This update includes the addition of 1417 KS sequences, representing a major expansion of the taxonomic and functional diversity represented in the webtool database. The phylogeny-based KS classification scheme now recognizes 41 class and subclass assignments, including new type II PKS subclasses. Workflow modifications accelerate run times, allowing larger datasets to be analyzed. In addition, default parameters were established using statistical validation tests to maximize KS detection and classification accuracy while minimizing false positives. We further demonstrate the applications of NaPDoS2 to assess PKS biosynthetic potential using genomic, metagenomic, and PCR amplicon datasets. These examples illustrate how NaPDoS2 can be used to predict biosynthetic potential and detect genes involved in the biosynthesis of specific structure classes or new biosynthetic mechanisms.The Natural Product Domain Seeker (NaPDoS) webtool detects and classifies ketosynthase (KS) and condensation domains from genomic, metagenomic, and amplicon sequence data. Unlike other tools, a phylogeny-based classification scheme is used to make broader predictions about the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes in which these domains are found. NaPDoS is particularly useful for the analysis of incomplete biosynthetic genes or gene clusters, as are often observed in poorly assembled genomes and metagenomes, or when loci are not clustered, as in eukaryotic genomes. To help support the growing interest in sequence-based analyses of natural product biosynthetic diversity, here we introduce version 2 of the webtool, NaPDoS2, available at  These enzymes produce structurally diverse polyketides ranging from lipids to macrolides, which represent an important source of compounds for pharmaceutical and other biotechnological applications . Polyketecursors . PKSs haecursors  of whiche domain , 14. Sime domain , while oe domain . A centre domain , 17, 18,http://napdos.ucsd.edu/napdos2/, includes many updated features that improve the usability of the tool for natural product discovery. Here, we report database and pipeline modifications that provide broader taxonomic coverage, better resolution among functionally characterized PKSs, a new subclassification scheme for type II PKSs, an increased capacity for processing large datasets, and the enhanced detection of eukaryotic KSs. Statistical validation tests have been used to select parameters for optimizing sensitivity and specificity, including both detection and classification accuracy based on query sequence length. The upgraded webtool was used to demonstrate the utility of NaPDoS2 for predicting biosynthetic potential in genomic, metagenomic, and amplicon datasets.The NaPDoS2 website, available at As in the original release , NaPDoS2A). This page provides the option to select classes of specific interest for more detailed investigation (B), which is particularly useful when large numbers of domains are detected. Each BGC represented in the database is linked to a summary page that includes a representative structure and the classification of each KS or C domain within that BGC (C). The independent classification of each domain is particularly useful given that a single gene or BGC can contain multiple domain types (A) and a downloadable documentation and tutorial file.User interface upgrades include the addition of a “Domain Classification Summary” page that lists the total number of domains detected in the query data as grouped by their NaPDoS2 classification A. This ptigation B, which that BGC C. The inin types . New webin types A and a dThe primary goals for NaPDoS2 were to expand the KS database and classification scheme to include biosynthetic functions that were not represented in the original release, to supplement poorly populated classes, and to provide greater taxonomic coverage. One thousand four hundred seventeen new KS sequences were added, raising the database total to 1877 (average length 418 ± 49 amino acids), an increase of 308% . Most ofThe ability to predict PKS and NRPS biosynthetic potential based on KS or C domains distinguishes NaPDoS2 from other bioinformatic tools. The domain classification scheme is derived from KS sequence phylogenies and their relationships to established biochemical functions, gene architectures, and structural features of the compounds produced. While not all classes and subclasses are monophyletic, functionally coherent clades form the basis of the classification scheme. Phylogenies generated from the updated KS database allowed us to establish 41 class and subclass assignments associated with type I and II PKSs and FASs . This recis-AT PKSs and several protist (Phyla Amoebozoa and Apicomplexa) and metazoan  PKSs, including those linked to characterized natural products from Caenorhabditis elegans  and 14 subclasses in the NaPDoS2 release. Example structures associated with each KS type are shown in With the classification scheme established, a simplified reference tree was generated using 414 sequences representing all class and subclass assignments . Broadly elegans  and Dictscoideum . The sevcis-AT class comprises KSs associated with the canonical assembly line PKSs  and now includes four subclasses of which two [olefin synthase and tandem enoyl-CoA hydratase (ECH)] are new to NaPDoS2  maintains a modular organization, with multiple enzymatic domains on a single protein, yet instead of functioning as an assembly line these enzymes catalyze an iterative series of elongation steps  derived from polyene chains , partially reducing, and NR iPKS subclasses (Iterative bclasses . These sbclasses . Their pbclasses . NR iPKSbclasses .trans-AT PKSs form multiple clades in the reference tree (trans-AT PKSs (0) typically follow an NRPS module, lack the catalytic histidine required for elongation, and are proposed to facilitate the transfer of the growing polyketide to the next module  were not assigned subclasses and are instead annotated as “type II aromatic unclassified” in NaPDoS2.Type II KSs function as a heterodimer, with the KSα subunit responsible for the iterative elongation of a nascent poly-β-keto chain, the KSβ subunit determining the chain length , and both subunits mediating the first ring cyclization reaction , 40. Thetrans-AT PKSs. These KSs are associated with HMGS or HCS (3-hydroxy-3-methylglutaryl coenzyme A synthase) gene cassettes that encode several monodomain proteins including a decarboxylating KS that can be resolved in the type II KS phylogeny . An. Antranshylogeny . Noniterhylogeny , 46. Thehylogeny , with thhylogeny . Similarhylogeny , 48, 49.hylogeny , 49. TheA and B), which encompasses enzymes that catalyze decarboxylating or nondecarboxylating Claisen-like condensation reactions for the synthesis and degradation of fatty acids and polyketides from all kingdoms of life . Negative controls (308 sequences) were selected from condensing enzyme families falling immediately outside the NaPDoS2 clades and similarly clustered at 50% amino acid identity (A). These negative controls, which include beta-ketoacyl-ACP synthases, ketoacyl-ACP synthases III, type III chalcone and stilbene synthases, thiolases, and sterol carrier protein-associated thiolases, were augmented with additional sequences (C) and KSs from type III PKSs retrieved from MIBiG 2.0 (D).Recognizing that computational prediction algorithms are seldom perfect, leave-one-out cross validation  and receidentity A. These equences  , demonstrating the superior performance of NaPDoS2 (AUC = 0.987) versus the original release (AUC = 0.978). ROC curves were further used to establish e-value cutoff points that maximize sensitivity and minimize false positives, identifying optimum values as 1e-8 for NaPDoS2 and 4e-11 for the original NaPDoS release. Although these cutoff values provided equivalent sensitivity (detection of true positives) for their respective algorithms, the highest achievable specificity obtainable for NaPDoS was 93.0%  versus 97.2% for NaPDoS2 . This improvement is most likely explained by the expanded database underlying the NaPDoS2 pipeline. Consequences of these statistically identified differences in real-world use cases are presented in the “NaPDoS2 applications” section below.Leave-one-out cross-validation scores were generated for positive and negative control sequences based on the e-values of their closest nonself match using both the original NaPDoS and NaPDoS2 KS reference databases. ROC curves were calculated from these scores to generate area under the curve (AUC) values A, demonsThe effects of partial KS sequences on detection and classification accuracy were evaluated using both full-length database sequences  and shorter overlapping subsets of 30, 50, 100, and 200 amino acids (aa) covering the entire length of these sequences. These subsets were designed to mimic domain fragments encountered in draft genomes, metagenomic assemblies, or KS amplicon sequences. Using the previously established 1e-8 cutoff for maximum sensitivity, NaPDoS2 detected 99% of the 200aa length subsequences as KS domains, of which 85% were correctly classified . DetectiBased on performance evaluation results, default NaPDoS2 parameters were set at an e-value of 1e-8 and a minimum alignment length of 200 amino acids; however, users may choose to adjust these settings for their individual datasets. Sensitivity declines for partial KS domains can be partially offset by decreasing BLAST stringency, at the cost of increasing false positives and misclassifications. PCR-generated amplicons can provide better sensitivity than similarly sized random sequence fragments but may still be too short to obtain accurate classifications. Confident assignments to poorly populated classes or subclasses are particularly challenging given the sequence diversity observed within highly populated classes and subclasses . Some amB).Large-scale performance evaluations targeting genomic, metagenomic, and amplicon sequence data from a variety of bacterial, fungal, metazoa, and environmental sources were conducted to demonstrate the utility of NaPDoS2 in identifying polyketide biosynthetic potential . These eFig. S3Salinispora genomes , 57Malas minimus , and sixconpicua , thus fuA single NaPDoS2 analysis can provide a simultaneous overview of both bacterial and eukaryotic polyketide and fatty acid biosynthetic potential in large metagenomic datasets. While it is not reliant on fully assembled BGCs, assembled contigs are recommended to avoid the reduced classification accuracies associated with short sequence reads  and S11.cis-AT modular  that functions as an aldolase/β-lactone synthase in salinosporamide A biosynthesis  and published reports. Sequences were extracted from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and KS domains identified using annotations from both published literature and MIBiG. In silico predictions were made using the NRPS/PKS prediction tool  according to experimentally verified functions and information derived from the associated gene or BGC. Metadata including BGC name, BGC type , MIBiG accession number, PubMed reference, source organism, and example product name and structure were recorded for each BGC. The complete NaPDoS2 amino acid sequence database (KS and C domains in FASTA format), BGC table, and domain metadata can be downloaded from the NaPDoS2 website.Experimentally validated PKS BGCs were selected from MIBiG (ion tool . KS domahttp://www.atgc-montpellier.fr/phyml/) with smart model selection enabled (Phylogenies generated from the 1877 KS database sequences (1417 new and 460 existing) were used to establish class and subclass assignments based on correspondence between functional annotation and tree topology. Sequences were aligned using MAFFT (v 7.017)  as imple enabled .The type II aromatic PKS subclasses were established using a concatenated alignment of the KSα and KSβ subunits and phylogenies generated using the methods described above. Subclasses were delineated based on phylogenetic groupings and features of the core polyketide structure including the length of the poly-β-ketoacyl intermediate, the carbon position of the first ring cyclization, and the type of starter unit. The accuracy of select NaPDoS2 KS classifications from the use case analyses were assessed by analyzing the associated contig with antiSMASH 6.0 , the NRPhttps://treeviewer.org/) and annotations added using Adobe Illustrator.A subset of 414 KS sequences representative of all class and subclass assignments were chosen to generate a KS reference tree  and S7. Database sequences and associated metadata are stored in a back-end MySQL database linked to the NaPDoS2 web portal through CGI-scripting as previously described . The traThe 1877 full-length amino acid sequences in the NaPDoS2 KS database were clustered at 50% identity using CD-HIT v 4.7 , yieldinDomain detection sensitivity and specificity were determined using BLASTP searches of full-length positive and negative control sequences against the NaPDoS and NaPDoS2 databases, excluding self-matches, to generate leave-one-out cross-validation values. Receiver operating characteristic (ROC) curves were constructed and AUC values calculated from these results using easyROC v 1.3 . EasyROChttps://github.com/spodell/NaPDoS2_website) was used to subdivide the 213 positive control KS sequences into test sets containing overlapping subsequences of 30, 50, 100, or 200 amino acids, each offset by a 10 amino acid sliding window start site. These size-selected test sets, which contained 8555, 8129, 7064, and 4934 subsequences, respectively, were analyzed using the NaPDoS2 workflow to assess the effects of query size on classification accuracy. Accuracy evaluations for each test set were based on the percentage of subsequences whose best nonself, BLASTP match had the same NaPDoS2 classification as the original, full-length sequence from which it was derived.A custom perl script .Accession information for all sequences and datasets analyzed is provided in Salinispora genome protein sequences .KS amplicon sequences were analyzed using minimum alignment lengths of 50aa in NaPDoS2 unless otherwise noted. Sequence accession and dataset references are listed in https://osf.io/uzhcp/. The code used to construct version two of the Natural Product Domain Seeker website can be found on the corresponding Github repository: https://github.com/spodell/NaPDoS2_website. All accession information and dataset references can be found in Relevant alignment files, phylogenetic tree files, webtool documentation file, example tutorials, sequence files, and other supporting information can be found on the corresponding OSF project page: Additional figures and tables referred to in the text , 84, 85.The authors declare that they have no conflicts of interest with the contents of this article.


Antimicrobial resistance is a global threat to human, animal, and environmental health. In pig production, antimicrobials and heavy metals such as zinc oxide are commonly used for treatment and prevention of disease. Nevertheless, the effects of antimicrobials and heavy metals on the porcine resistome composition and the factors influencing this resistance profile are not fully understood. Advances in technologies to determine the presence of antimicrobial resistance genes in diverse sample types have enabled a more complete understanding of the resistome and the factors which influence its composition. The aim of this review is to provide a greater understanding of the influence of antimicrobial and heavy metal usage on the development and transmission of antimicrobial resistance on pig farms. Furthermore, this review aims to identify additional factors that can affect the porcine resistome. Relevant literature that used high-throughput sequencing or quantitative PCR methods to examine links between antimicrobial resistance and antimicrobial and heavy metal use was identified using a systematic approach with PubMed (NCBI), Scopus (Elsevier), and Web of Science  databases. In total, 247 unique records were found and 28 publications were identified as eligible for inclusion in this review. Based on these, there is clear evidence that antimicrobial and heavy metal use are positively linked with antimicrobial resistance in pigs. Moreover, associations of genes conferring antimicrobial resistance with mobile genetic elements, the microbiome, and the virome were reported, which were further influenced by the host, the environment, or the treatment itself.The online version contains supplementary material available at 10.1186/s13567-023-01143-3. Antimicrobial resistance (AMR) is one of the great global concerns of the twenty-first century . The preBased on the requirements of the WHO global action plan, every member state had to develop its own action plan to tackle and prevent the spread and development of AMR. In the latest legislative changes in the EU, the prophylactic use of antimicrobials in livestock production was banned as of January 2022 and the prophylactic use of medicinal levels of zinc oxide was banned from June 2022 based on Regulation (EU) 2019/6 on Veterinary Medicinal Products and Regulation (EU) 2019/4 on Medicated Feed. The aim of these measures is not only to reduce the development and spread of AMR and environmental pollution, but also to suppress the use of antimicrobials in routine farm protocols and additionally promote animal health through farm management, biosecurity, hygiene, immunoprophylaxis and animal welfare measures on farms .Traditional methods to evaluate AMR such as antimicrobial susceptibility tests are dependent on bacterial growth and are mainly restricted to specific culturable species which allows AMR detection only within a limited spectrum. However, antimicrobial and heavy metal resistance are not limited to these bacteria and could be related to non-culturable microorganisms which may play a role in the spread of AMR . Novel aBoth methods, qPCR and metagenomics, have their advantages and disadvantages as reviewed by Waseem et al. . New devThe aim of the current review is to summarise the findings of studies investigating the association between AMU and HMU and AMR in pig production using such culture-independent approaches to identify factors and drivers that can impact AMR on pig farms.A systematic literature search approach was used to identify scientific literature investigating associations between the usage of antimicrobials and heavy metals on commercial pig farms with AMR in pigs. Moreover, this review only included studies that used culture-independent methods such as metagenomics and quantitative PCR, although additional use of culture-dependent methods was not an exclusion criteria.Literature was identified using the PubMed (NCBI), Scopus (Elsevier), and the Web of Science  databases. The key questions were formulated according to the PIO  criteria. To separate search terms within one PIO group, the Boolean operator “OR” was used Table , thus PIst of January 2000 until the 10th of February 2022 were included in the results as part of the literature search. All records found were imported into and further processed via EndNote X9 reference management software . The literature search resulted in the identification of 118, 145, and 180 records, obtained from the Web of Science, Pubmed, and Scopus databases respectively .Only records published in English from the 1Prior to literature thinning duplicates were removed using EndNote X9. In total 152 duplicates were removed, leaving 291 records which were further screened via two dependent screening steps to determine their suitability for inclusion in this review. The first screening step was based on available information in the record’s title and abstract, while the second screening step was based on full-text analysis of the records that were not removed in the first screening step.n = 44). According to the set inclusion criteria, all records that examined how the use of antimicrobials and heavy metals can affect the porcine resistome were classified as suitable. However, literature was excluded in the following cases: (1) no information on antimicrobial and heavy metal usage and the porcine resistome, (2) focus not on pigs, (3) focus on antimicrobial alternatives, (4) a setting other than pig production, (5) antimicrobials and heavy metals used for the purpose of growth promotion, and (6) literature that did not consider associations between antimicrobials and the porcine resistome. After literature screening, 32 records were left, of which 3 reviews were excluded which discussed findings of studies that were already included in the literature selection, and one study that used culture-dependent methods for AMR quantification. Consequently, 28 records remained. Further information on the literature selection can be extracted from the PRISMA flow-chart , while four of them were comparisons at an international level. The latter included comparison of a randomized trial in a research facility in the United States of America with AMR on three Chinese pig farms (n = 1), comparison of AMR on farms in nine European countries (n = 2) and comparison of AMR on a Chinese, a Danish, and a French pig farm (n = 1). The majority of single-country studies were conducted in the United States of America (n = 8), followed by studies in Europe (n = 6), China (n = 5), Canada (n = 3), Ecuador (n = 1), and Thailand (n = 1). No studies based in lower-middle or low income countries were identified, thus a geographical and economical bias as part of this review cannot be excluded. Details regarding the geographical distribution of the identified studies can be seen in Figure In total, 28 research studies were included in this literature review. Most studies were conducted in a single country (n = 19) reported a positive association between AMU or HMU, and AMR   or investigated AMR in the absence of AMU and HMU or only resistome development under AMU (n = 3). Studies investigated AMR prevalence by either using a metagenomics (n = 17), 16S rRNA gene amplicon sequencing (n = 1) or quantitative PCR (n = 8) approach, or two methods (n = 2), as can be additionally extracted from Additional file Although the methodologies of the 28 assessed studies differed greatly, the majority of studies n = 9 reporteStaphylococcus aureus ,12:i:-, often carrying multidrug resistance modules such as R-type ASSuT, encoding resistance for ampicillin, streptomycin, sulphonamides, and tetracycline [A study by Zhao et al.  quantifiacycline , 52. Moracycline –55. ThistetA and tetB. This may also explain why some studies such as that of Pollock et al. [Although the majority of studies using a metagenomics- or qPCR-approach reported associations between AMU and HMU and AMR, the observations were highly variable. One explanation for heterogeneity of changes in the resistome in response to heavy metal or antimicrobial treatment is a possible influence of the microbiome composition on the resistome . As mentk et al.  did not intl1 and ISCR1, and vertical gene transfer (VGT) in response to sulphonamide treatment. In contrast, Zhao et al. [Interestingly, Muurinen et al.  observedo et al.  reportedo et al.  observedo et al. . Taking o et al.  and Piepo et al. . Nevertho et al. , who obso et al. . Furthero et al.  did not o et al. .Bacillota corrig. phyl. nov. (formerly: Firmicutes) [The virome constitutes another factor that is seldom described in the literature as of yet, but potentially affects the resistome, or more precisely the bacteriophageome. For instance, lysogenic bacteriophages usually infect bacteria, integrate their genetic material into the bacteria chromosome and become dormant until the host bacterium experiences stress. In response to host stress, the phage switches to the lytic cycle and forces the host to reproduce and assemble new bacteriophages, which includes the excision of the prophage from the host chromosome for packaging. Afterwards, the host is killed by lysis . During micutes) , 58. Altmicutes)  observedThe investigation of AMR in livestock production via high-throughput methods, such as qPCR and metagenomics approaches has increased within the last decade. Reasons for this are the decreasing costs and further developments of next-generation sequencing and qPCR methods in recent years, which has allowed increased sample sizes, but also greater analysis depth of various samples deriving from different environments, humans, and animals , 59. TheHowever, despite these massive developments over a short period of time, these high-throughput methods still face many limitations, such as the loss of species or contamination of samples during DNA extraction . Addition = 7) tried to address this by calculating correlations. Mencía-Ares, et al. [Another still unsolved challenge is the accurate ascription of ARGs and MRGs to specific taxa. A number of studies , and feed, water, as well as the kind of antimicrobial compound or heavy metal used. Due to this complexity, comparison of farms or pig production facilities with each other to find a common solution against AMR, is extremely challenging. Consequently, each farm or pig production facility will need its own suite of measures to address AMR spread and development by reducing AMU and HMU, while maintaining animal health and welfare. Future studies should include analysis of the microbiome of different elements of the production cycle, the virome of the host, and MGEs in their analysis and try to set observations into context with environmental and host-derived factors to obtain deeper insights on the interplay between AMR key factors. Furthermore, a standardized protocol for analysis via e.g. metagenomics could be helpful in the future to allow better comparability of studies.Additional file 1. Methodologies used by studies investigating AMU and HMU associations with AMR in pig production. Table summarizing methodologies used by studies.Additional file 2. Key observations indicating associations between AMU and AMR in identified studies undertaken in pig production. Table of found AMU associations with AMR in pig production.Additional file 3. Key observations indicating associations between HMU and AMR in identified studies undertaken in pig production. Table of found HMU associations with AMR in pig production.


To analyse the early and mid-term outcome of patients undergoing conventionalaortic valve replacement (AVR) versus minimally invasive via hemi-sternotomyaortic valve replacement (MIAVR).n = 516) or MIAVR (n = 137) betweenAugust 2015 and March 2020. Using pre-operative characteristics, patientswere propensity matched (PM) to produce 114 matched pairs. Assessment ofperi-operative outcomes, early and mid-term survival and echocardiographicparameters was performed.A single centre retrospective study involving 653 patients who underwentisolated aortic valve replacement (AVR) either via conventional AVR(n = 57) and female(n = 57) patients were equal. PM MIAVR group mean age was71.1 (±9.5) years, and 47% of patients were female (n = 54)and 53% male (n = 60). Median follow-up for PM conventionalAVR and MIAVR patients was 3.4 years  and3.4 years , respectively. Larger sized aorticvalve prostheses were inserted in the MIAVR group versus conventional AVR group .Cardiopulmonary bypass (CPB) time was longer with MIAVR(94.4 ± 19.5 minutes) compared to conventional AVR . Aortic cross-clamp (AoX) time was also longer in MIAVR(71.6 ± 16.5 minutes) compared to conventional AVR . There were no differences in the early post-operativecomplications and mortality between the two groups. Follow-upechocardiographic data showed significant difference in mean aortic valvegradients between conventional AVR and MIAVR groups . There was nosignificant difference between conventional AVR and MIAVR in mid-termsurvival at 3 years .The mean age of the PM conventional AVR group was 71.5 (±8.9) years and thenumber of male (Despite the longer CPB and AoX times in the MIAVR group, there was nosignificant difference in early complications, mortality and mid-termsurvival between MIAVR and conventional AVR. However, the evidence for MIAVR is mostly based on observationalclinical studies which are not well matched, constitute smaller numbers and lackadequate clinical and echocardiographic follow-up. In this study, we aim toundertake a propensity-matched (PM) analysis of the peri-operative results, mid-termsurvival and echocardiographic outcome of patients, who underwent conventional AVRversus MIAVR with UHS at our institution.The concept of minimally invasive aortic valve replacement (MIAVR) was firstintroduced by Rao and Kumar.This is a retrospective study on prospectively collected data including all adultpatients (age >16 years) who underwent isolated AVR via a MIAVR with UHS orconventional AVR between August 2015 and March 2020. The exclusion criteriaincluded, emergency AVR, MIAVR with ART, Ozaki procedure, redo or concomitantsurgery. Preoperative characteristics, intraoperative and post-operative data suchas short-term complications, follow-up echocardiographic data as well as mid-termsurvival were collected and analysed. Follow-up involved searching our onlinedatabase system which stores statistics on the number of patients who are alive andthat have died as well as date of death. Echocardiographic follow-up data wasobtained using our Insignia online system which enables us to review imaging andaccompanying reports. PM was performed using pre-operative characteristics to matchthe two groups. Pre- and post-matched patient characteristics and follow-upechocardiographic data were analysed using IBM SPSS Statistics for Windows, version26  and Microsoft excel. Institutional approval wasobtained and patient consent was waived due to retrospective design.All patients who underwent MIAVR had the UHS surgical technique performed. Aftergeneral anaesthesia and establishment of sterile field, a UHS was performed into theright third or fourth intercostal space using the oscillating or reciprocating saw.Cardiopulmonary bypass (CPB) was then instituted between the ascending aorta andright atrium. After placement of aortic cross-clamp (AoX), antegrade bloodcardioplegia was then given into the aortic root and then directly into right andleft coronary artery ostia after aortotomy. Carbon dioxide was also used routinely.AVR was then performed under standard fashion with semi-continuous or interruptedpledgeted sutures.2, gradient mmHg), native aortic valve pathology, ejection fractioncategory, left ventricular ejection fraction, operative urgency, aortic valvehaemodynamic pathology and first operator grade. Regarding pre-operative variables,including body mass index, previous myocardial infarction and hypertension had<1% missing data and pre-op atrial fibrillation had <2% data missing for bothgroups. Variables including >1 coronary vessel disease and left main stem diseasehad <4% data missing for the conventional AVR group. About 32% of data wasmissing for the severity of aortic valve stenosis (EOA in cm2) variablein the conventional AVR group and 16% in the MIAVR group. Similarly, 30% of theconventional AVR group had missing data for the severity of aortic valve stenosisgradient (mmHg) variable and 16% was missing for the respective MIAVR group. Overallthough there was data missing it was not deemed significant enough to warrantinverse weighting of variables prior to analysis. Before matching, for pre-operativecharacteristics, all continuous data had the mean and standard deviation (SD), ormedian and interquartile range (IQR) calculated and proportions in percentages werealso calculated for nominal data. Nearest neighbour PM method was used to match 114MIAVR patients to 114 conventional AVR patients with the closest propensity scoreand these calculations were repeated  wasalso calculated for continuous data. For nominal data, the Chi-squared test wasused, as well as Fisher’s exact test. The Likelihood ratio was used where necessaryand p < 0.05 were deemed significant. The mid-term survival of the patients wasalso reported with Kaplan-Meier survival analysis . Atotal of 29 covariates were included in the LR model. Pre-operative characteristicswhich were inserted into propensity score matching included age, gender, body massindex, logistic euroscore, angina, previous myocardial infarction, previouspercutaneous coronary intervention, diabetic, hypertension, smoking status, actualcreatinine at time of surgery, dialysis for chronic renal failure, New York HeartAssociation classification score, chronic obstructive pulmonary disease/emphysema orasthma, neurological disease, neurological dysfunction, abnormal pre-operative heartrhythm, pre-op atrial fibrillation/flutter, ⩾1 coronary vessel disease, left mainstem >50% diameter stenosis, severity of aortic valve stenosis  and516 (79%) had MIAVR and conventional AVR, respectively. PM resulted in 114 patientsin each group for analysis. Pre-matched pre-operative data  showed t19 Despite this, earlypost-operative complications and survival were comparable between the two groups.There is no doubt that any kind of minimally invasive cardiac surgery is morechallenging when compared with conventional procedure via median sternotomy. This ismainly due to a limited incision and reduced operative space that lead to restrictedmanoeuvrability.20 Given that increased CPB and AoX times are known to beassociated with adverse early outcomes, our study with MIAVR did not support thisnotion. Whether this leads to enhanced systemic inflammatory response or degree ofmyocardial damage at molecular level remains to be explored and more work isrequired in this area. Despite the complexity of MIAVR operations and risk ofsubsequent complications, MIAVR did not increase early mortality compared toconventional AVR, reasons may be due to technical maturation of theprocedure.21 Though previous studies found reduced length of hospitalstay and post-operative complications with MIAVR, our post-operative findings weresimilar for both groups indicating similar safety for both operations.22 Someauthorities also believe that higher mid-term mortality during follow-up may occurdue to increased AoX time leading to inadequate cardio-protection and reduced LVfunction. We did not demonstrate this but larger studies with longer follow-up maybe required to further determine this.Our findings of longer CPB and AoX times with MIAVR compared to conventional AVR arein line with similar results in the literature.24A surprising finding is that larger valve prostheses were inserted in the MIAVR groupwith significantly lower mean aortic valve gradients compared to conventional AVR.The reason for this is not completely understood but the higher insertion of rapiddeployment valves (RDV) in our MIAVR series compared with conventional AVR may havecontributed to this.14 Hence, it is unsurprising tosee in the literature that mostly MIAVR are performed by consultants. However, ithas been reported that a MIAVR training programme can be established withoutcompromising patient safety.25 Masuda et al.26 suggestedapproximate 40–50 cases were required to overcome the learning curve which may notbe the ideal stage to train residents. Similar data were also reported by Murzi etal.27In our institution, nearly three quarters of the MIAVRs were performed by consultantsurgeons. However, an increasing trend was seen in trainees performing MIAVRs in ourunit with comparable results in pre-PM data.A steep learning curve for MIAVR has been reported by several authors, even amongstthe most experienced surgeons.One of the limiting factors of our study is that it is based on single centreretrospective data, analysing the results of only one type of MIAVR. Therefore,propensity matching was conducted to decrease selection bias. Furthermore, as aregional cardiac surgical centre, some patients had their follow-up echocardiographydone in the referral centres of which not all follow up data was accessible.Therefore, the echocardiographic analysis was performed with a smaller group ofpatients.Our results have shown that larger aortic valve prostheses are being implanted in theMIAVR group with lower mean aortic valve gradients. The complication rate, early andmid-term mortality were comparable to conventional AVR despite longer CPB and AoXtimes with MIAVR. Follow-up echocardiographic data currently shows no difference;however, analysis of long-term follow-up data with larger number of matched patientsis required to ascertain late outcomes.


The radiative cooling of a stored, initially rotationally hot OH Such comparison can be challenging, since experimentally molecular ions are usually produced by a violent act that leaves them in a superposition of excited states, which are dependent on the production process, the environment as well as the time scale. Given that the rates of many chemical reactions are, in general, highly dependent on temperature, it becomes important to be able to devise experiments where the temperature, or the actual internal energy distribution of the molecular ion reactant could be probed. In addition, time-dependent measurement of the state population would allow for a better understanding of the internal molecular dynamics, as well as a direct comparison with (or measurement of) state selective lifetimes or Einstein coefficients. Moreover, while most environments where molecular ions are involved in reactions  are usually defined by an equilibrium temperature, it is not always clear whether in an experiment the population of the molecular ion rotational states faithfully replicates the expected Boltzmann distribution at thermal equilibrium.Information on the internal energy states of molecular ions is a prerequisite for many experiments, as well as for comparison to theoretical calculations and astrophysical observations2. Among them, a few popular methods are the supersonic expansion techniques4 and the ion trapping methods where cooling is achieved through collision with a buffer gas5. At a different scale of size and beam energy, the heavy ion storage ring6, and later on, the electrostatic ion beam trap (EIBT)10 techniques have demonstrated their advantages for studying infrared active molecular ions. In such a setting, a beam of hot and fast (keV to MeV) molecular ions is injected into a storage ring or an EIBT, and relaxation of the internal degrees of freedom occurs through spontaneous emission of the electronic, vibrational, and rotational states. The asymptotic internal temperature of the molecular ions is limited by the blackbody radiation emitted by the wall of the ring or the trap. Recently, a new generation of cryogenically cooled storage rings13 and EIBT14 has reached a temperature of the order of 10–20 K, allowing for almost complete relaxation of the internal degrees of freedom. While typical radiative  electronic and vibrational relaxation times are of the order of ns to several seconds, the rotational cooling can extend to several minutes, and even hours. Although the asymptotic equilibrium temperature is known (regulated by the blackbody radiation), these techniques cannot be easily used to probe and follow the internal dynamics  over a large range of temperatures.Various methods have been developed to produce cold (i.e. ground state) molecular ions16. In such experiments, a tunable laser is merged with the stored beam at different times and various wavelengths. By measuring the photodetachment cross-section at different wavelengths and times, it is possible to extract the population changes of the rotational states. While such experiments can precisely measure the rotational population at relatively low temperatures (tens of K), they are more difficult to apply at higher temperatures when many rotational states are still populated16.A few experiments have successfully measured the rotational state population of stored molecular anions in cryogenic storage rings using near-threshold photodetachment16, it is more general, works over a larger range of temperatures, and provides a direct estimation of the rotational population during the entire storage time.In the following, we present a new method using OH23 and its structure, energetic and potential energy surfaces have been calculated in several theoretical works24, and references therein. OH25, and the rotational lines spacings are large, a feature particularly interesting for high resolution photoelectron spectra.The photodetachment of OH20) is produced in a Cs sputter ion source, accelerated to an energy of 4.2 keV and injected into an EIBT7. A schematic of the experimental setup is shown in Fig. 28. Once injected in the EIBT  allows for a precise calibration of the VMI setup . Using j-dependent photoelectron detachment model18, the threshold transition intensities j is OH20).In order to extract the time-dependent temperature of the stored OHT is the molecular rotational temperature and C are the rotational energy and the relative population of the j level of OH16a are the photon energy, the transition energies for P3 and P1 transitions and the Wigner factor, respectively. In this study, a is set to 0.215, and N is a normalization constant. Although the partition function is temperature dependent, it has only a constant value for a given temperature . Since we use only relative populations it does not affect the extracted temperature when the area under the distribution is normalized to unity (as was done in this work).Assuming a Boltzmann distribution j. In order to fit the experimental data shown in Fig. T as a free parameter , and shown as a green dotted line. As can be seen, the assumption that the electron kinetic energy is the result of a Boltzmann distribution for the rotational state of OHUsing Eqs.  and  2)2), the pT are plotted as a function of time in Fig. 15, where the molecular ions were produced in a similar ion source.The resulting fitted temperatures j is calculated using the following Master equation:j and To support the analysis of the data shown in Fig. 33.In order to solve Eq. , the ini33, within the experimental resolution.The solution of the differential equations  yields tTo provide an overall comparison between the photoelectron spectrum model and the Master equation Eq. , a time-In conclusion, the relaxation behavior of rotationally hot OHSupplementary Information.


Dopamine is a neurotransmitter that plays a crucial role in adaptive behavior. A wealth of studies suggests obesity-related alterations in the central dopamine system. The most direct evidence for such differences in humans comes from molecular neuroimaging studies using positron emission tomography (PET) and single-photon emission computed tomography (SPECT). The aim of the current review is to give a comprehensive overview of molecular neuroimaging studies that investigated the relation between BMI or weight status and any dopamine target in the striatal and midbrain regions of the human brain. A structured literature search was performed and a summary of the extracted findings are presented for each of the four available domains: (1) D2/D3 receptors, (2) dopamine release, (3) dopamine synthesis, and (4) dopamine transporters. Recent proposals of a nonlinear relationship between severity of obesity and dopamine imbalances are described while integrating findings within and across domains, after which limitations of the review are discussed. We conclude that despite many observed associations between obesity and substrates of the dopamine system in humans, it is unlikely that obesity can be traced back to a single dopaminergic cause or consequence. For effective personalized prevention and treatment of obesity, it will be crucial to identify possible dopamine (and non-dopamine) profiles and their functional characteristics. Endogenous dopamine enables signal transduction and modulation throughout the central nervous system, and contributes to synaptic plasticity. Dopamine plays a crucial role in adaptive behaviour, such as behavioural and cognitive control ,3,4,5,6,A wealth of evidence suggests obesity-related differences in neurocognitive measures of these dopamine-dependent processes in humans, reviewed in , indirecStructural dopamine alterations in human obesity have been demonstrated in molecular imaging studies, in particular in the domain of dopamine D2/D3 receptor availability and dopamine release, but dopamine synthesis capacity and dopamine reuptake transporters have also been under investigation. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are the most direct ways to investigate dopaminergic changes in humans in vivo. Although dopamine levels cannot be measured directly, certain molecular substrates of the central dopamine system can be approximated by means of highly-selective radiolabeled tracers . RadiotrExisting molecular imaging findings have been discussed in a number of excellent reviews that focus on different subsets of the four available dopamine domains ,16, 23,,25,26,27https://pubmed.ncbi.nlm.nih.gov/ last accessed on 28 August 2021) was performed on 9 December 2020 using the search terms “((Positron Emission Tomography) OR (Single-Photon Emission Computerized Tomography)) AND ((Obesity) OR (BMI)) AND (dopamine)” and resulted in 91 hits. On 28 August 2021 the search was repeated to check for new publications. Despite six more hits, no new empirical articles were retrieved. Titles and abstracts of all 97 hits were screened. Only empirical PET and SPECT studies in human volunteers that reported striatal and/or midbrain measurements of at least one dopamine radiotracer were included, and statistically tested for a correlation with BMI and/or a group difference between BMI groups with no known eating disorders. After screening, the full texts and references of the remaining 31 articles were assessed in detail. One article did not include dopamine measurements in the striatum or midbrain and was excluded -IBZM, or the PET-radiotracers [11C]-Raclopride, [18F]-Fallypride, or [11C]-(+)-PHNO -PHNO . Six stureported ,40,47,482 and compared the obese group to a non-obese control group (BMI < 30 kg/m2) consistently found lower binding -(+)-PHNO consistently report higher striatal D2/D3 receptor binding with higher-BMI-up-to-severely obese [18F]-Fallypride [11C]-Raclopride [2) and non-obese women using [11C]-Raclopride or the D2 receptor specific radiotracer [11C]-NMB -NMB ,37,47. S11C]-NMB  and Eise11C]-NMB , as well-NMB -Raclopride and [123I]-IBZM are similar in their characteristics, with a moderate affinity for D2 receptors and high correlations in binding potential between striatal subregions. These radiotracers are typically used to capture the binding potential of the striatum as a whole. [18F]-Fallypride binds to D2/D3 receptors with higher affinity and thus enables the imaging of D2 receptor binding of striatal subregions and extrastriatal regions [11C]-(+)-PHNO has a notably higher affinity for D3 receptors. As an agonist, [11C]-(+)-PHNO preferably binds to functionally active D2/D3 receptors (high affinity state) rather than the antagonists that bind to both high and low affinity state receptors. D3 receptors are most abundant in the ventral striatum and dopaminergic midbrain. As such, findings localized to these regions tend to be interpreted as D3-receptor-related effects. However, the ventral striatum also expresses D2 receptors abundantly and it has been shown that the [11C]-(+)-PHNO binding potential in this region still accounts much stronger for D2 rather than D3 receptor availability. Based on this, authors of a multi-tracer obesity study explain their positive correlation of BMI with [11C]-(+)-PHNO binding and the absence of a relationship with the D2-preferring radiotracer [11C]-Raclopride as a BMI-related difference in the density of active D2/D3 receptors, rather than of D3 receptors [11C]-NMB, which binds specifically to D2 receptors and is not replaceable by endogenous dopamine, tentatively suggest that positive findings with other radiotracers may reflect differences in dopamine levels. We return to this point below -PHNO study including a wide BMI range could address the open question if the consistent observations of a positive correlation with BMI reflect a linear relationship that also holds for those with more extreme obesity, or rather a nonlinear relationship.It is crucial to consider the characteristics of radiotracers when interpreting findings from PET and SPECT studies, in particular in the domain of D2/D3 receptors, as has been discussed extensively before . [11C]-R regions . In conteceptors . Becausent below . Future 18F]-Fallypride -IBZM, [11C]-Raclopride, [18F]-Fallypride, or [11C]-(+)-PHNO). Dopamine release is induced by a certain manipulation and assessed by contrasting the measurement following manipulation to a baseline or placebo measurement. A common manipulation is the administration of a stimulant drug, such as methylphenidate -(+)-PHNO measurements in a wide BMI range could provide new insights. Due to its preferred binding to the functional high-affinity state D2/D3 receptors, [11C]-(+)-PHNO is particularly sensitive to changes in endogenous dopamine and it can capture changes in both the striatum and midbrain.Drug-induced dopamine release did not vary as a function of BMI , nor werparisons . Peripheparisons . When adparisons . Followiparisons  observed2) and a negative relationship for severe obesity (>40 kg/m2). Although this is not supported by secondary analyses of the two studies that sampled the right-hand side of the putative inverted U-shaped curve -FDOPA and [18F]-FMT are used to assess aromatic L-amino acid decarboxylase (AADC), an enzyme part of the dopamine synthesis pathways  as an estimate of striatal dopamine tone. In line with the [18F]-FDOPA influx findings, EDVR was negatively correlated with BMI in all striatal subregions, whereas [18F]-FDOPA washout was positively correlated with BMI in the ventral striatum and putamen. These findings suggest that a lower obesity-related dopamine synthesis capacity goes hand in hand with lower striatal dopamine tone, which resonates with the idea that previous D2/D3 receptor-related findings might need to be interpreted in terms of dopamine tone [18F]-FMT) measured in the striatum and D2/D3 receptor binding potential ([11C]-Raclopride), but not with methylphenidate-induced dopamine release -FMT ,52. In aine tone  (discuss release . However18F]-FMT vs. [18F]-FDOPA), sample size (15 vs. 60), sex ratio (~50:50 vs. predominantly men), age range (20–30 vs. 30–42 years old) and inclusion of smokers (none vs. 18 out of 60). Furthermore, different instructions to control food intake prior to scanning were given  and found a positive correlation with BMI in the amygdala in a nonobese sample [Despite the great added value of PET and SPECT studies for approximating molecular targets in humans in vivo, the methods used are inherently limited. Due to their binding characteristics, most radiotracers are highly selective, binding with high affinity to only one or a few molecular substrates in specific regions of the brain. At the same time, the measurements of a given radiotracer do not allow us to quantify parts of the dopamine pathways unequivocally. As discussed by others  this come sample . Anothere sample . This waThe dopamine system is, of course, an integral part of the brain and the body as a whole. Of note in this context is that dopamine interacts with other neurotransmitter systems that have also been associated with obesity, such as the serotonin . LongitA major limitation of the current review, and the field in general, is the operationalization of obesity in terms of distinct BMI categories. Although BMI categories are well-defined and commonly used to determine adiposity and related health risks, it is also widely criticized for use as a single measure because of its heterogeneous nature ,92,93. TFinally, we emphasize the qualitative nature of the current review. Although the literature search closely follows existing guidelines for systematic reviews of non-intervention studies , not allBased on a wealth of evidence, a role for central dopamine differences in obesity seems undeniable. Currently, the evidence leans towards lower striatal D2/D3 receptor binding potential in extreme obesity and higher binding potential in overweight and moderate obesity, which could reflect differences in D2/D3 receptor availability, dopamine tone, or a combination of the two. Overweight to moderate obesity may also be characterized by a higher dopamine synthesis capacity in the striatum. Furthermore, obesity-related differences in dopamine release or transporter availability have not convincingly been shown in humans. The existence of a nonlinear relationship between severity of obesity and dopamine differences have been proposed by several researchers to explain seemingly inconsistent findings either within or between dopamine domains. Whether or not such nonlinear relationships exist, and for what parts of the dopamine pathways, remains to be tested. How to explain the existence of the proposed nonlinear relationships is also an important open question. Does it reflect a natural progression in the development of moderate to severe obesity? Is it the result of an interaction between differences in D2/D3 receptor density and tonic dopamine levels? And could it be that a nonlinear relationship captures distinct (dopamine) anomalies for different obesity profiles? Despite many observed associations between obesity and components of the dopamine system, it is unlikely that obesity can be traced back to a single dopaminergic cause or consequence. For effective personalized prevention and treatment of obesity, it will be crucial to identify possible dopamine (and non-dopamine) profiles and their functional characteristics. To identify helpful dopamine targets for different stages of obesity (development), large multi-tracer and multi-modal neuroimaging studies are required that better characterize overweight and obesity in terms of body shape, lean and fat mass, energy expenditure, metabolism, obesity onset and duration, diet, and physical activity, and can identify different obesity profiles. Longitudinal and intervention studies  are crucial for understanding the progression of dopaminergic changes that leads to an imbalance of the system, and the targets that can be used to reverse this imbalance at different stages.


NP contributions varying depending on the excitation wavelength. Finite difference time domain (FDTD) analyses and control measurements indicate that these effects can be modeled provided the local complex dielectric constant is strongly modified upon cross-linking. This suggests quantum hybridization at a molecule–band (q-MB) level. Given the vast number of molecules and nano-building blocks available, X-NS have potential to significantly increase the range of available 2D nanosheets and associated quantum properties.A central goal in molecular electronics and optoelectronics is to translate tailorable molecular properties to larger materials and to the device level. Here, we present a method to fabricate molecularly cross-linked, self-assembled 2D nanoparticle sheets (X-NS). Our method extends a Langmuir approach of self-assembling gold nanoparticle (NP) arrays at an air-water interface by replacing the liquid sub-phase to an organic solvent to enable cross-linking with organic molecules, and then draining the sub-phase to deposit films. Remarkably, X-NS comprising conjugated oligophenylene dithiol cross-linkers (HS-(C Bottom-up nanomaterial fabrication enables control over nano building-block structure and structure-property relationships, affording opportunities to engineer devices and materials with desirable electronic and optoelectronic behaviors. Here, the authors produce stable oligophenylene dithiol cross-linked gold nanoparticle nanosheets via interfacial self-assembly and show the emergent effects of the cross-linkers on the material’s electronic, optical and photoconducting properties. Incorporating molecules into larger structures that continue to reflect control over single molecule properties, however, is an open challenge in this direction. Studies of molecular electronic devices have revealed that molecule–electrode contacts play a crucial role in influencing device behavior2, leading to significant device-to-device variation3. Further, Fermi-level pinning of molecular states by the electrodes can dominate electronic device behavior, repressing the effects of desirable individual molecular properties6. For instance, thiol-based conjugated molecules such as oligophenylene dithiols (OPDs) have exhibited decreasing conductance in molecular junctions with an increase in molecule length, although longer molecules within this family exhibit narrower highest occupied—lowest unoccupied molecular orbital (HOMO-LUMO) gaps6. The decrease in conductance with molecular length is attributed to Fermi-level pinning owing to gold–thiol bonds at the electrode–molecule interface, and is in contrast to the behavior exhibited by highly conjugated molecular wires such as fused porphyrins, where conductance across junctions increases with molecular length8. Encouragingly, however, reports of nanostructured materials comprising nanoparticles cross-linked with molecules have found that properties of such nanoparticle-molecule materials depend strongly on both nanoparticle and molecular building blocks used11. Moreover, nanoparticle-molecule cross-linked systems have shown that the incorporation of nanoparticles in molecular junctions can increase reproducibility of assembled devices12, as well as increase conductance across double layers of molecular self-assembled monolayers13. With a vast range of nanostructures and molecules available, cross-linked nanoparticle-molecule systems comprise a promising platform for the development of molecular optoelectronics. To date, however, a major bottleneck in this regard has been a lack of versatile methods for assembling ordered, extended cross-linked nanoparticle structures with a variety of cross-linking molecules. Nanoparticles are commonly first self-assembled either on a solid substrate15 or at an air–liquid interface17, before introduction of a molecular cross-linker to facilitate molecular exchange and linking between adjacent nanoparticles11. On solid substrates, nanoparticle cross-linking within the monolayer is generally restricted to molecules of lengths close to that of the interparticle separation11 in order to preserve the nanoparticle monolayer order/configuration18. Conversely, although nanoparticles assembled at air–liquid interfaces are highly mobile and interparticle distance can be controlled through Langmuir methods 16, cross-linking at such interfaces remains a challenge due to difficulties in introducing cross-linkers into the liquid subphase as well as facilitating ligand exchange. Since water is a common choice of subphase in such systems, hydrophobic cross-linkers such as organic dithiols and conjugated molecular wires cannot be introduced, and cross-linking is again restricted until the nanoparticle film is collected upon a solid substrate.Synthetic control over molecular structure and strong structure-property relationships in molecules afford an opportunity in molecular optoelectronics, namely a possibility to engineer molecular devices and materials with electronic/optoelectronic behavior that is tailored from the bottom-upvia ligand exchange, as shown in Fig. 2)nSH for n = 2, 4, 6, 8) and oligophenylene dithiols (OPDs) (HS-(C6H4)n-SH for n = 1, 2, 3), as well as densely packed gold nanoparticle monolayers ligated with oligophenylene thiols (H-(C6H4)n-SH for n = 1, 2) are investigated. This comparison approach enables insight into the effects of varying molecular backbones and varying the maximum number of strong gold–thiol interaction . Structurally, these nanosheet materials are highly ordered, macroscopic in 2-dimensions and one nanoparticle thick. They exhibit electronic, optical and photoconducting properties that correlate with those of the gold nanoparticle and molecular cross-linker building blocks as well as reveal emergent effects. Remarkably, X-NS comprising conjugated OPD cross-linkers exhibit conductance that increases exponentially with molecule length due to electron delocalization, in contrast to previously reported OPD junctions using bulk electrodes6. Further, the rate of increase is 10-fold higher than that previously reported using similar molecules with terminal alkanethiol chains employed to isolate the conjugated molecular backbone from the substrate19. In addition, conjugated molecules in X-NS exhibit π → π* transitions that red shift with increasing molecular length due to electron delocalization, as in solution, but with 6 orders of magnitude higher absorbance extinction coefficients. We use finite difference time domain analysis to model the absorbance of X-NS using the complex dielectric functions of the nanoparticle and molecular cross-linker building blocks as inputs and attribute emergent X-NS behavior to quantum hybridized molecule-band (q-MB) states.The present work describes an extended Langmuir method that enables fabrication of molecularly cross-linked self-assembled nanoparticle sheets (X-NS) with a variety of aliphatic and conjugated organic dithiol cross-linkers of varying lengths. X-NS are fabricated by first self-assembling NP monolayers at an air–water interface and subsequently exchanging the water subphase for an organic solvent to enable in situ cross-linking with organic molecules n, nanoparticles generally remain isolated, except for X-NS obtained using the shortest alkanedithiols, namely butanedithiol and especially ethanedithiol , where Rn is the resistance of the n-alkanedithiol X-NS, n is the number of repeat units, and R0 and β are constants. This value of beta is larger than a typical value of ~1 per methylene unit reported by previous studies using one or a few molecules in break junctions or self-assembled monolayers on deposited electrodes25. In contrast, OPD cross-linked X-NS exhibit a reverse trend with DC resistances decreasing exponentially with increasing number of phenyl units. Again applying an exponential fit to resistance as above, this time data yields a negative value for β = −1.86 (R2 = 0.98) per phenylene unit . Decreasing gold Fermi level—molecular frontier orbital energy level barrier is also achieved in the present study without employing previously reported measures to decouple molecules from contacts26. Combined, these studies highlight critical roles that both the molecule and electrode–molecule architecture can play in governing the degree to which charge transport through molecular orbitals can be controlled by varying molecular structure, which is one of the central goals of molecular electronics. In particular, the present remarkable observation of a strong correlation between increasing delocalization at the molecular level and decreasing resistance at the X-NS level suggests that X-NS are a promising platform for the development of molecular electronic materials.To explore the effect of dithiol cross-linkers introduced during X-NS fabrication, we measured X-NS electrical and optical properties, using films fabricated using monothiols as controls. Figure 27. These results suggest that cross-linking is indeed occurring in our system , and film properties are significantly modified accordingly.To explore whether the di-thiols generate a degree of NP cross-linking or simply replace capping ligands, we also measured the resistance of films fabricated using mono-thiols, namely benzenethiol and biphenylthiol, as controls , but exhibit little further shifting within the series as the number of phenyl units is varied  provides further evidence for phenomena enabled by cross-linking in this system. Figure ied Fig. . In contied Fig.  compared* transitions  and the peak at 200–210 nm to the sulfur moiety absorption. Remarkably, the strengths of these molecular absorption peaks are prominent on the scale of the NP surface plasmon peaks even though the NPs are much larger, are metallic and respond collectively. For reference, molecular absorption peaks in NP films with corresponding oligophenylene monothiols are much weaker  analysis and compare absorption of systems with monothiol ligands vs. dithiol cross-linkers and phenyl- vs. biphenyl- molecular backbones. To highlight emergent effects, we rely on measured optical properties of building blocks, i.e., measured UV–Vis absorption spectra of molecules in solution and published complex dielectric constant of bulk gold, as inputs, and model corresponding properties of molecule-NP assemblies. Although this approach is approximate, for instance it neglects solvent contributions and variation in molecule response within the inter-NP region, the simplicity and effectiveness of the model provide useful insight into behavior exhibited by X-NS. Rearranging the Beer-Lambert law for a lossy medium, molecule absorbance is given by:31. Comprehensive 3D-FDTD simulations are conducted to solve Maxwell’s equations and determine overall film absorption and local fields. We first calculate the absorption spectra of NP films fabricated using mono-thiol molecules, which represents a simpler case where the short wavelength molecular peaks are much less pronounced. Numerically calculated absorption spectra for benzene- and biphenyl-mono-thiol NP films are shown in Fig. It is noted here that the K-K relations provide approximate values of n causing the main plasmon absorption peak to broaden and red shift significantly, thus making it impossible to model both short and long wavelength absorption peaks simultaneously. However, if, in addition to increasing conductivity, 6), then we obtain much better agreement between the model and observed spectra for both benzene- and biphenyl-dithiol X-NS  perpendicular to the beam path is 60 in solution, 10 in X-NS and 0.3 in hot-spots in X-NS 2/(size of the nanoparticle)2 ≈ (1 nm)2/(6 nm)2 fewer than total in X-NS, see Fig. 4–3 × 105 going from solution to X-NS. Using biphenyldithiol as an example, the absorbance at 300 nm decreases from 0.17 in solution . At 610 nm (~surface plasma resonance), field enhancement is concentrated in regions where NPs are closest, also known as hot-spots. These results combined with TEM structural data indicate that the optoelectronic response of X-NS is dominated by dithiols located in the regions of closest NP-NP approach, where dithiol—NP cross-linking is most likely.Figure −2), 520 nm (54.3 mW cm–2) and 638 nm (28.2 mW cm−2)  with a variety of aliphatic and conjugated dithiol cross-linkers of varying lengths. In particular, oligophenylene monothiols and dithiols (OPDs) as well as alkanedithiols are incorporated into this system and resulting electronic and optical properties of the films are characterized. Room temperature resistance measurements reveal exponentially decreasing conductance with increasing alkanedithiol lengths in X-NS, and conversely exponentially increasing conductance with increasing oligophenylene dithiol length. Additionally, UV–Vis measurements of OPD X-NS exhibit increased molecular absorbance in comparison to films fabricated with oligophenylene monothiols and molecules in solution. FDTD modeling of OPD X-NS suggest that the enhanced absorption in such films can be explained by a ~1036. 50 mL of 50 mM tetraoctylammonium bromide in toluene was added to 25 mL of 40 mM hydrogen tetrachloroaurate in water and stirred for roughly 20 min. The colored organic phase was then removed, and added to 25 mL of 0.4 g of sodium borohydride dissolved in water, and stirred under vigorous agitation for 18 h. The deep red organic phase was then separated again, and washed 3 times with 50 mL each of aqueous 0.1 M H2SO4, 1 MNa2CO3, and finally deionized water. Anhydrous magnesium sulfate was then used to remove excess water from the organic nanoparticle solution. The resulting stock Au NPs possessed diameters of ~ 5.8 nm ± 24% and tetraoctyl-ammonium bromide ligands in toluene. UV–Vis spectra of the stock Au NPs is shown in Supplementary Fig. Au NPs were synthesized according to a modified Brust method36 before use in X-NS fabrication. 0.042 g of dodecylamine (DDA) was added to 5 mL of stock NP solution, and left to sit for at least 30 min for ligand exchange to occur. Simultaneously, a dodecylamine/ethanol (nonsolvent) solution was prepared by adding 0.214 g of DDA to 10 mL of absolute ethanol. The nonsolvent solution was then slowly added to the NP solution while simultaneously stirring the mixture. Once a volume ratio of 5 (mL): ~3.5 (mL) toluene: DDA/EtOH was achieved, the NP solution was centrifuged for 10 min at 2400 rpm (900 g). The precipitate was then collected, while more ethanol nonsolvent was added to the supernatant for further size-selection of the nanoparticles, collecting precipitates at volume ratios of 5 (mL): ~7 (mL) and 5 (mL): ~10 (mL) of toluene: ethanol. Note that the amount of ethanol nonsolvent added prior to each centrifugation step can be adjusted to tune NP precipitate sizes and yields. UV–Vis spectra of size-selected NPs are shown in Supplementary Fig. As synthesized Au NPs were subsequently size-selected36. A Teflon ring  is positioned within a glass well (~6 cm diameter and ~1.5 cm height) and slowly filled with deionized water until the water surface exhibits a slightly convex surface curvature  capping groups to move on the water surface and form a relatively dense and ordered monolayer Fig. , cross-lAt this stage molecular cross-linkers are introduced into the methanol subphase Fig. . For croFollowing NP cross-linking, the methanol/cross-linker subphase is once again exchanged by flowing neat methanol (~30 min at ~1.2 mL/min) to remove excess dithiols, and the subphase is then drained (~1 mL/min), depositing the film on a desired substrate located at the bottom of the well Fig. . FollowiSupplementary Information


The nanofibrous mat shows hydrophilic characteristics and good antifouling performance. Under mild NIR irradiation, ICG could convert NIR light into thermal energy that elevates the surrounding temperature above 45 °C. This thermal energy also markedly accelerates the DOX release from the nanofibrous mat due to softening of the nanofibers, indicating the drug release could be controlled and switched on/off by light-triggering. Moreover, this light-triggered thermal energy and releasing behavior contribute to enhancing the cell lethality. Intracellular DOX distribution confirms the more drugs release upon light irradiation. All results demonstrate the developed light-triggered drug release nanofibers as LDDS are biocompatible and antifouling as well as has the superior combinational chemotherapy/photothermal therapy.Localized drug delivery systems (LDDS) have gained great interests because they can directly treat the tumors and minimize systematic toxicity, and maximize drug action by controlling release precisely at the tumor site. However, the resistance of the non-specific adsorption of biomolecules is also important to alleviate the inflammatory reactions and avoid the decrease in performance of LDDS. In this study, we develop a near infrared (NIR) light-triggered nanofibrous delivery system consisting of zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine)- In the past few decades, the delivery methods of drugs have attracted greater attention because they influence not only the therapeutic efficacy of a drug, but also the pharmacokinetics, pharmacodynamic, distribution, metabolism, and systemic toxicity . Combineco-NIPMAAm) hydrogel containing gold nanorods (AuNRs)   was synthesized via ring-opening polymerization (ROP) and atom transfer radical polymerization [P1 has the shorter PMPC segment, whereas P2 has the longer PMPC length. We varied the length of the MPC segment to investigate its hydrophilicity and resistance to protein adsorption. The purity and compositions of synthesized PCL-b-PMPC were characterized by 1H NMR spectra -rization  as depicrization  and used spectra .b-PMPC (P1 and P2) and PCL were fabricated by electrospinning (ES) with polymer concentration of 16 wt% for PCL-b-PMPC and 25 wt% for PCL in methanol/chloroform solvent mixture, respectively. b-PMPC and PCL formed continuously and bead-free nanofibers with average diameters of 177 ± 32 nm for P1, 163 ± 35 nm for P2, and 2.49 ± 0.5 μm for PCL. The insert SEM images show a smooth and nonporous surface of all nanofibers. Compared to PCL for fabrication of nanofibers, PCL-b-PMPC required lower solution concentration and had smaller fiber diameters. This might be due to the presence of electrostatic interaction between zwitterionic groups and increase in the electrical conductivity of the polyelectrolyte solution that enabled the formation of uniform and thinner fibers [b-PMPC and PCL nanofibers were conducted by water contact angle measurement , which can easily and strongly adsorb on a wide range of material surface, was used as the model protein. In this study, the adsorbed BSA protein was quantified by using the BCA assay and qualitatively evaluated by CLSM. As shown in 2). Belfort et al. indicate non-polar surfaces destabilize proteins to change their conformations, resulting in strong protein–surface interaction [2 for P1 and 6 μg/cm2 for P2), indicating incorporation of PMPC moiety can effectively improve the protein resistance by increasing hydration capacity and simulating cell membrane structure [b-PMPC nanofibers exhibited very little protein adsorption in comparison with the PCL nanofibers. The extent of protein resistance was increased with PMPC content. Therefore, P2 nanofibers with excellent antifouling property was chosen to encapsulate drugs for further study.To investigate the antifouling property of PCL-eraction . Howevertructure ,16,17. TP2ID represents both ICG- and DOX-loaded nanofibers, and P2D and P2I represent the DOX-loaded and ICG-loaded nanofibers, respectively. The photographs of the P2D, P2I, and P2ID nanofibers are shown in P2ID), 206 ± 41 nm (P2I), and 183 ± 51 nm (P2D). It can be observed that fibrous morphology was unaffected by the drug-loaded, but the fiber diameters were increased. The existence of both DOX and ICG in P2ID nanofibers was confirmed by UV-visible spectrum , respectively.Drug-loaded nanofibers were prepared by electrospinning of DOX- and/or ICG-added polymer solution. spectrum  and fluospectrum c. The coP2ID nanofibers under NIR light at different time intervals were monitored by the temperature increment in aqueous solution. As shown in P2 nanofibers, which was in the absence of ICG. However, in the presence of ICG, NIR light gradually elevated the solution temperature of P2ID nanofibers to 45.5 °C with the same photothermal effect as free ICG to 46.4 °C. This indicated the ICG in nanofibers could provide a significant photothermal effect and the temperature above 43 °C was sufficient to induce an irreversible damage to cancer cell [P2ID nanofibers after five on/off cycles of NIR irradiation. The temperature elevations of P2ID nanofibers exhibited no visible decrease and the temperature change was reproducible during five cycles, indicating the P2ID nanofibers have excellent photostability. The softened fibrous structures of P2ID nanofibers after NIR irradiation were observed by FE-SEM  than that of P2ID fibers (227 ± 39 nm). These results demonstrated the drug release process could be successfully manipulated by ICG within nanofibers via photothermal effect. This NIR light remote system can achieve precisely and actively controlled drug release by irradiation.The NIR light-dependent release behaviors of urements . The P2IPCL and P2 nanofibers were investigated against L929 cells. Cells seeded on culture plate without any treatment acted as the control group. As shown in PCL and P2 nanofibers had good L929 cell compatibility with no cytotoxicity, which was suitable for biomedical applications. In order to evaluate the combined therapeutic effect of P2ID nanofibers for cancer cells under NIR light irradiation, in vitro cytotoxicity of free DOX, P2ID, P2D, and P2I nanofibers with or without 808 nm laser irradiation were examined using HeLa cells via MTT assay. The drug-loaded nanofibers contained 27 μg/mg of DOX and/or 27.5 μg/mg of ICG. As shown in P2 nanofibers incubated with HeLa cells for 24 h was around 90%, indicating good biocompatibility and no significant cytotoxicity of P2 nanofibers. The cell viability of drug-loaded nanofibers  in the dark also showed high cell survival due to the less amount of drug release. Before confirming the anticancer efficacy of P2ID nanofibers for the application of combination therapy, the cells treated with NIR laser light at a power density of 0.47 W/cm2 for 5 or 10 min showed no phototoxicity in our experimental conditions. Then, P2ID nanofibers were used to evaluate the combined chemotherapy and photothermal therapeutic effect. The results showed the viability of HeLa cells had an irradiation-dependent nature, from 90.3% to 51.8% and 22.2% for 5 and 10 min irradiation, respectively. The significantly enhanced cell lethality can be attributed to the fast release of DOX triggered by photothermal heating resulted from ICG under irradiation. Moreover, ICG converted light energy into heat under NIR irradiation, which could provide hyperthermia effect on killing cancer cell. On the other hand, the cells cultured with P2D nanofibers under irradiation showed no apparent cell death, indicating the slow release of DOX in the absence of ICG even under NIR irradiation. For P2I nanofibers, the cell viability was about 95.2% and 69.3% after 5 and 10 min laser irradiation, respectively. The less cytotoxicity of P2I fibers compared to that of P2ID fibers suggested only the hyperthermia effect from ICG had low therapeutic effect on killing cells; however, combinational chemotherapy/photothermal therapy had dramatically enhanced the therapeutic efficiency.Good biocompatibility is an essential factor for biomedical applications. Thus, the cytotoxicity of plain P2ID nanofibers in response to light irradiation in HeLa cells. A blue-fluorescent dye DAPI was used to stain the cell nuclei. HeLa cells treated with P2ID nanofibers in the absence of NIR light irradiation showed less DOX fluorescence existence in the cytoplasm and cell nuclei after 20 h incubation  was used to investigate the subcellular localization of the released DOX from cubation b. Howeveb-PMAC copolymer, electrospinning technique, and dual indocyanine green (ICG) and doxorubicin (DOX) encapsulation for combination therapy. This light-triggered LDDS system (P2ID nanofibrous mat) can effectively convert light to thermal energy and simultaneously accelerate the release of encapsulated DOX when activated by NIR light. The developed P2ID mat also exhibits excellent antifouling property and biocompatibility. Intracellular DOX distribution confirms more drug release due to the light triggered release. Moreover, the P2ID mat can generate heat and release anticancer drugs, thus causing significant death of cancer cells. We demonstrate that the light-triggered P2ID LDDS presents excellent antifouling property, superior combinational chemotherapy/photothermal therapy on cell growth inhibition, and an effective treatment with low systemic toxicity for treating cancers.In this work, we prepared a NIR light-triggered localized drug delivery system (LDDS) based on zwitterionic PCL-


Resistance to antibiotics is considered one of the most urgent global public health concerns. It has considerable impacts on health and the economy, being responsible for the failure to treat infectious diseases, higher morbidity and mortality rates, and rising health costs. In spite of the joint research efforts between different humans, animals and the environment, the key directions and dynamics of the spread of antimicrobial resistance (AMR) still remain unclear. The aim of this systematic review is to examine the current knowledge of AMR acquisition, diversity and the interspecies spread of disease between humans, animals and the environment. Using a systematic literature review, based on a One Health approach, we examined articles investigating AMR bacteria acquisition, diversity, and the interspecies spread between humans, animals and the environment. Water was the environmental sector most often represented. Samples were derived from 51 defined animal species and/or their products A large majority of studies investigated clinical samples of the human population. A large variety of 15 different bacteria genera in three phyla  were investigated. The majority of the publications compared the prevalence of pheno- and/or genotypic antibiotic resistance within the different compartments. There is evidence for a certain host or compartment specificity, regarding the occurrence of ARGs/AMR bacteria. This could indicate the rather limited AMR spread between different compartments. Altogether, there remains a very fragmented and incomplete understanding of AMR acquisition, diversity, and the interspecies spread between humans, animals and the environment. Stringent One Health epidemiological study designs are necessary for elucidating the principal routes and dynamics of the spread of AMR bacteria between humans, animals and the environment. This knowledge is an important prerequisite to develop effective public health measures to tackle the alarming AMR situation. Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria [tet (M) and tet (Q), and the integron-associated sulfonamide resistance gene, sul1, are the top three widely transferred mobile ARGs on the bacterial species level [The O’Neill report reviewed the problem of antimicrobial resistance (AMR) in 2016, proposing recommendations from both economic and social perspectives. The landmark report estimated that, by 2050, ten million lives will be lost annually due to AMR . Antimicbacteria . The tetes level . IncF plIn spite of the increasing prevalence of AMR bacteria-associated infections, the development of new antibiotics is currently stagnant. Although humans, animals and the environment are closely linked and interconnected in complex ways, the direction and dynamics of AMR spread between hosts, and commensal and pathogenic bacteria, still remain unclear ,10,13. Rwww.jpiamr.eu (accessed on 15 August 2019)) stated the following critical knowledge gaps: How different sources contribute to antibiotics and antibiotic resistant bacteria in the environment, the environmental attribution (anthropogenic inputs in particular) on the evolution of resistance, the impacts of exposure to resistant bacteria from the environment on human and animal health, and the efficacy of technological, social, economic and behavioral interventions, to reduce environmental antibiotic resistance. Similarly, Woolhouse et al. [In 2017 in Gothenburg, Sweden, the Joint Programming Initiative on Antimicrobial Resistance  and pathogenic bacteria ,10,21. AHowever, many studies attribute blame to certain compartments, usually without providing scientific evidence, instead of applying a holistic approach to investigate the spread of AMR. We hypothesized that there is no substantial evidence-based knowledge on the routes and directions of the spread of AMR between all three compartments; therefore, we undertook this review to summarize the current knowledge on acquisition, diversity and the interspecies spread of AMR between humans, animals and the environment.TM qualitative analysis software, assessed the investigated fields in humans, animals, the environment and the geographical location. Further, we examined the investigated bacteria and resistance genes, and the methods of AMR susceptibility testing. Attention was paid to studies on the direction of transmission and spread. Most of the studies investigated only one or two of the three compartments–either human and animal, human and environment or animal and environment compartments–but only few of them investigated all three compartments, in accordance with a HSES approach; this might give more insights into AMR dynamics. The study selection process can be seen in A detailed plan of the systematic review is provided in the Online Groupings of compartments were consensually made between the reviewers during the review process. They included the broader environmental compartment with highly specific sections, such as water or soil, but also included less specific sections, such as the closer animal environment. The specification of water sources was straightforward, as was the animal sector, for which most of the animal species could be identified. Foods sourced from animals were identifiable as from specific animal species, apart for a few specified exceptions. Human compartments were distinguished as of clinical or non-clinical origin, with some unknown sources. Bacterial genera and phyla could all be identified and were listed in the order of frequency of reporting.From an initial dataset of 9115 different publications that fit our search criteria , we finally included a total of 89 different publications into our systematic review see . The 89 Included publications cover 92 different countries (including oceans but excluding metagenomic datasets), with China (20%) and the United States (20%) most represented see .In total, 60 studies investigated water, in particular wastewater/sewage . Water wSamples were derived from 51 defined animal species and/or their products  from individuals of different ages, health status and country of origin. A large majority of studies (65%) investigated clinical samples of the human population see . ClinicaA large variety of 15 different bacteria genera see  in threeMore than 95% of the 89 publications fulfilling the criteria for study inclusion, following the consensus, did not address the spread of AMR in detail. In AMR spread between the three compartments occurs in different ways. Transmission can occur horizontally or vertically through ARGs, and it can also occur through AMR bacteria themselves exchanging between the different compartments. However, the latter form of transmission is controversial, as the exchange of AMR bacteria themselves between the different compartments does not necessarily lead to colonization of these “new” hosts, which is required for sustained conservation of ARGs. Furthermore, the expression of ARGs is dependent on extrinsic factors, as described below, and might not be conserved after an exchange into another compartment.Scientific evidence for many hypotheses concerning AMR transmission dynamics between the different compartments is insufficient and often limited to descriptive or comparative studies, e.g., through comparison of the prevalence of AMR bacteria and/or ARGs in different compartments. A higher prevalence of AMR bacteria/ARGs in certain compartments is often attributed to the emergence of this specific antibiotic resistance within the compartment. Furthermore, the detection of similar or identical resistance genes within different compartments is often considered as proof of AMR transmission. However, the latter scenario could also occur if the involved compartments are subjected to the same or similar selection pressure and/or previously unknown driver. The investigation of mobile genetic elements, as well as their transmission abilities, e.g., filter matings for the investigation of plasmid transmission ,28,29, lGatica and colleagues  reportedDifferent publications ,41,42,43Numerous publications ,49,50,51The high need for the better understanding of such factors, as detailed below, is best addressed using a One Health/HSES approach.Seasonality: According to Hu and colleagues [lleagues  no signilleagues  confirmelleagues . The mecTime frame and geographical location: Different studies [ studies ,46 descr studies ,52,53,54 studies ,55,56 an studies ,58. Thes studies ,43,44.Sampling site within the same compartment: Ibekwe and colleagues [E. coli isolates were more diverse in surface water than in sediment, with more complex AMR profiles found in isolates from surface water than from sediments. The authors suggested that sediment host resident bacteria populations, whereas more transient populations are present in surface water [lleagues  demonstrce water .Age categories: Mutters and colleagues [Staphylococcus aureus (MRSA) carriage, depending on the age and production category of pigs; here, porkers showed the highest rate, followed by piglets and sows. Ma and colleagues [lleagues  describelleagues  also shoGenetic variation: It is hypothesized that a limited gene pool with low inter-individual variability, as is present, for example, in the chicken industry, where there are only a few genetic lines of primary breeding flocks utilized, might be associated with a lower ARG-gene pool and lower resistome variability, respectively [ectively ,35,45.E. coli (blaCTX-M-1) isolates from poultry. Antunes and colleagues [Atterby and colleagues  investiglleagues  report sMutters and colleagues  observedFitzpatrick and Walsh  attributInadequate prescribing and the overuse of antibiotics in livestock production and agriculture are considered the most important factors behind the emergence of antibiotic resistance, but numerous studies demonstrate that AMR is much more complex and many additional factors influence its presence and spread ,49,50,51It is striking that many studies are focused on the responsibility of certain compartments for the current AMR problem, instead of providing more evidence-based data. Many studies stated that the high usage of antibiotics in veterinary medicine is one of the main drivers of emerging antibiotic resistance , and resLimitations of the study methodology: The primary objective of this study was a qualitative systems analysis. We did not perform a formal analysis concerning the risk of bias in the included studies because we performed no quantitative meta-analysis of the data. We limited our search to one database (Pubmed). The comparability between different countries may be difficult as antibiotic resistance dynamics can vary between different countries, as well as between the antibiotics used/prescribed. Limitations may also include risks of bias, such as selection bias, attrition bias, publication bias, inadequate blinding, selective outcome reporting, and inconsistencies that may lead to clinical and statistical heterogeneity.Most of the limitations encountered in the publications analyzed for this review involved the study design . In many studies, isolates are poorly described ,26, withAnother problem involved the separation of the different compartments. Some studies ,54,63 inThe limited sample size ,53,64,65Another bias seen in the publications included in this review involved the populations studied. Most of the studies including animal samples, investigated animal species in terms of their high relevance for human beings, such as pets or livestock and their products. Although this allows statements about transmission dynamics between different compartments that are in close relation, this could lead to a spatial bias; this is because AMR emergence in each compartment is no longer independent of the other compartments due to their closely linked habitats.Most of the studies are limited to a subset of resistance genes ,67 or phDespite the strict inclusion criteria chosen for this systematic review , many of the remaining publications only assessed the prevalence of phenotypical and/or genotypical resistance, without an integration of epidemiological analyses ,68,70. AFurthermore, the approaches chosen for the investigation of source tracking/phylogeny analysis, were often of insufficient experimental quality. Many studies used the PFGE, however, the authors applied this method using only a single restriction enzyme ,71 for tIn the studies included in this review, the One Health principle was often overlooked when analyzing and discussing the data. Compartments and interfaces between them were not considered all together; often only two were compared, while the third one was disregarded ,47.Best practices for integrated One Health studies on AMR should thus include the following minimal standards: (a) State a clear hypothesis of the study; use standard epidemiological study design  with statistical power calculation depending on the research question; (b) Comprehensive reporting of spatio-temporal metadata, that allows the relation of causal pathways between the different compartments ; (c) Clarify the study objective for specific resistance genes or the whole resistome. Relate phenotypic and genotypic resistance; (d) Attempt an assessment of an incremental benefit of knowledge, cost and disease burden from an integrated study design .There is evidence that antibiotic resistance is closely linked to specific hosts and/or compartments ,45, whicOnly very few studies investigated the environmental compartment in detail when looking at the spread and transmission dynamics of antibiotic resistance. However, the environment might be an important component of this system, especially considering it as an interface between humans and animals. This further enforces the need for expanding the One Health to a HSES concept, allowing a better investigation of all influencing factors and their complex interplay; this should be supported by various studies ,59,75,76A similar systematic review has been performed for integrated studies on antimicrobial resistance genes in Africa and provides guidance for prospective integrated cohort studies . A furth