higher biosecurity standards were associated with lower relative ARG abundances in poultry and higher relative ARG abundances in pigs. Lower absolute ARG levels in dust were driven by, among others, summer season and certain bedding materials for poultry, and lower animal density and summer season for pigs. This study indicates different pathways that contribute to shaping the dust resistome in livestock farms, related to dust generation, or affecting the bacterial microbiome. Farm dust is a large reservoir of ARGs from which transmission to bacteria in other reservoirs can possibly occur. The identified determinants of ARG abundances in farm dust can guide future research and potentially farm management policy.

Any antimicrobial use (AMU) in humans and animals selects for antimicrobial resistance (AMR) and responsible AMU should therefore be promoted both in human and veterinary medicine. Insight into current AMU in companion animal clinics is necessary to be able to optimise antimicrobial (AM) prescribing behaviour. The objective of this study was to describe systemic AMU in 44 Dutch companion animal clinics over a 3-year time period (2012-2015), using retrospectively collected data. The number of Defined Daily Doses for Animals (DDDAs) per month and per clinic were calculated from prescription data for total, 1st, 2nd and 3rd choice AMU (classification according to Dutch policy on veterinary AMU). Time trends, seasonality and the influence of potential determinants (e.g., the number of dogs, cats and rabbits per clinic and other clinic characteristics) were explored using statistical modelling. Overall, the findings show that total AMU decreased over time and a shift in used classes of antimicrobials towards more 1st choice AMs was visible. Mean total AMU decreased from 1.82 DDDA/year in 2012-2013 to 1.56 DDDA/year in 2014-2015. Aminopenicillins, with and without clavulanic acid, accounted for the largest group of antimicrobials used; 38.7% (2012-2013), 40.2% (2013-2014) and 39.3% (2014-2015) of total AMU, respectively. Strong seasonal differences in AMU were found, with highest AMU in July-August and lowest in February-March. The distribution of different animal species per clinic appeared to affect AMU as well. In clinics with a larger proportion of dogs, 2nd choice AMU was significantly higher, whereas in clinics with a larger proportion of rabbits, 2nd choice AMU was significantly lower. Despite the decrease of AMU during the study period, there is still room for improvement left, especially with regard to the antimicrobial classes prescribed. According to Dutch classification of veterinary AMU, 1st choice AMs should be used as empirical therapy. A decrease in 2nd (might select for ESBL-producing bacteria) and 3rd choice AMU (i.e. fluoroquinolones and 3rd generation cephalosporins) should be aimed for.

Background Hyperammonemia can result in hepatic encephalopathy, which in severe cases eventually can lead to coma and death. In dogs, congenital portosystemic shunts (CPSS) are the most common cause for hyperammonemia. Conservative treatment consists of a protein modified diet, nonabsorbable disaccharides, antibiotics, or some combinations of these. Sodium benzoate (SB) and sodium phenylbutyrate (SPB) both are used in the acute and long-term treatment of humans with hyperammonemia caused by urea cycle enzyme deficiencies. Both treatments are believed to lower blood ammonia concentrations by promoting excretion of excess nitrogen via alternative pathways. Objectives To evaluate the efficacy and safety of PO treatment with SB and SPB on hyperammonemia and clinical signs in CPSS dogs. Methods Randomized, double-blind, placebo-controlled crossover trial. Concentrations of blood ammonia and bile acids were measured in CPSS dogs before and after a 5-day treatment with SB, SPB, and placebo. A wash-out period of 3 days was used between treatments. A standard questionnaire was developed and distributed to owners to evaluate clinical signs before and after each treatment. Results Blood ammonia concentrations were not influenced by any of the treatments and were comparable to those observed during placebo treatment. In addition, SB and SPB treatment did not result in improvement of clinical signs. Adverse effects during treatment included anorexia, vomiting, and lethargy. Conclusions and clinical importance Based on our results, we conclude that SB or SPB are not useful in the conservative treatment of hyperammonemia in dogs with CPSS.

Over the course of more than 500 million years, the kidneys have undergone a remarkable evolution from primitive nephric tubes to intricate filtration-reabsorption systems that maintain homeostasis and remove metabolic end products from the body. The evolutionarily conserved solute carriers organic cation transporter 2 (OCT2) and organic anion transporters 1 and 3 (OAT1/3) coordinate the active secretion of a broad range of endogenous and exogenous substances, many of which accumulate in the blood of patients with kidney failure despite dialysis. Harnessing OCT2 and OAT1/3 through functional preservation or regeneration could alleviate the progression of kidney disease. Additionally, it would improve current in vitro test models that lose their expression in culture. With this review, we explore OCT2 and OAT1/3 regulation from different perspectives: phylogenetic, ontogenetic, and cell dynamic. Our aim is to identify possible molecular targets both to help prevent or compensate for the loss of transport activity in patients with kidney disease and to enable endogenous OCT2 and OAT1/3 induction in vitro in order to develop better models for drug development.

Objectives The occurrence and zoonotic potential of antimicrobial resistance (AMR) in pigs and broilers has been studied intensively in past decades. Here, we describe AMR levels of European pig and broiler farms and determine the potential risk factors. Methods We collected faeces from 181 pig farms and 181 broiler farms in nine European countries. Real-time quantitative PCR (qPCR) was used to quantify the relative abundance of four antimicrobial resistance genes (ARGs) [aph(3′)-III, erm(B), sul2 and tet(W)] in these faeces samples. Information on antimicrobial use (AMU) and other farm characteristics was collected through a questionnaire. A mixed model using country and farm as random effects was performed to evaluate the relationship of AMR with AMU and other farm characteristics. The correlation between individual qPCR data and previously published pooled metagenomic data was evaluated. Variance component analysis was conducted to assess the variance contribution of all factors. Results The highest abundance of ARG was for tet(W) in pig faeces and erm(B) in broiler faeces. In addition to the significant positive association between corresponding ARG and AMU levels, we also found on-farm biosecurity measures were associated with relative ARG abundance in both pigs and broilers. Between-country and between-farm variation can partially be explained by AMU. Different ARG targets may have different sample size requirements to represent the overall farm level precisely. Conclusions qPCR is an efficient tool for targeted assessment of AMR in livestock-related samples. The AMR variation between samples was mainly contributed to by between-country, between-farm and within-farm differences, and then by on-farm AMU.

Significance Neuroblastoma is a childhood malignancy that originates from neural crest cells. We present a single-cell transcriptome and localization atlas of the developing adrenal gland and identify seven different cell clusters that make up the adrenal medulla. Their transcriptomic profiles were used to generate gene signatures that were compared to neuroblastoma samples. The neural crest-derived “SCP” gene signature score anticorrelates with disease severity based on staging and poor prognosis (molecular) markers, while a high score was associated with higher overall survival rates., Neuroblastoma is the most common extracranial solid tumor and accounts for ∼10% of pediatric cancer-related deaths. The exact cell of origin has yet to be elucidated, but it is generally accepted that neuroblastoma derives from the neural crest and should thus be considered an embryonal malignancy. About 50% of primary neuroblastoma tumors arise in the adrenal gland. Here, we present an atlas of the developing mouse adrenal gland at a single-cell level. Five main cell cluster groups (medulla, cortex, endothelial, stroma, and immune) make up the mouse adrenal gland during fetal development. The medulla group, which is of neural crest origin, is further divided into seven clusters. Of interest is the Schwann cell precursor (“SCP”) and the “neuroblast” cluster, a highly cycling cluster that shares markers with sympathoblasts. The signature of the medullary SCP cluster differentiates neuroblastoma patients based on disease phenotype: The SCP signature score anticorrelates with ALK and MYCN expression, two indicators of poor prognosis. Furthermore, a high SCP signature score is associated with better overall survival rates. This study provides an insight into the developing adrenal gland and introduces the SCP gene signature as being of interest for further research in understanding neuroblastoma phenotype.

Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.

Delayed sleep–wake phase disorder (DSPD) is the most frequently occurring intrinsic circadian rhythm sleep–wake disorder, with the highest prevalence in adolescence. Melatonin is the first-choice drug treatment. However, to date melatonin (in a controlled-release formulation) is only authorised for the treatment of insomnia in children with autism or Smiths-Magenis syndrome. Concerns have been raised with respect to the safety and efficacy of melatonin for more general use in children, as melatonin has not undergone the formal safety testing required for a new drug, especially long-term safety in children. Melatonin is known to have profound effects on the reproductive systems of rodents, sheep and primates, as well as effects on the cardiovascular, immune and metabolic systems. The objective of the present article was therefore to establish the efficacy and safety of exogenous melatonin for use in children with DSPD, based on in vitro, animal model and clinical studies by reviewing the relevant literature in the Medline database using PubMed. Acute toxicity studies in rats and mice showed toxic effects only at extremely high melatonin doses (>400 mg/kg), some tens of thousands of times more than the recommended dose of 3–6 mg in a person weighing 70 kg. Longer-term administration of melatonin improved the general health and survival of ageing rats or mice. A full range of in vitro/in vivo genotoxicity tests consistently found no evidence that melatonin is genotoxic. Similarly long term administration of melatonin in rats or mice did not have carcinogenic effects, or negative effects on cardiovascular, endocrine and reproductive systems. With regard to clinical studies, in 19 randomised controlled trials comprising 841 children and adolescents with DSPD, melatonin treatment (usually of 4 weeks duration) consistently improved sleep latency by 22–60 min, without any serious adverse effects. Similarly, 17 randomised controlled trials, comprising 1374 children and adolescents, supplementing melatonin for indications other than DSPD, reported no relevant adverse effects. In addition, 4 long-term safety studies (1.0–10.8 yr) supplementing exogenous melatonin found no substantial deviation of the development of children with respect to sleep quality, puberty development and mental health scores. Finally, post-marketing data for an immediate-release melatonin formulation (Bio-melatonin), used in the UK since 2008 as an unlicensed medicine for sleep disturbance in children, recorded no adverse events to date on sales of approximately 600,000 packs, equivalent to some 35 million individual 3 mg tablet doses (MHRA yellow card adverse event recording scheme). In conclusion, evidence has been provided that melatonin is an efficacious and safe chronobiotic drug for the treatment of DSPD in children, provided that it is administered at the correct time (3–5 h before endogenous melatonin starts to rise in dim light (DLMO)), and in the correct (minimal effective) dose. As the status of circadian rhythmicity may change during long-time treatment, it is recommended to stop melatonin treatment at least once a year (preferably during the summer holidays)., Highlights • Melatonin improves sleep onset without serious adverse effects in youths with DSPD. Change th text after the fourth bullet into: Melatonin is an efficacious and safe chronobiotic drug for the treatment of DSPD in youths. • Melatonin for indications other than DSPD, dose not cause relevant adverse effects. • Long term melatonin treatment does not impair sleep, puberty, and mental health. • Melatonin is an efficacious and safe chronobiotic drug for the treatment of DSPD in youths. • Melatonin should be administered at the correct time and in the minimal effective dose.

Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine., Pre-clinical cell culture models capturing the heterogeneity of childhood kidney tumours are limited. Here, the authors establish and characterise an organoid biobank of tumour and matched normal organoid cultures from over 50 children with different subtypes of kidney cancer.