For the low-molecular-weight sample, a mono-exponential decay is observed, which becomes slightly faster with shear, indicating restrictions of the polymer chain motion. For the high-weight sample, a much faster bi-exponential decay is observed, indicative of entanglements. Both components in this decay become longer with shear. This implies that the free polymer segments between entanglements become effectively longer as a result of shear.The local dynamics in polymer melts and the impact of external shear in a Couette geometry have been investigated using rheological nuclear magnetic resonance (NMR). The spin-spin relaxation time, T The dynamics and ordering of macromolecules under shear is of general interest for the understanding of the dynamic behavior as well as in the processing of polymer materials; a combination of both orientation and deformation of the polymer is expected . Common The combination of various nuclear magnetic resonance (NMR) experiments with flow or shear, termed rheo-NMR, is one intriguing way to obtain non-destructive insight into flow pattern or into molecular information of materials under shear and flow conditions . It has 2) has been widely used to characterize polymer chain mobility, because it is sensitive to the slow motions of polymer chain segments [2 because of the lowered residual dipolar couplings. The basis of the present investigation is the shear-induced changes of T2.A typical property of polymers is the presence of entanglements, which manifest themselves in heterogeneity of the polymer mobility which can be observed in relaxation NMR and other methods. Transverse or spin-spin relaxation  of relatively narrow molecular weight distribution has been investigated under shear in situ. The experiments have been performed on a Bruker Avance I NMR spectrometer  using a 7 T widebore magnet operating at a Larmor frequency of 300 MHz for protons. The spectrometer is equipped with a Bruker Micro 2.5 microimaging system with three orthogonal gradients of 1 T/m maximum gradient strength for each channel. This is applied to control the adjustment of the Couette cell and to check the flow profile using flow NMR using the same cell in an imaging probe head with a birdcage resonator with 10 mm inner diameter. For better sensitivity, linewidth and radio-frequency performance, a 10 mm Bruker high-resolution NMR probe head has been used in conjunction with an in-house-built rheo-NMR accessory. In the 10-mm NMR tube, a 7.7 mm rotor made from Vespel has been inserted. This rotor is connected to a drive shaft inserted from the top of the magnet containing ceramic ball bearings for smooth and centric rotation. From the top the shaft is driven by a servomotor, at a rotation rate which is controlled from an in-house-built rheo-NMR controller, which had been used previously for high-temperature investigations .2, the Carr-Purcell-Meibom-Gill (CPMG) sequence has been applied [To measure the transverse relaxation time, T applied ,16. ThisPoly(dimethylsiloxane) (PDMS) samples of 410 g/mol and 63,000 g/mol have been purchased from Alfa Aesar  and used as received without any further purification or treatment. According to the supplier, the materials exhibit a polydispersity below 1.15. After inserting the samples in the Couette cell, they were weakly stirred to remove any possible air bubbles and then rested for at least 30 min before the start of the experiments. Flow profiles for the two samples that had been investigated are depicted in Rheological tests have been performed on a Haake MARS II rheometer  applying a cone-and-plate geometry with a gap of 0.034 mm. No shear-rate dependence of the viscosity has been observed for either sample; the viscosities are 1.8 mPa·s and 9470 mPa·s for the low-molecular weight and high-molecular weight samples, respectively. In shear-step experiments, relaxation effects have been observed for the sample of 63,000 g/mol only, as can be seen in 2, is more than one order of magnitude longer for the sample of low molecular weight, indicating much more molecular motion. This sample is more of an oligomer. The other significant difference is that the low-molecular-weight sample exhibits a mono-exponential decay indicative of homogeneous molecular mobility. In contrast, the high-molecular-weight sample exhibits a bi-exponential decay, clearly showing heterogeneity of the molecular motion, which is explained by the presence of entanglements. The molecular weight is well above the reported entanglement molecular weight of PDMS of about 35,000 g/mol [2.In 00 g/mol . Entangl2 is observed, while for the sample of higher molecular weight, a significant extension of the decay is observed. In Besides the fact that the low-molecular-weight sample exhibits a mono-exponential decay and the high-molecular-weight sample exhibits a bi-exponential decay, there is an opposite trend in the effect of shear. For the low-molecular-weight sample, a slight shortening of T2 for the low-molecular-weight sample, which does apparently not exhibit any entanglements, indicates that the polymer chains align under the shear present, which leads to a closer packing of the polymer chains and thus restricted mobility of chain segments. On the other hand, for the sample in which signatures of entanglements are observed, T2 increases, showing that there is more polymer chain mobility; the chain segments, which can freely move, become longer. Two scenarios are feasible: either polymer chains are pulled out of entanglements or entanglements are squeezed together and become more localized. Both scenarios lead to effectively longer chain segments between the entanglements and thus an overall larger mobility. However, as there is no change in the intensity fractions for the two components observed, the fraction of entangled polymer remains constant and thus the second scenario describes the finding better. This will be the subject of further investigations.The slight reduction of T2, has been observed for two samples of different molecular weight as a function of the shear rate applied in a Couette cell. A low-molecular-weight sample shows a shortening of T2 that can be interpreted as chain ordering. This effect is not observed for a polymer of high molecular weight exhibiting signatures of entanglements. In that case, with shear an increase of the chain mobility is concluded from the NMR relaxation. The fact that the relative amplitudes of the two relaxation components remain unchanged implies that the entanglements become more localized and the effective chain segments, which can move freely, become longer. The chain ordering as observed for the low-molecular-weight sample is not excluded, however the dominating effect for the chain dynamics is the larger chain segments moving freely.Rheo-NMR has been applied as a measure of polymer chain mobility to investigate the effect of continuous external shear on the polymer chain dynamics. The spin-spin relaxation time, T


The recent advancements in rapid and affordable DNA sequencing technologies have revolutionized diagnostic microbiology and microbial surveillance. The availability of bioinformatics tools and online accessible databases has been a prerequisite for this. We conducted a scientific literature review and here we present a description of examples of available tools and databases for antimicrobial resistance (AMR) detection and provide future perspectives and recommendations. At least 47 freely accessible bioinformatics resources for detection of AMR determinants in DNA or amino acid sequence data have been developed to date. These include, among others but not limited to, ARG-ANNOT, CARD, SRST2, MEGARes, Genefinder, ARIBA, KmerResistance, AMRFinder, and ResFinder. Bioinformatics resources differ for several parameters including type of accepted input data, presence/absence of software for search within a database of AMR determinants that can be specific to a tool or cloned from other resources, and for the search approach employed, which can be based on mapping or on alignment. As a consequence, each tool has strengths and limitations in sensitivity and specificity of detection of AMR determinants and in application, which for some of the tools have been highlighted in benchmarking exercises and scientific articles. The identified tools are either available at public genome data centers, from GitHub or can be run locally. NCBI and European Nucleotide Archive (ENA) provide possibilities for online submission of both sequencing and accompanying phenotypic antimicrobial susceptibility data, allowing for other researchers to further analyze data, and develop and test new tools. The advancement in whole genome sequencing and the application of online tools for real-time detection of AMR determinants are essential to identify control and prevention strategies to combat the increasing threat of AMR. Accessible tools and DNA sequence data are expanding, which will allow establishing global pathogen surveillance and AMR tracking based on genomics. There is however, a need for standardization of pipelines and databases as well as phenotypic predictions based on the data. The science of infectious disease, along with other medical and biological specialties, is undergoing rapid change brought on by the advent of affordable whole genomic sequencing (WGS) technologies –3. TheseSalmonella genomes alone are in the public domain with hundreds being added weekly. A complete genomic DNA sequence represents the highest practicable level of structural detail on the individuating traits of an organism or population. As such, it can be used to provide more reliable microbial identification, definitive phylogenetic relationships, and a comprehensive catalog of traits relevant for epidemiological investigations. This is having a major impact on outbreak investigations and the diagnosis and treatment of infectious diseases, as well as the practice of microbiology and epidemiology  between the presence of known AMR genes or mutations and Minimum Inhibitory Concentration (MIC) of several antimicrobials at or above the epidemiological cut-off value or clinical breakpoint for resistance. High sensitivity of >87%, defined by the ability to correctly identify AMR determinants associated with an antimicrobial resistance phenotype (true positive rate) and high specificity of >98%, defined by the ability to correctly identify the absence of AMR determinants in an antimicrobial susceptible phenotype (true negative rate), have been observed depending on the bacterial species analyzed . –18. Enteanalyzed –21. The Campylobacter in the USA, where WGS revealed that the rising trend was driven by nine different resistance alleles, six of which had never been detected in Campylobacter previously and would not have been found easily using PCR  with much greater precision compared to phenotypic tests . It has in silico AMR detection is performed by using a search algorithm to query input DNA or amino acid sequence data for the presence of a pre-determined set of AMR determinants contained in AMR reference databases , bitbucket (https://bitbucket.org/account/user/genomicepidemiology/projects/DB) and similar.The high level of agreement between phenotype and genotype coincides with the development of new and updated versions of bioinformatics tools to predict AMR, and the maturation of well-curated AMR gene databases. In principle, atabases . This caThe various bioinformatics software can process sequence data either as reads or as assemblies . Generalin silico AMR prediction is critically dependent on the availability of accurate AMR databases. AMR reference databases can be subdivided into solutions specialized for detection of resistance to specific antimicrobials and/or in specific bacterial species or in solutions allowing detection of virtually any possible AMR determinant in any DNA/amino acid sequence. Besides their focus area, AMR reference databases have important differences which users need to acknowledge for choosing the optimal fit-for-purpose database. First, AMR reference databases differ for criteria of inclusion of entries. For example, entries in CARD must have been published in scientific literature. In ResFinder, publication is not a strict requirement. Genes must have a GenBank number and expert review of the GenBank entries. Also, the types of entries differ across databases, with most databases including AMR genes and only a few databases including mutations of chromosomal genes mediating AMR. Finally, the available AMR databases differ regarding the format of the entries , the possibility of download, and the availability and frequency of curation  organized by the European Commission Joint Research Center, the challenges of designing a benchmarking strategy for assessing bioinformatics tools to detect AMR determinants was discussed . Here, sn others . In contn others .The quality and type of sequence data are also important factors. This also needs to mimic a real-life scenario where genomes will differ in error rates, read lengths, and read quality and may be raw reads or assemblies. The robustness of bioinformatics tools will differ in performance when dealing with low quality genomes and assemblies compared to optimal conditions , 65.Prior to executing a benchmarking exercise, the reference AMR classes need to be determined as to whether all known or acquired determinants will be included, or only specific mechanisms such as certain enzymes, efflux pumps, mutations/single nucleotide polymorphisms (SNPs), upregulated or downregulated genes or porins. Ideally, the bioinformatics tools should enable the detection of all known determinants if used for surveillance or guiding clinical treatment unless the scope is different and agreed upon .Since the main objective of a benchmarking exercise is to assess the ability of the bioinformatics tool to provide reliable analysis of AMR gene content, it is vital that the concordance is high between the reference result and the expected outcome . The senAssessing the performance of bioinformatics tools is often based on a comparison between the genotypic and phenotypic results and a calculation of the specificity, sensitivity, positive predictive (PPV) and negative predictive values (NPV), accuracy [Simple Matching Coefficient (SMC)] and performance [Matthew's Correlation Coefficient (MCC)] followed by a comparison of these parameter's between the different bioinformatics tools , 66.Surprisingly, only a few studies have benchmarked bioinformatics tools against each other to detect AMR determinants. 24 used two previously published pair-end Miseq datasets , 8 of 19Salmonella genomes and 164 E. coli genomes of which a large proportion harbored upregulated ampC-mediated resistance to extended spectrum cephalosporins. ResFinder provided the highest accuracy, SMC and performance, MCC predicting resistance in the E. coli genomes and GeneFinder for Salmonella genomes. The correlation to phenotypic susceptibility testing was for Salmonella spp. Ninety percent for all bioinformatics tools but higher for GeneFinder specifically for fluoroquinolones. The accuracy, SMC revealed to be lower in E. coli than testing Salmonella for all bioinformatics tools due to the bias of the E. coli dataset containing a high number of upregulated ampC genotypes not predicted by any of the bioinformatics tools (Another study (ENGAGE)  evaluatecs tools . Hunt etcs tools . The ARIcs tools  and SRSTcs tools .Following the benchmarking described above, both the ResFinder and the KmerResistence bioinformatics tools have been updated. Thus, the Resfinder tool now includes a number of chromosomal point mutations such as those to detect resistance to colistin, fluoroquinolones, etc. Overall, the benchmarking exercises revealed that all bioinformatics tools evaluated performed almost similarly good but were affected by the type and quality of input data.In an assessment of the accuracy of NCBI's AMRFinder, a 2018 study by Feldgarden et al compared it with a 2017 version of ResFinder . AMRFindhttps://www.globalmicrobialidentifier.org/).Standardization of WGS procedures from DNA preparation to the final genome is paramount to ensure reliable prediction of AMR determinants for surveillance and clinical purposes. To ensure the production of reliable high quality genomic data, laboratories routinely performing WGS should participate in laboratory proficiency testing (PT) or external quality assurance systems (EQAS) , 68. Forin silico prediction of wgMLST and AMR determinants. The genomes and analysis were submitted to DTU for quality control assessment using closed genomes of the test strains as a reference. The quality control assessment was facilitated by an in-house developed PT QC pipeline measuring a large number of parameters. These included the numbers of reads after trimming, unmapped reads, map to the total reference DNA, reference chromosome, reference plasmids; proportion of reads that map to reference chromosome; coverage of the reference chromosome and reference plasmids; depth of coverage of total DNA, reference chromosome, and reference plasmids; Phred quality score (Q score), total size and proportion of assembly map to the reference DNA, number of contigs including above a length above 200 bp, N50, and NG50. Underperformance was observed and reported in each trial mainly caused by laboratory contamination or poor performance.In 2014, GMI launched its first pilot PT in WGS lead by the DTU and US FDA to trial test the WGS platforms, procedures, test material and the functionality of the assessment pipeline . In 2015An important element of genomics as a tool for AMR surveillance and diagnostics is that, once data quality standards are met, the data set is platform-independent, discrete and portable. The analytical outputs and data sharing then become the most important considerations . A plethConcurrently with the vast amount of genomic data being produced, traditional antimicrobial susceptibility testing is being conducted in parallel on a large scale. Up until recently, it was only possible to submit and store DNA sequence data in the International Nucleotide Sequence Database Collaboration (INSDC), whereas all AST data was stored separately in closed local or national repositories. Furthermore, not all genomic data is submitted to the online open genomic repositories of INSDC and shared globally due to difficulties to submit, a lack of appreciation for its value, access to local or national repositories, fear of being data being published by others, or privacy of the data . Nonethehttps://www.ebi.ac.uk/ena/pathogens/home. Subsequently, the data could be visualized by using a developed Notebook tool integrated the Pathogen Data Portal to query and display all typing data including distribution of the phenotypic AST data enable a potential real time monitoring of AMR (At European Nucleotide Archive (ENA), a mechanism to host and link submitted genomic and AST data has been developed by the EU COMPARE partners and EMBL-EBI . Brieflyg of AMR . The advg of AMR –90.The National Center for Biotechnology Information (NCBI) is the US member of the INSDC and part of the United States National Institutes of Health, and houses hundreds of thousands of bacterial genomes from around the world. Sequences are submitted from global research studies, but the majority are from national public health surveillance programs with systematic sampling schema. With the expansion of WGS capacity, the number of genome submission is expected to rise soon to over 100,000 annually from US sources alone.https://www.ncbi.nlm.nih.gov/pathogens/) was developed. This resource is designed for exploring the genomic features of various bacterial pathogens. These include major foodborne and zoonotic pathogens, such as Salmonella enterica, Escherichia coli, and Campylobacter spp. Included in these datasets is a variety of metadata, including strain ID, source, date collected, geographical location, antimicrobial resistance, and more. This page was established in collaboration with GenomeTrakr, an international consortium of laboratories organized by the U.S. Food and Drug Administration (FDA) that collect and sequence bacterial strains from a variety of food and environmental sources . This suite of data dashboards is focused exclusively on analysis and visualization of AMR genes extracted from the complete genomes at the NCBI. ResistomeTracker was developed for the U.S. National Antimicrobial Resistance Monitoring System (NARMS) to better understand the epidemiological aspects of resistance by making the large amounts of resistome data accessible to a broad user audience. This includes the identification of new resistance determinants, differences in the prevalence of resistance genes among various food commodities, and geographical spread over time. Additionally, continuous updates to ResistomeTracker enable users to detect early resistance threats. ResistomeTracker allows for user-directed queries of the data that are informative for individual interests. Because it is linked directly to the NCBI pathogen database, it allows the user to begin a query with a specific resistance allele, and end with a phylogenetic analysis of related strains. It currently is focused on foodborne bacteria, but can be modified to exploit and genome for resistance gene content.To help make the resistance information accessible, the US Food and Drug Administration developed a tool called ResistomeTracker  coordinates the Antibiotic Resistance Laboratory Network (ARLN) to rapidly detect emerging resistance threats in healthcare, food and the community. Among many activities, this comprehensive network performs WGS for numerous pathogens, including all isolates of Salmonella in 2013 and has incorporated WGS data for Salmonella and Campylobacter in its reports since 2014. Online tools enable users to examine resistance trends at the genetic level using various query filters. These tools provide graphical visualizations of the genotypes behind changing resistance patterns over time by source and serotype.The National Antimicrobial Resistance Monitoring System (NARMS) is a long-standing program focused on bacteria transmitted commonly through food . NARMS iAs national resistance surveillance matures to better fit the One Health model, animal pathogens and environmental testing are beginning. In the US, the Department of Agriculture National Animal Health Laboratory Network (NAHLN) and the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) are starting to gather resistance information and WGS data on pathogens from food animals and companion animals, respectively. The US Environmental Protection Agency (EPA) conducts periodic water surveys that includes detection of resistance genes. While in the early stages, national public health surveillance programs using DNA sequence information will continue to expand and permit new associations to be inferred from resistomic analyses of the data.https://eur-lex.europa.eu/eli/dir/2003/99/oj) for Member States (MSs) to monitor AMR phenotypically by MIC determination in Salmonella, Campylobacter, and E. coli obtained from healthy food-producing animals and from food. The monitoring also include a specific monitoring of extended-spectrum beta-lactamase (ESBL)-, AmpC- and carbapenemase-producing Salmonella and indicator commensal E. coli stipulated in the Commission Implementing Decision 2013/652/EU of 12 November 2013 (http://data.europa.eu/eli/dec_impl/2013/652/oj). The data collection on human diseases including AMR from MSs is optimal and based on either MIC or disk diffusion and conducted in accordance with Decision 1082/2013/EU (http://data.europa.eu/eli/dec/2013/1082/oj).In Europe, its mandatory by law, Directive 2003/99/EC (Salmonella and indicator commensal E. coli from healthy food-producing animals and from food, has expressed an interest to replace the mandatory phenotypic MIC determination with WGS due to this already been implemented locally in the specific MSs. Thus, in the preparatory work of updating the Commission Implementing Decision 2013/652/EU coming into force in 2021, the preliminary draft of the technical specifications on harmonized monitoring of resistance in zoonotic and indicator bacteria from food-producing animals and food from EFSA suggested to allow replacing MIC determination with WGS combined with using the CGE ResFinder tool till 2025 (Salmonella and indicator commensal E. coli from healthy food-producing animals and from food and considered to be expended replacing all phenotypic MIC determinations as well as species identification. The resulting AMR determinant profile will be submitted to EFSA and used to predict the phenotype which will be reported in the European Union summary report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food. It will be optional for the individual MSs to also submit the DNA sequences and metadata data to ENA. It's believed that all MSs by 2015 have acquired WGS and conducing bioinformatics analysis of DNA sequences of single isolates for monitoring purposes.A number of MSs providing data for the specific monitoring of AmpC- and carbapenemase-producing ill 2025 . From 20https://www.danmap.org/, leading to a narrow pathogen spectrum that does not capture all relevant AMR genes. The majority of AMR genes may be present in the commensal bacterial flora of healthy humans and animals or the environment.Current AMR surveillance often focuses on few pathogens mainly based on passive reporting of phenotypic laboratory results for a few selected specific pathogens as in the Danish monitoring system, DANMAP Metagenomics techniques, using short-read next-generation sequencing data, benefit from the ability to quantify thousands of especially transmissible resistance genes in a single sample without any prior selection of which genes to look for. Moreover, it can provide additional information about the presence of bacterial species, pathogens and virulence genes and the data can be re-analyzed, if novel genes of interest are identified.It was recently shown that metagenomics is superior to conventional methods for AMR surveillance in pig herds , useful Metagenomics will sequence all DNA present in the sample including food and host DNA, which may result in low sensitivity. Quantitative PCR procedures, including large scale capture PCR methodologies have been developed, likely providing higher sensitivity . HoweverIn the future the application of metagenomics directly on samples from healthy and clinical ill individuals and animals as well as potential reservoir might results in the ultimate One Health surveillance of AMR allowing determination of all resistance genes and their context in all reservoirs. However, as for single isolates different pipelines and databases are also used for such metagenomics studies and there is a need for global standardization.ad hoc gene detection was the norm and PFGE was the typing tool of choice.An important advantage of using WGS technologies in detecting and tracking AMR is the opportunity to expand it to align with a One Health surveillance framework and allowing for exact comparisons across reservoirs. This cannot be done using WGS only on the phenotypic antimicrobial class level, but at the exact genetic mechanism level. This One Health goal has so far been impeded by the high cost of testing animal and environmental samples using classical methods based on metabolic and biochemical characterization. As the NGS technology becomes more affordable, it will become more common to use metagenomics to explore the potential role of different environments in the ecology of resistance. Thus, One Health monitoring is now poised to evolve into nucleotide surveillance of complex microbial ecosystems. And to the extent that the data can be generated and reported without delay, it appears that something analogous to a “weather map” of infectious diseases and resistance is possible. This was not practicable in the past, where The advancement in whole genome sequencing and the application of online tools for real-time detection of AMR determinants is essential for control and prevention strategies to combat the increasing threat of AMR. We identified a number of accessible tools available in the prediction of AMR determinants to support expanding to establish global pathogen surveillance and AMR tracking based on genomics. In addition, we identified a number of preceding requirements for a successful transition such as curated AMR databases ensuring a high concordance between pheno- and genotypes, benchmarking designs, PT schemes, sharing options etc. There is however, a vital need for standardization of pipelines and databases as well as phenotypic predictions based on the genomic data.RH and PM conceived, outlined and critically revised the manuscript. All authors wrote, read and accepted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antibody-mediated rejection (AMR) is one of the main problems after transplantation, partly due to the unclear aspect of the pathogenesis of AMR. Allograft rejection caused by antibodies can be mediated by different mechanisms. Classically, antibodies, produced by B-cells, induce acute rejection through the fixation and activation of the complement cascade, resulting in tissue injury . SearchiOncotarget, Wang et al. [HVEM/LIGHT/BTLA/CD160 polymorphisms and AMR after kidney transplantation. The authors used next generation sequencing (NGS) to evaluate the association between 41 single nucleotide polymorphisms (SNPs) of HVEM/LIGHT/BTLA/CD160 genes in 200 renal recipients, 69 with AMR and 131 that were considered stable. Wang et al did not found any significant association between these polymorphisms and AMR in renal transplantation.There have been many attempts to associate solid organ allograft outcomes with specific genetic variants . In factg et al.  explore et al, and in view of the relevant role of HVEM/LIGHT/BTLA/CD160 pathway in the immune response, we retrospectively analyzed the presence of SNPs in LIGHT/BTLA genes by NGS, due to their importance as HVEM ligands. For this purpose, 46 heart transplant recipients were analyzed, 23 with AMR and 23 matched controls without AMR. In this study, we have identified 7 SNPs in LIGHT/BTLA genes (Table Based on the study of Wang et al. and our research group in renal and cardiac transplant respectively, there is strong evidence suggesting the involvement of this pathway in immune regulation and graft rejection [HVEM/LIGHT/BTLA/CD160 polymorphisms in CR, without ruling out their possible role in AMR. It should be recalled that these genes are expressed in other cell types like B cells, NK cells, DCs, and macrophages, which are involved in AMR [HVEM/LIGHT/BTLA/CD160 polymorphisms in the progression of AMR and CR.Despite the negative results obtained by both Wang ejection , 7. A poejection . However


Here, we studied the effects of molecular chain length on the contact line movement in water/n-alkane/solid systems with different surface wettabilities. We used n-heptane (C7), n-decane (C10), and n-hexadecane (C16) as alkanes and α-quartz as the solid surface. We calculated the time-variation contact line moving velocity and also analyzed the jump frequency and the mean distance of the molecular displacement occurring within the contact line zone by molecular-kinetic theory. Molecular dynamics simulation results show that the contact line velocity decreases with increasing the chain length, originally caused by the decreasing the jump frequency and mean distance. These variations with the molecular chain length are related to the more torsions and deformations of the molecules with a longer chain length. In addition, the moving mechanism of the contact line on the same solid surface does not change at different molecular chain lengths, implying that the moving mechanism mainly depends on the three-phase wettability.The movement of the contact line in liquid-liquid-solid systems is a major phenomenon in natural and industrial processes. In particular,  The phenomenon of water displacing oil occurs in many processes such as dish and cloth washing and petroleum recovery , where tn-alkanes are widely occurring in the oil, soil pollution, and chemical industries [n-Alkane molecules have flexible chains that may be oriented [n-alkane chains [n-alkanes are presented as the mixtures of homologues with various molecular lengths. Therefore, studying the effects of molecular chain length on the contact line movement is crucial. Suggestions in regulating and controlling the contact line movement can be given from the perspective of changing the parameters of the molecular-kinetics theory in systems with different molecular chain length. Previous investigations have evidenced the parallel orientation of alkane chains [The dustries ,16. n-Aloriented  under sooriented . The cone chains . In indue chains  and the e chains ,20. HoweTherefore, we study here the effects of chain length on the contact line movement at a liquid-liquid-solid system with different surface wettabilities. The molecular dynamics simulation method is adopted, which is a widely used method capturing the movements of the atomic particles controlled by Newton’s laws of motion and is used in many applications ,22,23,24n-alkane/solid three-phase systems using the LAMMPS software. The two liquids are placed on a 28.4 nm × 6 nm α-quartz surface, as shown in n-Heptane (C7), n-decane (C10), and n-hexadecane (C16) are used as the alkane phase. The displacing side contains 16,245 molecules of H2O with transferable intermolecular potential with 3 points (TIP3P) model. Simulations are performed using periodic boundary conditions in the directions parallel to the silica surface, and using reflective boundary conditions in the directions vertical to the silica surface [α-quartz surface, the non-bridging oxygen atoms which are bonded to only one Si atom are attached to the hydrogen atoms [n-alkane and H2O molecules. This allows us to better assess the morphology of the molecules near the three-phase contact lines.Molecular dynamics simulations are implemented in the water/ surface . For theen atoms . The surF is added to drive the systems, then simulations are performed to reach a steady state in the systems. In the simulations, the time step is set to 1.0 fs and the moving process of the three-phase contact line is recorded with a period of 10,000 steps (0.01 ns). The system temperature is of 300 K in the NVT ensemble for different n-alkane systems.The simulations are firstly run for enough time to reach the equilibrium in the canonical (NVT) ensemble. Then a 6 nm × 6.4 nm wall is added from the left of the displacing side to drive the movement . We prevα-quartz atoms. Here, the cutoff radius (rcut) is set at 1.0 nm, which is acceptable and widely used [ijr is the distance between atoms i and j, ε is the energy parameter and σ the length scale.iq and jq are the charges of atoms i and j, Q is the energy-conversion constant, ε0 is the dielectric constant in vacuo.We adopt several models to simulate the interactions among atoms. For the added wall, we applied the adaptive intermolecular reactive empirical bond-order (AIREBO) potential model, whose form and parameters are given by Stuart et al. . We usedely used . The lonα-quartz atoms and atoms in liquid molecules are modeled using the hybrid potential model. The L-J potential parameters between the crossing atoms are calculated using the Lorentz-Berthelot mixing rule. The charges of n-alkane atoms are calculated by the DMol3 module of Materials Studio 7.0. These potential parameters have been verified [The interactions between verified ,22.ε in Equation (1). For simplicity, the constant E enables us to selectively increase or decrease the interactions between different types of atoms by writing the energy parameter in Equation (1) as E·ε. We assign different types of interactions as Ewater-solid and Ealkane-solid to represent the water-solid interactions and the n-alkane-solid interactions. In our simulations, the solid surface is both physically and chemically uniform. Therefore, the wettability provides a useful way to test the quality of the surface. Here, we consider the Ewater-solid of 1 and 0.2, the Ealkane-solid is 1 in both cases, and the resulting three-phase wettability (contact angle of water side) is 45° and 90°, respectively. The equilibrium contact angles are obtained by linear fitting of the n-alkane/water interface points [7, C10 and C16, the wettabilities are almost the same in different chain length systems; thus, we can neglect the small differences.We have found that the moving contact line has several typical mechanisms at hydrophilic, intermediate, and hydrophobic systems. Noteworthy, in the hydrophobic system the contact line is hard to be driven . Thus, he points . Since tz-direction by dividing the simulation regions into bins. The bin size Δz was σ/5 [σ is the length scale in Equation (1). We use the oxygen atoms to represent the water molecules because one water molecule has only one oxygen atom. Similarly, the carbon atoms are adopted to represent the alkane molecules. For oxygen atoms, the σ is 3.15 Å and for carbon atoms the σ is 3.50 Å. So the bins should be 0.63 Å and 0.70 Å, separately. z coordinates of H2O and n-alkane molecules are stored in bins. Then we calculated the number density distribution based on Equation (3):n(b) represents the number of elements in the bth bin and S is the projected area of the elements on the x-y plane.The characteristics of the liquid molecules near the solid surfaces are crucial in understanding the contact line movement. In particular, liquid molecules can be adsorbed onto the solid surface to form a high-density zone. Here we obtained the molecular density distribution along the  was σ/5 . Here σ z = 11.34 Å, z = 13.23 Å, and z = 16.39 Å, corresponding respectively to the distance 0.14 nm, 0.33 nm, and 0.65 nm away from the O-surface. This finding is in agreement with the results from Emami et al. [i et al. . The firi et al. . The secn-alkane molecules, we find that there are several peaks for the n-alkane adsorption, as shown in n-alkane/solid interactions. In addition, the chain-like structure of n-alkane is likely to modify molecular distribution in a confined space, particularly at the nano-scale level [n-alkane molecules and the solid surface. For the bulk phase out of the layers, the density distribution is nearly uniform. Furthermore, for a longer chain length, the number of alkane molecules per Å2 is smaller at the same z coordinate since the molecular volume is larger.For the le level . When thF. If F is too low, the system is static. If F is too high, a steady velocity cannot be reached by the simulation due to continuous interfacial deformations till the contact line disappeared. So a reasonable F is crucial to allow the contact line and water/n-alkane interface to reach a stable state, in which cases the contact line can achieve a steady velocity. Here, we defined a tripping force, which is the minimum force moving the contact line to reach a steady state. We firstly study the tripping forces in different systems, which are necessary to the first step of the contact line movement. The three-phase contact line is driven by an external driving force n-alkane interface points and the solid surfaces. Then we use these coordinates to calculate the displacements of the contact line. The average velocity is used because of the weak fluctuation in the velocity caused by the thermal effects of the molecules. The effects of chain length, from C7 to C16, on the time-variation of the velocity are shown in We analyzed the velocities of the three-phase contact line. The coordinates of the three-phase contact line at different times can be obtained by intersecting the fitting line of the water/−3 eV/Å is adopted to obtain the effects of the molecular chain length. Here, the time-variation velocity decreases with increasing the molecular chain length. However, the trend that the velocity increases firstly and then reaches to a steady value  is almost the same at different chain length, indicating that the moving mechanism of “Roll up” is the same at all of these cases [The contact line velocities in the hydrophilic cases (wettability of 45°) are shown in se cases .−3 eV/Å. Similarly, we observe that the time-variation velocity decreases with increasing the molecular chain length. The trend that the contact line velocity has a maximum value at the start-up stage and then decreases to a steady value  is almost the same at different chain length, indicating the same moving mechanism of “Piston” at all of these cases [For the intermediate cases (wettability of 90°), the results of the contact line velocities are shown in se cases .To further explain these phenomena, we analyze the driving force and the resistant force of the three-phase contact line at different chain length. The driving force in all systems is the unbalanced Young force generated by the external force . The resv and the dynamic contact angle dθ [Bk and T are the Boltzmann constant and the absolute temperature, respectively. n-alkane interfacial tension . K0 and λ represent the jump frequency and the mean distance of the molecular displacement occurring within contact line zone, respectively.In order to further explain the effects from a quantitative perspective, we try to obtain the parameters of molecular-kinetics theory at different molecular chain lengths. The molecular-kinetics theory  can descangle θd , as giveK0 and λ are directly obtained by reducing Equation (4) to a single exponential form, results are shown in K0 and λ are the basic characteristics of the system and are affected by the properties of the liquids and the substrate, which have nothing to do with the driving forces. An example of water/n-decane/solid system with the wettability of 90° is shown in λ in K0 changes for about 2 orders of magnitude, which verified the different mechanisms at different surface wettabilities [λ decreases as the molecular chain length increases; namely, from 0.65 nm to 0.48 nm at the wettability of 45°, and from 4.5 nm to 1.43 nm at the wettability of 90°. Additionally, the frequency K0 will also decrease with increasing the molecular chain length; from 918 × 106 Hz to 835 × 106 Hz at the wettability of 45°, and from 16.55 × 106 Hz to 1.7 × 106 Hz at the wettability of 90°. These decreases are resulted from the more torsions and deformations of the molecules at the nano-scale. The lower frequency and mean distance imply a larger resistance and show us why the velocity is slower at a longer molecular chain system. These results give us more physical explanations about the effects of the molecular chain length on the contact line movement in a liquid-liquid-solid system.In this work, we adopt the method by Renate Fetzer et al.  to obtaibilities . The mean-alkane/solid systems via molecular dynamics simulations. The time-variation contact line velocities are calculated and the key parameters (jump frequency and the mean distance) obtained by the molecular-kinetics theory are analyzed to get a deep understanding of the contact line movement from the physical viewpoint. Results show that a longer chain length makes the contact line harder to move and increases the tripping force, and that the time-variation contact line velocity decreases at the same driving force. In addition, a longer molecular chain length decreases the jump frequency and the mean distance owing to the more torsions and deformations, resulting in a larger contact line friction and therefore slows the contact line movement. However, the molecular chain length will not affect the moving mechanism of the contact line, implying that the moving mechanism mainly depends on the three-phase wettability. This work is helpful to give suggestions in regulating and controlling the contact line movement in systems with different molecular chain length.Surfaces with different wettabilities are adopted to study the effects of the molecular chain length on the contact line movement in water/


ColIV) and applied them for ultrasound molecular imaging of endothelial injury at early stage. Our results showed that the MBColIV, with a typical multi-peak particle distribution, was successfully constructed, which was confirmed by Alexa Fluor® 555-labeled secondary antibody. Ex vivo adhesion of microbubbles revealed that MBColIV can effectively and specially bind to the surface of balloon-injured carotid artery. The in vivo animal experiments showed ultrasound molecular imaging signals from carotid artery-injured rats administrated with MBColIV were significantly higher than those administrated with isotype control microbubbles. Histological staining of the left carotid common artery revealed that collagen IV was obviously exposed after endothelium denudation in balloon-injured artery. In conclusion, our current study provides an effective approach to detect vascular injury at the early stage and a potential platform for image-guided therapy to vascular injury.Cardiovascular intervention is a common treatment procedure for many cardiovascular diseases. But restenosis often occurs after these procedures, greatly discounting their long-term therapeutic effects. Early detection of endothelial denudation is helpful for the diagnosis and prevention of restenosis. Here, we fabricated targeted microbubbles by conjugating anti-collagen IV antibodies to the surface of biotinylated microbubbles (MB Cardiovascular diseases from atherosclerosis are responsible for the major cause of mortality worldwide , could improve long-term survival and reduce late myocardial infarction and recurrent unstable angina requiring rehospitalization for patients with non-ST-elevation myocardial infarction  and applied them for ultrasound molecular imaging of endothelial injury at early stage . Biotinylated MBs (MBbiotin) were obtained by mechanically vibrating for 30 s. The resulting MBbiotin were washed with phosphate buffered saline (PBS) solution twice to eliminate excess unincorporated lipids by centrifuge at 400 g for 3 min. Three microgram of avidin per 107 MBs was then incubated with the MBbiotin dispersion for 30 min at room temperature, followed by rinsing three times to remove unreacted avidin . Then either 2.5 μg of biotinylated goat anti-rat Type IV Collagen antibody or biotinylated goat IgG isotype control antibody  was added to per 108 MBs. After incubation for 30 min at room temperature, MBColIV or isotype control MBs (MBCtrl) were collected by centrifugation. Non-targeted MBs (MBN) without conjugated primary antibody were also prepared.Biotinylated, lipid-shelled MBs were prepared as previously described .To confirm the conjugation of Type IV Collagen antibody to the surface of MBs, Alexa Fluor® 555-labeled anti-goat secondary antibody  was added to MBColIV, MBN were used as the blank control. The morphology of the MBs were examined by using bright-field and fluorescence inversion microscopy . Particle size, size distribution and concentration of MBs were determined by using the Accusizer 780 Optical Particle Sizer  with a 0.5 μm diameter detection limit.To analyze the characteristics of the MBAll animal procedures were performed in accordance with the principles outlined in the Guide for the Care and Use of Laboratory Animals and approved by Animal Care and Use Committee. Adult male Sprague–Dawley rats , weighing 400–500 g, underwent the rat carotid artery balloon injury as previously described  and transferred to liquid nitrogen for flash-freeze. Vessels were sectioned to obtain arterial cross-sections across the length of the artery by cryostat microtome  and were examined histologically using routine hematoxylin-eosin (H&E) staining for morphometric analysis. Digital images were collected with bright-field by inversion microscopy .Bilateral carotid arteries were harvested following in situ perfusion fixation were completed, the LCCA was harvested and then opened longitudinally. The side of adventitia was adhered to a coverslip. Then MBs were added into a 6-well plate (2 × 107 MBs per well) and filled with PBS. Due to the static flotation nature of MBs, the coverslip with the artery coated faced downward to maximize interaction between the tissue and MBs for 5 min at room temperature. After that, the free MBs were removed by rinsing five times with PBS. To further determine the specificity of MBs adhesion, vessel tissues were pretreated with 25 μg/mL goat anti-rat Type IV Collagen antibody to block available receptors prior to MBColIV incubation. Digital images were obtained with bright-field by inversion microscopy  to count the number of attached MBs in five random fields of view.Attachment ability of MBs to collagen IV was evaluated by counting the number of adhering MBs. Briefly, after the rat carotid artery injury model and the 8 MBs/ml. Then 200 μl MBs suspension was injected intravenously through tail vein following by flushing with 50 μl PBS. Four minutes after MBs injection, 100 frames of images were captured to obtain a signal from adherent and freely circulating MBs. A continuous high-power destructive pulse  was then applied for 2 s to destroy these MBs. After 2 s, to allow the freely circulating MBs to replenish, another 100 frames of images were acquired, in which the ultrasonic signals were from any residual freely circulating MBs and tissue. To minimize the bias and test the specificity of these molecular imaging signals only resulting from adherent targeted MBs, MBColIV, and MBCtrl were administered in random order to all rats. Another 30-min delay was allowed to clear MBs from the preceding imaging session. As a control, ultrasound molecular imaging was also performed in normal rats to further assess the specificity of MBs adhesion. The acoustic imaging signals were analyzed by using commercially available analysis software . As previously described  using a L11-3 linear array transducer. Contrast imaging mode was applied in the ultrasound molecular imaging experiments. All imaging parameters were kept constant throughout the whole procedure as follows: frequency 5.6 MHz, depth 2 cm, gain 45 dB, frame rate 10 Hz, dynamic range 115 dB, and mechanical index 0.085. In order to reduce motion interference, both the transducer and the rats were fixed to maintain the same long axis cross section of carotid artery. The concentration of MBs suspension was adjusted to 2 × 10in situ perfusion fixation with PBS and 4% paraformaldehyde. Then artery samples were transferred to paraformaldehyde overnight, followed by ethanol dehydration and paraffin embedment. Artery samples were sectioned to obtain arterial cross-sections. After the process of deparaffinage and rehydration, slides were blocked and then incubated with the primary antibody goat anti-rat Type IV Collagen , followed by being rinsed with PBS thrice and then incubated with horseradish peroxidase-conjugated secondary antibodies. Finally, slides were incubated with diaminobenzidine for horseradish peroxidase reaction. In order to exhibit the tissue morphology of vascular wall, slides were counterstained with hematoxylin. Immunohistochemical images were acquired by using inversion microscopy  under bright field.Immunostaining analysis for collagen IV was performed to confirm that collagen IV was exposed through endothelium denudation. Briefly, LCCAs from normal artery group and balloon-injured artery group were excised, followed by t-test, and one-way ANOVA were used to compare among more than two groups. If the differences among these groups were significant for one-way ANOVA, the difference between the two groups was conducted by Student-Newman-Keuls test. The P < 0.05 indicated statistically significant difference.Data were analyzed by the statistic software SPSS 20.0 . As values were normally distributed, statistic difference between two groups was determined by Student's ColIV appeared a typical multi-peak particle distribution. There was no significant difference between MBColIV and MBN for the particle size and distribution . The mean diameter of MBColIV and MBN were 1.92 and 1.89 μm, respectively. The results demonstrated that the conjunction of avidin and biotinylated antibody to MBs surface did not change the particle size and distribution of MBs   Figure .in vivo, ultrasound molecular imaging was performed in the balloon-injured carotid artery group (n = 8) and the normal carotid artery group (n = 7). As shown in Figure ColIV were hardly attached to the normal artery wall  was higher than MBCtrl (0.57 ± 0.26 dB) in balloon-injured carotid artery group (P < 0.01)   Figure . Moreove) Figure .ColIV. In addition, collagen IV was also a component of the basement membrane for SMCs in tunica media, which had been reported in previous studies (Shekhonin et al., To confirm the results of ultrasound molecular imaging, LCCAs of the normal group and balloon-injured group were harvested and analyzed for the exposure of collagen IV after endothelium denudation. The results of immunohistochemical staining examination were presented in Figure Microbubble-based ultrasound molecular imaging is a promising approach in the disease diagnosis at the early stage. This is especially the case for vascular disorders since the microscale-targeted bubbles only exist in the blood and are not extravasate from the circulation. This feature makes it greatly standing out from other nanoparticle-based molecular imaging modalities, such as quantum dot-based optical molecular imaging and iron nanoparticle-based magnetic imaging resonance (Lobatto et al., ColIV by biotin-avidin bridging method. As shown in Figure in vitro tool in our static binding experiments such as culture plates, pre-coated with Matrigel, which has been taken to examine the binding of targeted particles to collagen IV (Dong et al., ex vivo binding experiments (Figure ColIV binding to the tissue of injured artery was nearly four times higher than MBCtrl, indicating MBColIV we obtained can be used as a potential ultrasound molecular imaging probe.In our study, we fabricated the targeted MBs Figure . The numin vivo in order to determine whether MBColIV could adhere to the site of endothelial denudation and be visually detected by a clinical ultrasound scanner or not. Surprisingly, we found stronger ultrasound molecular imaging signals from injured vascular wall after MBColIV injection but not MBCtrl, the former being nearly five times higher than MBCtrl. Interestingly, no obvious ultrasound molecular imaging signals were detected from the normal arterial wall after MBColIV or MBCtrl injection. These results suggested that MBColIV had a highly specific binding ability to these collagen IV targets and could function as an ultrasound molecular imaging probe to detect vascular injury. To take the drug-loaded advantages of MBs into consideration, it is easy to design the MBColIV into drug-loaded targeted MBs through carrying drugs (Lammertink et al., ColIV could be further functionalized as theranostics agent and would play a great role for ultrasound imaging-guided treatment of vascular injury.In our present study, we used intravenously injection for ultrasound molecular imaging There are several limitations in our study. The rat carotid artery balloon injury model described in our study imitate vascular injury by angioplasty. However, in clinical practice, restenosis occurs in patients who have occlusive atherosclerotic lesions after dilating the narrow lumen through angioplasty. Besides, to evaluate non-culprit stenosis severity, the instantaneous wave-free ratio, which is helpful for guiding PCI strategy, has been regarded as the promising method and used in patients with acute coronary syndrome and multivessel disease (Indolfi et al., ColIV by conjugating anti-collagen IV antibodies to the surface of MBs and confirmed the feasibility to apply them for ultrasound molecular imaging of endothelial injury at early stage. Our study provides an early diagnosis tool for restenosis due to endothelial damage and a potential platform for image-guided therapy for vascular injury.In summary, we have successfully fabricated MBXM and BZ initialed the project. XM and FY conducted the experiment and data analysis. BZ and FY provided equipment, materials, and experimental methods. XM wrote the manuscript. FY proofread the manuscript. All authors agreed with the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The relationships between dexamphetamine-induced dopamine release, dexamphetamine-induced positive psychotic symptoms using the Positive and Negative Syndrome Scale (PANSS), and childhood trauma using the Childhood Trauma Questionnaire (CTQ) were assessed using linear regression and mediation analyses, with childhood trauma as the independent variable, dexamphetamine-induced dopamine release as the mediator variable, and dexamphetamine-induced symptoms as the dependent variable. There was a significant interaction between childhood trauma and ventral striatal dopamine release in predicting dexamphetamine-induced positive psychotic symptoms , but a mediation analysis was not significant . There were no significant effects of dopamine release and childhood trauma on change in negative (p = 0.280) or general PANSS symptoms (p = 0.061), and there was no relationship between ventral striatal baseline D2/3R availability and positive symptoms (p = 0.368). This indicates childhood trauma and dopamine release interact to influence the induction of positive psychotic symptoms. This is not consistent with a simple sensitization hypothesis, but suggests that childhood trauma moderates the cognitive response to dopamine release to make psychotic experiences more likely.Childhood trauma is a risk factor for psychosis. Amphetamine increases synaptic striatal dopamine levels and can induce positive psychotic symptoms in healthy individuals and patients with schizophrenia. Socio-developmental hypotheses of psychosis propose that childhood trauma and other environmental risk factors sensitize the dopamine system to increase the risk of psychotic symptoms, but this remains to be tested in humans. We used [ It is a complex disorder with a multi-factorial aetiology involving both neurobiological alterations and environmental risk factors5. However, the relationship between neurobiological and environmental risk factors, and how they interact to increase the risk of schizophrenia and other psychotic disorders, is currently not known.Schizophrenia is among the leading causes of global disease burden and is associated with high mortality and morbidity11. Supporting the theory, administration of drugs that increase dopamine levels such as amphetamine or cocaine induces psychotic symptoms in both healthy people14 and patients with schizophrenia15. Molecular imaging studies have characterized the nature of the in vivo dopaminergic changes in schizophrenia, showing higher dopamine synthesis capacity and amphetamine-induced dopamine release with large effect sizes in patients with the disorder6. To our knowledge, seven of eight imaging studies found that schizophrenia is associated with increased striatal dopamine release induced by amphetamine23, and a further study found greater striatal dopamine release induced by acute stress exposure24. In addition, a number of these studies report a direct correlation between the magnitude of amphetamine-induced dopamine release in the striatum and amphetamine-induced psychotic symptoms in patients with psychosis18. However, individuals vary in the degree to which amphetamine induces psychotic symptoms14, suggesting that other factors may moderate individual vulnerability.The dopamine hypothesis has been a leading neurobiological theory of schizophrenia for several decades28). Large population-based studies also found positive dose–response relationships between trauma and subclinical psychotic symptoms as well as the persistence of psychotic experiences in the general population34, suggesting that childhood trauma may increase the risk for developing psychotic symptoms as well as a psychotic disorder. However, not everyone exposed to childhood trauma develops psychosis, suggesting biological vulnerability is also relevant.One of the most consistent environmental risk factors associated with schizophrenia is experiencing trauma in childhood and adolescence. Childhood traumas, such as neglect, physical, sexual and psychological abuse, increase the risk for psychosis in adulthood with odds ratios of 2.8–3.6  studies show that humans who reported lower maternal care during childhood have greater stress-induced dopamine release43, and participants who have experienced a greater number of traumatic events in childhood and higher levels of perceived stress have elevated levels of amphetamine-induced dopamine release44, both in the ventral striatum. In addition, increased dopamine synthesis capacity has been found in adults exposed to childhood adversity 45. However, to our knowledge, the relationships between childhood trauma, amphetamine-induced dopamine release and induced psychotic symptoms remain poorly understood. In particular, it remains unknown whether childhood trauma and amphetamine-induced dopamine release are associated with induced positive psychotic symptoms in isolation, whether childhood trauma interacts with amphetamine-induced dopamine release to induce positive psychotic symptoms (moderation effect) or whether childhood trauma influences induced psychotic symptoms through amphetamine-induced dopamine release (mediation effect).It has been proposed that childhood trauma, and other environmental stressors, sensitizes the mesostriatal dopamine system to the effects of later challenges, such as amphetamine, subsequently leading to the development of psychotic symptoms2/3R) availability. We focused on the ventral striatum as this region has previously been shown to be sensitive to the effect of childhood adversity on the magnitude of amphetamine-induced dopamine release46. We hypothesized that the interaction and/or mediation between childhood trauma and dopamine release capacity would predict dexamphetamine-induced positive psychotic symptoms, in light of the sensitization of the mesostriatal dopamine system described above38, and evidence showing that dopamine release in isolation is not correlated with an increase in amphetamine-induced positive symptom scores in young adults with hearing impairment47.In view of this, we tested the relationships between childhood trauma and dexamphetamine-induced striatal dopamine release to predict dexamphetamine-induced positive psychotic symptoms using both moderation and mediation models. We also tested whether these relationships were specific to striatal dopamine release capacity, or whether they were also related to baseline striatal dopamine-2/3 receptor  history of substance abuse/dependence as determined by the SCID-CV; (4) history of head injury with a loss of consciousness; (5) a family history of any psychiatric disorder in first- or second-degree relatives; (6) contraindications to PET scanning ; (7) positive urine drug screen, including for cannabis50.The study was approved by the West London & GTAC NHS research ethics committee (12/LO/1955). Participants were recruited via online and newspaper advertisements. All participants gave informed written consent to take part in the study after its full description. The inclusion criteria for the study were (1) age above 18 years and (2) capacity to give written informed consent. The exclusion criteria were (1) any past or current major medical condition; (2) history of a psychiatric disorder as determined by the Structured Clinical Interview for DSM-IV Axis 1 Disorders, Clinician Version (SCID-CV)11C]-(+)-4-propyl-9-hydroxynaphthoxazin (PHNO) PET scans on separate days: the first following a single oral dose of placebo, and the second following a single oral dose of dexamphetamine (500 µg/kg) in a fixed order. On the first scan day, participants completed the Childhood Trauma Questionnaire (CTQ). On both scan days, positive (psychotic) symptoms, negative symptoms and general psychopathology symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS) at baseline (pre-dosing with placebo or dexamphetamine), 60 min post-dosing, 120 min post-dosing, and after the scan (275 min post-dosing). Participants were blind to placebo/dexamphetamine administration. The rater was blind to childhood trauma load and dexamphetamine-induced dopamine release. All participants provided urine samples prior to each scan to screen for current recreational drug use. Participants were asked to abstain from smoking, drinking coffee, eating and using alcohol overnight (minimum 10 h) prior to their PET scans.The timeline of scanning day is shown in Fig. 52, a reliable and valid measure of childhood trauma in patients with psychosis53. The CTQ is a retrospective self-completed questionnaire covering the following five domains of childhood trauma: sexual abuse, physical abuse, emotional abuse, physical neglect and emotional neglect52. Sexual abuse is defined as “sexual contact or conduct between a child younger than 18 years of age and an adult or older person”. Physical abuse is defined as “bodily assaults on a child by an adult or older person that posed a risk of or resulted in injury”. Emotional abuse is defined as “verbal assaults on a child’s sense of worth or well-being or any humiliating or demeaning behaviour directed toward a child by an adult or older person”. Physical neglect is defined as “the failure of caretakers to provide for a child’s basic physical needs, including food, shelter, clothing, safety, and health care”. Emotional neglect is defined as “the failure of caretakers to meet children’s basic emotional and psychological needs, including love, belonging, nurturance, and support”. In the short version of the CTQ, each domain includes five items  “I got hit so hard by someone in my family that I had to see a doctor or go to the hospital”; (2) “I was punished with a belt, a board, a cord, or some other hard object”; (3) “I believe that I was physically abused”; (4) “I got hit or beaten so badly that it was noticed by someone like a teacher, neighbor, or doctor”; (5) “People in my family hit me so hard that it left me with bruises or marks”). Participants have to rate the frequency to which they have been exposed to each item . For each participant, the sum of all the 25 items in the CTQ was used in the analyses.Childhood trauma was assessed with the self-completed CTQ 25-item short form54. Each of the 30 items is accompanied by a specific definition as well as detailed anchoring criteria for all seven rating points. These seven points represent increasing levels of psychopathology from 1 (absent) to 7 (extreme)54. The symptoms assessed for the positive dimension are delusions, conceptual disorganization, hallucinatory behaviour, excitement, grandiosity, suspiciousness/persecution and hostility. The symptoms assessed for the negative dimension are blunted affect, emotional withdrawal, poor rapport, passive/apathetic social withdrawal, difficulty in abstract thinking, lack of spontaneity & flow of conversation, stereotyped thinking. The symptoms assessed for the general dimension are somatic concern, anxiety, guilt feelings, tension, mannerisms & posturing, depression, motor retardation, uncooperativeness, unusual thought content, disorientation, poor attention, lack of judgement & insight, disturbance of volition, poor impulse control, preoccupation and active social avoidance. For each participant, the sums of the seven sub-items for positive symptoms, seven sub-items for negative symptoms and 16 sub-items for general symptoms were calculated and used in the analyses for the positive, negative and general symptoms, respectively.Positive, negative and general psychopathology symptoms were assessed during both placebo and dexamphetamine scans using the PANSS. The PANSS is an established clinician-rated scale used for measuring the severity of psychotic and related symptoms based on a 30-item standardized interview11C]-(+)-PHNO. All participants (n = 24) had a baseline PET scan and a PET scan following administration of dexamphetamine on a separate day in a fixed order. For the dexamphetamine PET scans, 500 µg/kg dexamphetamine was administered orally 3 h before [11C]-(+)-PHNO administration, so that the expected time of peak drug levels coincided with the scan time55. Previous studies have shown robust changes in dexamphetamine-induced dopamine release using this approach56. Dynamic emission data were acquired continuously for 90 min after the administration of the radiotracer. The dynamic images were then reconstructed using a filtered back-projection algorithm into 31 frames  with a 128 matrix, a zoom of 2.6 and a transaxial Gaussian filter of 5 mm.PET images were acquired using a Siemens Biograph HiRez XVI PET scanner . A low-dose computed tomography scan was first obtained for attenuation and model-based scatter correction followed by the injection of a single intravenous bolus of 0.020–0.029 µg/kg -(+)-PHNO:Bavail is the proportion of D2/3Rs available to be bound by PHNO (i.e. the fraction of receptors not bound by endogenous synaptic dopamine), fND is the free fraction of PHNO in the brain and 1/KD the affinity of ligand for the target. BPND was obtained by kinetic modelling with a simplified reference tissue model63, using the whole cerebellum as a reference region due its low content of dopaminergic neurons64. For each subject, we measured the magnitude of dexamphetamine-induced dopamine release bilaterally in each striatal sub-region. Specifically, this was quantified as the reduction in BPND from the baseline condition (BPNDBase) to the post-dexamphetamine condition (BPNDAmph), expressed as a percentage of BPNDBase:Regional time activity curves (TACs) were obtained by applying individual parcellations to the realigned dynamic images. Our outcome measure of interest was non-displaceable binding  to investigate whether clinico-demographic factors reported to impact dopamine release, such as gender, nicotine, alcohol, cannabis morphine or stimulants use70, were also related to CTQ scores in our sample. Our primary hypothesis was that dexamphetamine-induced positive psychotic symptoms could be predicted by the interaction between childhood trauma and dexamphetamine-induced dopamine release, in accordance with previously described models71.Statistical Package for the Social Sciences (SPSS) version 24 was used for all statistical analysis . A two-tailed significance alpha level of 0.05 was used for all tests. In order to examine potential confounds, we used Pearson’s correlation coefficient, independent ND), childhood trauma (CTQ score), and the interaction between these variables (ΔBPND × CTQ score). Our primary hypothesis was that the coefficient term (β) associated with the interaction term would be significantly greater than zero.First, we tested whether childhood trauma or dopamine release capacity were correlated with dexamphetamine-induced symptoms using Pearson’s correlation coefficient. Next, we investigated whether the interaction between these two variables was a significant predictor of positive psychotic symptoms, by running a linear regression model, which included dexamphetamine-induced change in positive psychotic symptoms as the dependent variable, and the following predictor (independent) variables: ventral striatum dopamine release capacity .Second, we tested the specificity of the results by conducting additional multivariate regression models. To test whether our hypothesized interaction effect was specific for dopamine release , we repeated our models after substituting baseline Dhttp://www.processmacro.org/index.html)72. Effect sizes were estimated using 5000 bias corrected bootstrap samples. Mediation was deemed to be significant if 0 was not contained within the 95% bootstrap confidence intervals of the indirect effect, testing the combined effects of paths a (CTQ to dexamphetamine-induced dopamine release) and b (dexamphetamine-induced dopamine release to dexamphetamine-induced positive psychotic symptoms).Third, in order to examine whether the relationship between childhood trauma and positive psychotic symptoms is mediated by dopamine release, we performed a mediation analysis with childhood trauma as the independent variable, ventral striatal dexamphetamine-induced dopamine release as the mediator variable, and dexamphetamine-induced positive symptoms as the dependent variable, using the ‘Model 4’ template implemented in the PROCESS (version 3) SPSS package (11C]-(+)-PHNO PET scan and a scan after oral dexamphetamine on a separate occasion  age = 23.4 (4.20) years). One male participant was excluded before data analysis due to a positive urine drug test for cannabis, as cannabis has been shown to influence D2/3 binding potential68. Participant demographics, PANSS positive scores and substance use are shown in Table ND relative to the baseline scan (t(23) = 14.29, p < 0.001), indicating significant dopamine release following dexamphetamine (Table t(22) = 6.32, p < 0.001) (Table 73 or 34.8 (9.73)74). However, they are lower than those reported in patients with psychosis (mean (SD) of 40.7 (11.4)75) or depression (mean (SD) of 40.5 (16.1)73 or 41.63 (10.23)74). The peak increase in PANSS positive was at 60 min post-dosing, with a mean (SD) increase of 1.44 (1.55). This is in line with increases reported in a previous study in healthy volunteers using amphetamine, which found a mean (SD) increase in PANSS of 1.6 (0.5) at 60 min post amphetamine dose76. We found no relationship between CTQ and age , and no differences between men and women’s CTQ scores (t(22) = −0.50, p = 0.621). All participants abstained from drugs, alcohol and nicotine on scan days, and an ANCOVA analysis showed no relationship between CTQ scores and current alcohol use  = 1.19, p = 0.294), past ecstasy  = 0.39, p = 0.543), cocaine  = 0.11, p = 0.741) and cannabis  = 0.37, p = 0.700) use. There were not enough participants who were smokers (n = 2) to make analysis of this a co-variable meaningful. No participants had a history of amphetamine use, heroin use or morphine use (see Table Twenty-four participants underwent a baseline (placebo) 1C-(+)-PHr = 0.348, p = 0.096, n = 24), nor between ventral striatum dopamine release capacity and dexamphetamine-induced positive psychotic symptoms . Unexpectedly, we found a negative correlation between childhood trauma load and ventral striatal dopamine release   = 6.915, p-value vs. constant model = 0.002). Importantly, the regression coefficient associated with the interaction term in this model was significantly greater than zero, indicating that the interaction between childhood trauma and dexamphetamine-induced dopamine release predicted dexamphetamine-induced positive psychotic symptoms  p = 0.004), nor did restricting the analysis of symptom induction to the combined ‘delusions’, ‘hallucinations’ and ‘suspiciousness’ items of the PANSS  p = 0.023).The general linear model GLM1) including dexamphetamine-induced positive psychotic symptoms as the dependent (outcome) variable , with three independent (predictor) variables  was superior to the null-model in predicting symptom scores , Sobel test’s p = 0.158). This indicates that there was no significant indirect effect of childhood trauma on dexamphetamine-induced positive symptoms (i.e. an effect mediated through dexamphetamine-induced dopamine release).The mediation analysis testing whether dopamine release mediates childhood trauma in predicting positive symptoms is shown in Fig. 2/3R availability  80. Human PET studies have also shown increased stress-induced dopamine release in the ventral striatum in people with history of low maternal care43, as well as increased amphetamine-induced dopamine-release in individuals with a greater number of traumatic events in childhood44 and individuals with hearing impairment47. Furthermore, positive correlations between amphetamine-induced dopamine release in the ventral striatum and plasma cortisol levels have also been observed in healthy individuals81. Oswald et al. found that the relationship between childhood traumatic exposure and dopamine release was mediated by perceived stress in adulthood, where higher levels of perceived stress were associated with higher ventral striatal dopamine responses to amphetamine44. This observation may explain the apparent inconsistency between our findings and these previous findings if there is lower perceived stress in adulthood in people with high CTQ scores in our sample. However, as we did no measure perceived stress in adulthood, this remains to be tested and highlights that it would be useful to measure perceived stress as well in future studies. Nevertheless, the interaction between childhood trauma load and ventral striatal dopamine release on the induction of psychotic symptoms indicates that a history of greater childhood trauma increases an individual’s vulnerability to the psychotogenic effects of striatal dopamine release, in line with cognitive schema-interpretation model of psychosis4.The interaction between childhood trauma load and ventral striatal dopamine release is in line with the large body of literature showing the role of the ventral striatum in stimulant-induced dopamine release after exposure to environmental stressors. However, we did not find any positive relationship between history of childhood trauma and induced striatal dopamine release—instead the relationship in our sample was negative  and change in positive symptoms97, whilst another study using [18C]-fallypride found an association between schizotypal traits and amphetamine-induced dopamine release in the head of the caudate but extending into the ventral striatum98. Two other studies found that patients with schizophrenia had a similar range of BPND reduction in the ventral striatum and sensorimotor striatum at 6–10 h following dexamphetamine administration99, and that patients with schizophrenia had mean (SD) percentage reduction of 17.6% (7.8) in the ventral striatum and 24.4% (7.2) in the sensorimotor striatum22. To our knowledge, none of the other studies of amphetamine-induced dopamine release have examined sub-regional differences in striatal dopamine release in schizophrenia21. The ventral striatum is the most sensitive region to amphetamine-induced dopamine release102. Interestingly, a study examining amphetamine sensitization in healthy individuals found that an initial dose of amphetamine caused dopamine release in the ventral striatum whilst further doses at 14 and 365 days elicited increased dopamine release in the ventral striatum relative to the initial dose, with sensitized dopamine release progressively extending to the dorsal caudate and putamen103. Furthermore, a longitudinal study of people scanned in the prodrome and then again when acutely psychotic showed progressive increases in dopamine synthesis capacity in the dorsal striatum104. Thus, these findings could suggest that the initial dopaminergic response is in the ventral striatum which then extends to the dorsal striatum with the development of a psychotic disorder, but further studies are needed to confirm this hypothesis. Lastly, the regional specificity of the findings to the ventral striatum might also reflect tracer-specific factors. [11C]-(+)-PHNO is more sensitive to amphetamine-induced dopamine release in the ventral striatum than D2/3R antagonist tracers such as [11C]-raclopride56, a property that may be related to its high affinity for the D3R, which is more abundant in the ventral compared to the associative striatum and [11C]-(+)-PHNO.Our finding that the effect is specific to the limbic striatum contrasts with those of a recent meta-analysis indicating that the main locus of increased dopamine synthesis capacity in schizophrenia is the associative striatum, and not the ventral (limbic) striatum105. Lastly, it is possible that a third variable that correlates with both dopamine release and CTQ mediates the relationship we observe. However, we found no evidence that clinico-demographic factors that have been reported to be related to dopamine release such as gender, nicotine, alcohol, cannabis morphine or stimulants70 are related to CTQ scores in our sample.The first limitation of this study is that all individuals were healthy volunteers, limiting the generalizability of these findings to patients with psychotic disorders. This may account for the relatively small increase in PANSS score following dexamphetamine. Further studies are therefore needed to examine the relationships between childhood trauma, dopamine release and positive psychotic symptoms in clinical populations, and to examine stress-induced dopamine release. Second, the study was cross-sectional and exposure to childhood trauma was assessed retrospectively via questionnaires, and could be influenced by recall bias. However, retrospective assessment of childhood trauma has been shown to be reliable in both healthy population and patients with psychosisChildhood trauma and ventral striatal dopamine release interact to influence the emergence of positive psychotic symptoms following dexamphetamine. This is not consistent with a simple dopamine sensitization model for the link between childhood trauma and psychosis risk, but could, instead, suggest that childhood trauma moderates the cognitive response to dopamine release to make psychotic experiences more likely.Supplementary informationSuppl.Figure1


Cross-border healthcare may promote the spread of multidrug-resistant microorganisms (MDRO) and is challenging due to heterogeneous antimicrobial resistance (AMR) prevention measures (APM). The aim of this article is to compare healthcare workers (HCW) from Germany (DE) and The Netherlands (NL) on how they perceive and experience AMR and APM, which is important for safe patient exchange and effective cross-border APM cooperation.n = 574) in hospitals in DE (n = 305) and NL (n = 269), using an online self-administered survey between June 2017 and July 2018. Mann-Whitney U tests were used to analyse differences between answers of German and Dutch physicians (n = 177) and German and Dutch nurses (n = 397) on 5-point Likert Items and Scales.A survey was conducted amongst HCW (p ≤ 0.001). German HCW were more optimistic about their potential role in coping with AMR (p ≤ 0.001), and scored higher on feeling sufficiently equipped to perform APM (p ≤ 0.003), although the mean scores did not differ much between German and Dutch respondents.Similarities between DE and NL were a high awareness about the AMR problem and the perception that the possibility to cope with AMR is limited (30% respondents perceive their contribution to limit AMR as insufficient). Especially Dutch nurses scored significantly lower than German nurses on their contribution to limit AMR  in their daily working routines. The observed differences reflect differences in local, national and cross-border structures, and differences in needs of HCW, that need to be considered for safe patient exchange and effective cross-border APM.The online version of this article (10.1186/s13756-019-0577-4) contains supplementary material, which is available to authorized users. Avoiding antimicrobial resistance (AMR) as well as limiting the spread of multidrug-resistant micro-organisms (MDRO) through AMR prevention measures (APM) is essential for the quality, safety and durability of healthcare and societal health , 2. CoreNational borders are no barrier for the spread of MDRO. Since the 2011 EU directive on the application of patients’ rights in cross-border healthcare, cross-border mobility of both patient and healthcare workers (HCW) between Germany (DE) and The Netherlands (NL) has steadily increased –19. As aPrevious studies performed within the EUREGIO have focused on differences in the organization of healthcare . The study was ethically approved by the ethical committee of the University of Twente (BCE18321).The study was performed in six hospitals, which were purposively sampled based on their location in north-eastern NL and north-western DE. The heterogeneous sample consisted of one large university hospital on each side of the border (DE: + − 1500 and NL: + − 1300 beds), as well as one smaller Dutch general hospital (+ − 700 beds) and three smaller German university hospitals (+ − 400–800 beds). Microbiological diagnostics was locally organised in all except one German hospital. In all participating hospitals, local guidelines on antibiotic prescribing were available in the form of (online) formularies. Expert consultations on medical microbiology, infectious diseases and hygiene were available by phone or in person. Because HCW are mainly responsible to perform APM, they were selected as the key-stakeholders. HCW consisted of physicians and nurses of relevant AMR departments (e.g. not psychiatry).After demographic questions, the survey addressed a variety of AMR-topics, mostly based on a valid and reliable AMR questionnaire . First, Screening diagnostics: the process of finding out if a patient carries resistant bacteria .Infection diagnosis: the diagnosis of an infection (present/absent).Treatment: the choice of antibiotics that meets both the patient’s diagnosis and the local antibiotic guidelines.Infection control: the implementation of suitable hygiene measures for infection and transmission prevention .APM were introduced to respondents as follows:The perceived importance of APM was questioned with one item. The perceived influence and perceived availability of resources, knowledge, and social support of colleagues and supervisor on APM was questioned with five items. The perceived influence and perceived availability of resources, knowledge, and social support of colleagues and supervisor were later combined for interpretation into a scale of “feeling sufficiently equipped” for the specific APM.The survey was originally designed in Dutch for the regional hospital, which was used as a pilot-test for the survey. Tests that were held with a nurse and physician to ensure comprehension and clarity of the questions resulted in small adaptions in wording. After translation by an official translation service to German, the German research team members adapted wordings to better fit the clinical context and jargon. The full survey can be found in Additional file The survey was developed and administered in Qualtrics, and consisted of 5-point Likert items . Respondents were informed of the voluntary nature of their participation and confidentiality was guaranteed.The survey was distributed by email or personal communication followed by snowball sampling with local differences due to practical matters . Reminders were sent twice, but could not be tailored to non-responders.p-values for the Mann-Whitney U tests are two-tailed  and a p-value < 0.05 was considered significant. Possible influence of demographic differences between the German and Dutch groups were considered by comparing Mann-Whitney U tests results with results of Analyses of Covariance (ANCOVA) on ranked responses for each item and scale with age, gender and years of hospital experience as covariates.Descriptive analyses were performed in SPSS (v24). As physicians and nurses have different responsibilities related to AMR , resultsp ≤ 0.001). German physicians were significantly younger (p ≤ 0.001). Dutch nurses were significantly more often female (p ≤ 0.001), were significantly older (p = 0.002), and had significantly more experience in the current hospital (p = 0.005). Completing the survey took respondents 16 min on average. The respondents of the two hospitals with the highest number of responses represented response rates of less than 19%.Respondent characteristics are presented in Table Results of the survey are presented in Table p ≤ 0.001), their hospital (p = 0.043) and their patients (p = 0.002), although German respondents also scored relatively high (lowest mean importance score is 4.1). Thus, both German and Dutch respondents perceived AMR as a problem on various levels, and Dutch respondents do so slightly more than German respondents.Most of the respondents (≥59%) perceived AMR as a problem for the general population, nursing homes, their hospital and their patients. Dutch respondents scored higher than German respondents on statements of AMR being a problem for the general population (p ≤ 0.001) and the admission of nursing homes (p = 0.006). Dutch respondents scored higher on the statement of the use of antibiotics by patients (p = 0.011) as a leading cause of AMR than German respondents.German respondents scored higher than Dutch respondents on statements of the leading causes of AMR being the use of antibiotics in farming animals (p ≤ 0.001) and on statements about antibiotic prescriptions according to guidelines (e.g. antibiotic prescriptions should be based on lab results (p ≤ 0.001), I am sufficiently informed about the diagnostic policy (p = 0.002), and broad spectrum antibiotics should not be provided when there is doubt of an infection (p ≤ 0.001)).German respondents scored higher on the statement that antibiotics are prescribed at the request of patients (p ≤ 0.001). This difference was mainly apparent for nurses, where the means differed more than for physicians .Notably, only 19% of all respondents totally agreed that he/she can sufficiently contribute to limit AMR, and 30% respondents perceive their contribution to limit AMR as insufficient. This is especially true for nurses (lower means than physicians in both countries). German respondents scored higher on the item about being able to sufficiently contribute to limit AMR than their colleagues from The Netherlands (All APM were deemed very important to limit AMR by most (≥67%) respondents . German respondents scored the importance of all APM higher than respondents from The Netherlands, although scores for APM importance were high for both groups (lowest mean importance of Dutch respondents was 4.5).p = 0.005, infection diagnosis p ≤ 0.001, and treatment p ≤ 0.001), although the mean scores did not differ much between German and Dutch respondents.German respondents scored also higher on the feeling of being equipped at their hospital for specific APM (screening diagnostics Both German and Dutch nurses scored feeling sufficiently equipped lower than physicians (lower mean scores) for most APM, although this was not statistically tested. This is less apparent when comparing the means in both groups (physicians-nurses) for infection control.The comparison of unadjusted (Mann-Whitney U tests) and adjusted  test results can be found in Additional file This study aimed to compare German and Dutch HCW in their perceptions of AMR and prevention measures. This was done in order to create understanding of the problem urgency and to learn how HCW perceive their potential contribution to tackle the AMR problem through daily work routines. Understanding and comparing HCW’ perspectives on AMR and APM between countries where patient and HCW mobility is promoted, is essential for safe patient and HCW exchange, and effective cross-border cooperation.S. aureus from cases of bacteraemia: DE: 9.1% vs. NL: 1,5% and VRE/E. faecium: DE: 16.5% vs. NL: 1,4%) [Especially Dutch nurses felt less able to contribute sufficiently to limit AMR, as reflected in their lower mean score. The resistance rates of several MDRO are higher in German hospitals than in Dutch hospitals  . These lL: 1,4%) , 48, whiL: 1,4%) . Dutch HL: 1,4%) –55. ThusAs the awareness in both Dutch and German HCW in this study is considerably higher compared to similar studies , 56, andAstonishingly, only few HCW from both countries perceived their contribution to limit AMR as sufficient. Although German respondents felt slightly more optimistic about their contribution to limit AMR than their Dutch colleagues, their mean score is far from optimistic (3.5).Therefore, AMR prevention strategies in both countries should primarily focus on the awareness of how HCW can contribute to preventing the (cross-border) spread of MDRO. Studies have shown that improved APM over time, which can only be realized by empowered individual HCW, have led to a regional/national stabilisation or even reduction of MDRO prevalence , 59, 60.Special attention is required for empowering nurses in APM, since nurses are less confident about their role in diagnostics, diagnosis and treatment, as also reflected in this study’s results , 61–64. Germany and The Netherlands both have very developed healthcare systems, but the two systems differ considerably from one another in organisational, regulatory and financial structures , 73. PreFurthermore, AMR outcomes and APM cooperation in a cross-border setting are not only influenced by HCW’ perceptions and actions, but also by the complex interplay of organisational, regulatory and financial structures that shape a healthcare system . These seHealth has the potential to support and improve synchronisation AMR education, communication, and surveillance and performance feedback in a cross-border region, as has been successfully shown before in AMR studies , 78–81. This study used a purposive sample of hospitals in the EUREGIO and thus might not represent other cross-border regions, since every cross-border region has its own healthcare system structure and dynamics and its own AMR biotope , 83.Response rates were low, even for the two hospitals that provided the most responses (≤19%). This is most likely attributable to the fact that AMR and APM are not HCW’ core business. Therefore, only HCW with an interest in AMR/APM might have participated (i.e. selection bias), which might have influenced the results to be more positive than they actually are. HCW that do not have that much AMR/APM experience will likely answer more negatively on questions such as feeling sufficiently equipped . This wFurthermore, German and Dutch respondents varied significantly on nearly all demographic characteristics. However, the analyses adjusted for age, sex and years of hospital experience showed that only for a small number of questions the observed differences in HCW’ perspectives could be  explained by demographic differences.Other limitations relate to the use of Likert items. Central tendency bias might have occurred by respondents avoiding choosing the extreme response categories (scores 1 & 5) . We do nThe survey used was based on a validated questionnaire, used elements from health authorities’ recommendations on APM , 3–8, 38Despite these limitations, we do believe that this survey proved useful for a primary identification of HCW’ perspectives. This study can be seen as an essential step towards safer patient exchange and improved cross-border cooperation, since the cross-border AMR problem has, to our best knowledge, not been studied before from the HCW’ perspective.Both German and Dutch HCW are aware of the AMR problem, but both perceive their influence to limit AMR as insufficient. HCW do acknowledge the importance of APM  they perform in their daily working routines to limit AMR, but do not feel sufficiently equipped to do so. Therefore, AMR strategies should not primarily focus on emphasizing the relevance of APM, but should rather focus on empowering HCW in their working routines by providing them with the tools, knowledge and skills they need to limit AMR.Because of robust national healthcare structures, adaptive solutions are essential to tackle the challenges caused by AMR on a regional level. APM should be tailored to work in regional or even local settings, and need to be implemented by committed HCW. Thus, developing and implementing (cross-border) APM requires a comprehensive understanding of the contexts in which they will be implemented and the people that will execute the strategies (i.e. HCW). The similarities and differences between German and Dutch HCW as found in this study, can serve as a primary identification of factors that need to be considered for cross-border APM cooperation.Additional file 1:Survey on AMR and APM. AMR/APM survey (DOCX 15 kb)Additional file 2:Table S2a. Statement responses of (i) all respondents, (ii) German/Dutch physicians, and (iii) German/Dutch nurses, including p-values of differences between nationalities. Table S3a. APM responses of (i) all respondents, (ii) German/Dutch physicians, and (iii) German/Dutch nurses, including p-values of differences between nationalities. Full results on AMR (2a) and APM (3a) in the form of percentages per answer category. (DOCX 33 kb)Additional file 3:Table S2b. Ranked ANCOVA  results of (i) all respondents, (ii) German/Dutch physicians, and (iii) German/Dutch nurses, including p-values of differences between nationalities. Table S3b. Ranked ANCOVA  results of (i) all respondents, (ii) German/Dutch physicians, and (iii) German/Dutch nurses, including p-values of differences between nationalities. Comparison of unadjusted (Mann-Whitney U tests) adjusted ) results. (DOCX 32 kb)