However, most molecules in GDB17 are too complex to be considered for chemical synthesis. To address this limitation, we report GDBChEMBL as a subset of GDB17 featuring 10 million molecules selected according to a ChEMBL-likeness score (CLscore) calculated from the frequency of occurrence of circular substructures in ChEMBL, followed by uniform sampling across molecular size, stereocenters and heteroatoms. Compared to the previously reported subsets FDB17 and GDBMedChem selected from GDB17 by fragment-likeness, respectively, medicinal chemistry criteria, our new subset features molecules with higher synthetic accessibility and possibly bioactivity yet retains a broad and continuous coverage of chemical space typical of the entire GDB17. GDBChEMBL is accessible at  Innovation at the level of chemical structures is an essential part of drug discovery. Novelty often results from chemical intuition however this approach is increasingly difficult as the number of known molecules increases. Novelty is similarly limited in virtual combinatorial libraries  with high confidence datapoints  . We then sum all shingle values and divide the sum by the total number of shingles in the molecule (Equation 1).The frequency of occurrence of the 636,979 molecular shingles up to a diameter of six bonds found in our ChEMBL subset follows a power law distribution . To compThe histogram of CLscore for the 457,139 ChEMBL reference molecules is approximately Gaussian with a peak at CLscore = 3.9 . DrugBan9 molecules. Uniform sampling finally yields a final set of 10 million molecules evenly distributed across molecular size, stereochemical complexity and polarity, forming the database GDBChEMBL  and keeping molecules with CLscore ≥ 3.3, a cut-off value which retains 78.3% of our ChEMBL subset, eliminates 84.3% of GDB17. The remaining 26.2 billion molecules are then binned in triplet value bins considering heavy atom count (HAC 1-17), stereocenter count  and heteroatom count . There are 538 different triplet value bins, which are occupied by 1 to 1.6 × 10DBChEMBL .As a consequence of uniform sampling, the heavy atom count (HAC) profile of GDBChEMBL resembles that of FDB17 and GDBMedChem and is relatively flat compared to the very steep peak at HAC = 17 in the parent database GDB17 . UniformGDBChEMBL displays a very broad distribution in terms of hydrogen bond donor atoms HBD, , hydroge3 atoms than ChEMBL  in GDBChEMBL than for GDB17, FDB17, or GDBMedChem, reflecting the correlation between CLscore and SAscore noted above . SimilarBL fsp3, . As a coBL fsp3, . DespiteBL fsp3, .http://faerun.gdb.tools, each point represents one or more molecules present at the corresponding position and can be color-coded according to a molecular property selected from the faerun menu.To gain an overview of GDBChEMBL we computed Molecular Quantum Number (MQN) fingerprint values  from the frequency occurrence of circular substructures, called molecular shingles, in a subset of the database ChEMBL consisting of compounds active on single protein targets with high confidence datapoints. This selection reduced GDB17 by 84.3%, leaving 26.2 billion molecules, which we sampled uniformly across molecular size, stereochemistry and heteroatoms to form GDBChEMBL comprising 10 million molecules.Property profiles, chemical space maps and similarity searches show that GDBChEMBL is very different from our earlier GDB subsets FDB17 and GDBMedChem and spans chemical space more continuously. At the same time, the correlation between CLscore and the synthetic accessibility score (SAscore) implies that GDBChEMBL molecules will be on average easier to synthesize than molecules from FDB17 and GDBMedChem, which have significantly lower CLscore and higher SAscores. We anticipate that the requirements for GDBChEMBL molecules to share a minimum number of substructures with molecules of known bioactivities from ChEMBL will also facilitate target prediction and the selection of interesting GDB molecules for synthesis and testing.https://www.ebi.ac.uk/chembl/. Data points for extraction of molecular shingles were selected by applying the same restrictions that were used for extraction of training data for our Polypharmacology Browser PPB2  database was downloaded from https://github.com/reymond-group/GDBChEMBL.Scoring of GDB17 molecular structures was achieved by decomposition to molecular shingles in the exact same way as described for ChEMBL reference shingle extraction. For a specific query structure, all shingles are uniquely counted, then looked up in the ChEMBL reference database and upon match, logarithmic abundancy is summed up. The final CLscore is given by the ratio of total logarithmic abundancies of matched unique shingles to total unique shingles in the query structure. All respective scripts are accessible at: All 166.4 billion molecular structures of GDB17 were decomposed to unique substructures in the same way as described for ChEMBL reference molecules. Only structures with CLscore ≥3.3 were stored. The final GDBChEMBL was obtained by distribution of all filtered 26.2 billion structures to 538 property triplet bins . Property information was gathered using RDKit. Bins with 5+ hetero atoms and/or 8+ chiral atoms were merged. The actual even sampling was performed by sorting all property bins by size and defining target structure count as 10 million. Iteratively, remaining target count was divided by count of remaining bins, keeping all bins of size smaller than the current number to sample randomly. For each step, number of previously selected structures was subtracted from target count until random sample per remaining bins was lower than bin size. At this point, sample size was kept constant for all further bins.Property color coded 3D maps for GDBChEMBL, FDB17, and GDBMedChem were generated using FUn (doc.gdb.tools/fun), an in-house developed framework for interactive visualization of chemical spaces on the web. Datasets were given as plain text, consisting of the four columns (space-separated): SMILES-string, numeric ID, 42 MQN descriptors (semicolon-separated) and further molecular properties used for map coloring (semicolon-separated). Next, the preprocessing toolchain was used to project the 42-dimensional MQN-space to 3D by applying Principal Component Analysis (PCA) and to generate all further files needed for visualization. Finally, the Underdark server was run using docker with Faerun visualization containers mapped.https://github.com/spotify/annoy) which were created for the 42-dimensional MQN property space, as well as for 256-bit ECfp4. A third search option, MQN-MHFP6, initially searches using the MQN Annoy tree followed by resorting after Jaccard distance to query molecule in the MHFP6 fingerprint space (https://github.com/reymond-group/mhfp). The search tool is available at: gdb.unibe.ch/tools.For better accessibility, GDBChEMBL is provided as a web-based interactive similarity search tool. The implementation uses HTML, Bootstrap, JavaScript, and the python Flask framework. Search times were reduced using Annoy trees (Approximate Nearest Neighbors Oh Yeah, All datasets generated for this study are included in the article/supplementary material.SB designed and realized the study and wrote the paper. J-LR designed and supervised the study and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neisseria gonorrhoeae strains, it successfully predicted their AMR phenotypes based on a pan-genome analysis. Furthermore, it identified 328 genetic alterations in 23 known AMR genes and discovered many new AMR-associated genetic alterations in ciprofloxacin-resistant N. gonorrhoeae, and it clearly indicated the genetic heterogeneity of AMR genes in different subtypes of resistant N. gonorrhoeae. Additionally, PARMAP performed well in predicting the AMR phenotypes of Mycobacterium tuberculosis and Escherichia coli, indicating the robustness of the PARMAP framework. In conclusion, PARMAP not only precisely predicts the AMR of a population of strains of a given species but also uses whole-genome sequencing data to prioritize candidate AMR-associated genetic alterations based on their likelihood of contributing to AMR. Thus, we believe that PARMAP will accelerate investigations into AMR mechanisms in other human pathogens.Antimicrobial resistance (AMR) has emerged as one of the most urgent global threats to public health. Accurate detection of AMR phenotypes is critical for reducing the spread of AMR strains. Here, we developed PARMAP  to predict AMR phenotypes and to identify AMR-associated genetic alterations based on the pan-genome of bacteria by utilizing machine learning algorithms. When we applied PARMAP to 1,597  Neisseria gonorrhoeae strains. However, a comprehensive tool that integrates SNPs and gain/loss of genes in the pan-genome to predict AMR phenotypes and to prioritize candidate AMR-associated genomic alterations (based on their likelihood of contributing to AMR) is still lacking  has emerged as one of the most urgent global threats to public health . Many ba lacking .Current approaches for AMR prediction commonly make use of SNPs derived from comparisons of a newly assembled genome against the genome of a reference strain . Manson Burkholderia pseudomallei obtained ceftazidime resistance by loss of a penicillin-binding protein (PBP). Additionally, many higher-order computational approaches have been applied for cell type classification in research on single-cell genomics. These approaches include the uniform manifold approximation and projection (UMAP) technique, which is a novel manifold learning technique for dimension reduction  algorithm consistently outperformed support vector classification (SVC), random forest (RF), and logistic regression (LR), with area under the curve (AUC) scores >0.98 for resistance to each of the antibiotics investigated in N. gonorrhoeae strains. Moreover, PARMAP analysis revealed the genetic heterogeneity of ciprofloxacin resistance genes in N. gonorrhoeae. It identified 5,830 AMR-associated genetic alterations by deducing the genetic content variability, and 328 of the genetic alterations were associated with 23 known AMR genes. To test the robustness of our method, we applied PARMAP to predict the AMR phenotypes in Mycobacterium tuberculosis and Escherichia coli. As expected, it performed well in predicting AMR phenotypes related to various antibiotics in both species. These results demonstrate that PARMAP enables precise AMR prediction in a population of strains and prioritizes candidate AMR-associated genetic alterations based on whole-genome sequencing (WGS) data. Therefore, we believe that it will be useful for mechanistic studies on AMR phenotypes in a wide range of pathogens.In this study, we present PARMAP , an integrative computational framework for predicting AMR phenotypes and for identifying AMR-associated genes based on the pan-genome of bacteria by incorporating machine learning algorithms. PARMAP accurately predicted the AMR phenotypes of N. gonorrhoeae dataset, we downloaded the WGS data of 1,597 strains derived from three countries in a previous study   was used v8.2.12 , with a  v8.2.12 .p-value cutoff of 0.05 using the Benjamini–Hochberg procedure  was performed on the gene allele features using the scanpy package . All strrocedure . Thereafci denotes the number of strains with the gene allele in the ith cluster, and si denotes the total number of strains in that cluster.where The maximum proportion of strains with a particular gene allele in the resistant clusters was defined as follows:m denotes the number of resistant clusters.where Finally, AMRS was defined as follows:pj denotes the proportion of clusters with a particular gene allele in the jth susceptible cluster, and n represents the total number of susceptible clusters.where We then selected the informative gene allele features based on the AMRS cutoff of 0.9.Antimicrobial resistance prediction models were established by learning from the matrices of gene allele features (filtered based on the AMRS cutoff) and the phenotype of each strain using a set of machine learning algorithms, comprising GDBT, LR, RF, and SVC. Briefly, the strains were randomly divided into the training dataset (80%) and the testing dataset (20%) by the train_test_split function in the scikit-learn package . The AMRFirst, the machine learning models were trained using the training dataset . An in-sample testing dataset with the same sample size as the independent testing dataset  was then randomly selected from the training dataset using train_test_split in the scikit-learn package . The indUsing the train_test_split function in the scikit-learn package, the strains were randomly divided into the training dataset (80%) and the testing dataset  100 times. Feature selection and AMR prediction were performed independently in each permutation. The AUC and Recall values related to five-fold cross-validation and the independent testing were then calculated. Finally, boxplots were used to evaluate the robustness of the PARMAP algorithm.in silico 3D models were established using the Iterative Threading Assembly Refinement (I-TASSER) platform  were performed using SciPy .https://github.com/452990729/PARMAP) under GNU General Public License v3.0.PARMAP is an open-source package freely available in the GitHub repository  pan-genome construction, (ii) feature selection, and (iii) AMR prediction .de novo assembled draft genomes or genomes from other sources  genome assembly, (b) gene prediction and multiple sequence alignment, and (c) characterization of the pan-genome. First, gene prediction was performed to annotate  sources . We only sources . Next, m sources . We iden sources . We then sources .To extract the fine-grain genetic variations indicative of AMR evolution, we divided each gene cluster of the pan-genome based on the gene alleles present, i.e., the exact amino acid sequence variants, with each gene allele representing a potential AMR feature. We then established a gene allele–strain (GS) matrix showing whether each gene allele was present or absent in each specific strain . Our appTo develop a model for AMR prediction, we took advantage of several machine learning algorithms. To this end, all the strains were segregated into resistant and susceptible groups based on minimum inhibitory concentration (MIC) data or predefined AMR phenotypes from previous studies. Thereafter, 80% of the strains were randomly defined as the training dataset for feature selection and model training, while the remaining 20% were defined as the independent testing dataset . Next, tN. gonorrhoeae has substantially compromised antibiotic effectiveness  pan-genome construction, (ii) feature selection, and (iii) AMR prediction. We applied PARMAP to investigate AMR-associated genotype–phenotype relationships in 1,597 sequenced via horizontal gene transfer  of the ACNB gene, may disrupt the function of the ACNB protein based on 3D structural modeling of the ACNB protein  to predict AMR. We found that the ensemble methods (RF and GDBT) achieved better results than the LR and SVC algorithms. In particular, our analysis confirmed that the GDBT model was the most accurate model for predicting AMR in a population of strains of human pathogens.Furthermore, using data on We are aware that PARMAP does not account for non-protein-coding genes in the pan-genome construction, which limits its predictive power. Therefore, PARMAP cannot identify non-protein-coding genes related to AMR, such as 23S rRNA and 16S rRNA (rrs). However, only 84 (2.8%) of the 3,044 AMR entries in the CARD database are for non-coding genes, including 23S rRNA and rrs, and the resistance to 45 (97.8%) antibiotics is conferred by protein-coding genes . TherefoN. gonorrhoeae and other pathogens such as M. tuberculosis and E. coli. It both recovers known AMR genes and reveals potential novel AMR genes. The PARMAP framework integrates a pan-genome analysis and machine learning methods to provide a comprehensive tool for analyzing the associations between genotypes and phenotypes. We believe that PARMAP will provide vital information for mechanistic investigations of AMR in N. gonorrhoeae and other pathogens.Numerous methods have been developed to predict AMR in different pathogens, which have various advantages and disadvantages . Future All datasets presented in this study are included in the article/JZ and XL designed the project and wrote the manuscript. XL conducted the data curation, model training, and implementation of the computational framework. JL and YH provided technical support and helpful discussions. JZ convinced the data analysis results. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antimicrobial resistance (AMR) in bacteria is a complex subject, why one need to look at this phenomenon from a wider and holistic perspective. The extensive use of the same antimicrobial classes in human and veterinary medicine as well as horticulture is one of the main drivers for the AMR selection. Here, we applied shotgun metagenomics to investigate the AMR epidemiology in several animal species including farm animals, which are often exposed to antimicrobial treatment opposed to an unique set of wild animals that seems not to be subjected to antimicrobial pressure. The comparison of the domestic and wild animals allowed to investigate the possible anthropogenic impact on AMR spread. Inclusion of animals with different feeding behaviors  enabled to further assess which AMR genes that thrives within the food chain. We tested fecal samples not only of intensively produced chickens, turkeys, and pigs, but also of wild animals such as wild boars, red foxes, and rodents. A multi-directional approach mapping obtained sequences to several databases provided insight into the occurrence of the different AMR genes. The method applied enabled also analysis of other factors that may influence AMR of intestinal microbiome such as diet. Our findings confirmed higher levels of AMR in farm animals than in wildlife. The results also revealed the potential of wildlife in the AMR dissemination. Particularly in red foxes, we found evidence of several AMR genes conferring resistance to critically important antimicrobials like quinolones and cephalosporins. In contrast, the lowest abundance of AMR was observed in rodents originating from natural environment with presumed limited exposure to antimicrobials. Shotgun metagenomics enabled us to demonstrate that discrepancies between AMR profiles found in the intestinal microbiome of various animals probably resulted from the different antimicrobial exposure, habitats, and behavior of the tested animal species. The emergence of antimicrobial resistant (AMR) bacteria is one of the most important public health challenges worldwide . EstimatSalmonella might threaten public health through food of animal origin or direct contact with an animal or with animal husbandry facilities [The wide use of similar antimicrobials in human and veterinary medicine contributes to the selection of AMR bacteria and their spread in nature. Thus, the environment polluted with municipal sewage, manure and slurry becomes a reservoir of AMR genes and a risk element in its further transmission . Animalscilities , 9, 13.Several aspects have an impact on the occurrence and spread of AMR in animals. Animal behavior influences potential exposure to the acquisition of resistant bacteria. Differences in diet may alter the intestinal microbiome. Feed may be a source of compounds affecting gut resistome and serve as a vector of AMR determinants , 15. BeaFor effective combat of increasing bacterial AMR in times of intensification of animal production, the growing popularity of game meat and expansion of the human population, it is particularly important to assess the role of different animal species in AMR dissemination .Herewith we used a powerful shotgun metagenomic tool to prove the hypothesis that diverse antimicrobial exposure, habitats, and feeding behavior of different animal species lead to discrepancies between AMR profiles found in their intestinal microbiome.To investigate the AMR epidemiology we selected intensively produced farm animals: chickens, turkeys, and pigs, which are often exposed to antimicrobial treatment opposed to a unique set of wild animals such as wild boars, red foxes, and rodents that seems not to be subjected to antimicrobial pressure.Sus scrofa allowed to compare the same species living in free and farmed conditions. The study of red foxes (predators) and rodents as their presumed prey was another benefit that allowed us to look into feasible AMR genes flow within the trophic chain.The rationale for examining poultry was short fattening time/life span and AMR group treatment practiced during breeding. The inclusion of pigs and wild boars representing Our objective was to investigate and quantify the scale of the AMR phenomenon in several species of domestic and wild animals. We applied shotgun metagenomics of total DNA isolated from intestinal content of animals to explore the abundance of different resistance genes, and to examine bacterial and plasmid composition. We also looked at the possible diet of selected animals in the context of AMR spread.Gallus gallus, n = 10), turkeys , pigs  and also wild animals represented by wild boars , red foxes  and rodents: eight forest mice (Apodemus flavicollis), one field mouse (Apodemus agrarius) and one field vole . All samples were derived from healthy animals. In case of farm species, samples were collected close to slaughter and constituted fraction of samples tested within the EU-monitoring (2017–2018). Wildlife samples came from animals covered by rabies and Leptospira control programs.A total of 60 samples of intestinal content from different farm and wild animals originating from 2016 and 2018 were selected among the numerous samples available at the National Reference Laboratory for Antimicrobial Resistance (NRL) Poland. Samples included farm animals therein extensively produced poultry: chicken broilers . Slaughter animal samples were collected within 2013/652/EU: Commission Implementing Decision of 12 November 2013 on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria. Red fox and wild boars samples collected under the Polish Regulation Ordinance of the Minister of Agriculture and Rural Development of December 17, 2004 regarding the definition of disease entities, the manner of conducting control and the scope of control tests of animal infections.Leptospira. All procedures were carried out according to the ethical standards for the use of animal samples and were approved by the Local Ethics Committee for Animal Experimentation in Lublin, Poland (Resolution No. 30/2016). Animals were caught in their natural foraging areas (forests and meadows) using Sherman traps. Live animals were transported to the laboratory and euthanized on the same day with Isofluranum. The anesthetic was dosed according to the manufacturer's recommendations. All efforts were made to minimize animals suffering.Rodents were captured during 2016 and 2017 for the purpose of grant on Upon arrival at NRL samples were frozen and stored at −80°C until further processing in autumn 2018.All samples were only thawed once just before DNA extraction. Total DNA was extracted from each sample with a QIAamp Fast DNA Stool Mini Kit  according to a published protocol  with modhttp://www.ebi.ac.uk/ena/data/view/PRJEB40824).DNA was shipped on dry ice for library preparation and sequencing at the National Food Institute, Technical University of Denmark. DNA libraries prepared with a Nextera Library Preparation Kit (Illumina) were subsequently sequenced with the NextSeq platform (Illumina), using 2 × 150 paired-end sequencing per flow cell. A high output flow cell was used with a triple-capacity FC-404-2004 NextSeq 500/550 High Output v2 kit (300 cycles).The reads were deposited at the European Nucleotide Archive (ENA) (http://www.ncbi.nlm.nih.gov/genbank/). The selected databases were mapped in the following order: ResFinder and Plasmid (version 20180226) in option fullmode, and Bacteria (version 20180226), Bacteria_draft (version 20180226), Vertebrates_mammals (version 20180306), Vertebrates_other (version 20180306), Invertebrates (version 20180306), Plant (version 20180306) with bestmode approach.BBduk (BBMap software) was applied for raw read trimming  and BWA-Bestmode mapping, based on the highest alignment score, assigned the read-pair to only one reference sequence in one of all specified bestmode databases. In case of equal alignment scores the read-pair was assigned to the database defined as first. Fullmode option as previously described was applied for AMR genes and plasmid databases to enable read mapping to multiple databases .The results of ResFinder read mapping for individual genes were aggregated to clusters based on 90% identity as described previously . Based o6 and the results presented as reads per million (RPM).To validate our data, AMR results obtained for broiler chickens and pigs were compared to the data of Polish broiler and pig samples included in the EU-funded EFFORT project . In the Muridae and Soricidae families were considered the host material, similarly in poultry samples reads mapped to the Anatidae and Phasianidae families were disregarded.Relative abundance values were visualized in heat maps. Reads mapped to Vertebrates_mammals, Vertebrates_other, Invertebrates and Plants databases were considered as potential diet components of the tested animals. Examples of crops, fodder plants, wild plants, different animal species and insects were selected for the analysis. As for rodents, reads assigned to the The limitation of the method is that mapping to highly homologous sequences might result that the reads are assigned to related species. To reduce the bias of possible incorrect mapping when one genus was represented by several species, the sums of those reads were shown on heat maps.https://www.rproject.org/) using the library(vegan), library(pheatmap), library(ggplot2), library(reshape2), library(RColorBrewer), library(plyr), and library(grid) packages. The exception was Analysis and visualization of results on graphs and heat maps were carried out in the open source RStudio 3.5.3 version for Windows (The entire sequencing dataset yielded on average over 21.6 billion base pairs (bp) per sample. From over nine hundred million reads (ranging from ~111 to ~196 million reads per sample) 0.03% were attributed to AMR genes . In totatet(Q) being the most abundant gene in all tested animals except rodents. The tet(X) gene that confers resistance to tetracyclines and tigecycline was also noted (mef(A), lnu(C), and erm(B) in farm species while mdf(A) was more abundant in foxes and wild boars (ant(6)-Ia, aph(3′)-III, and aph(3′)-IIIa genes prevailed. Determinants encoding AMR towards beta-lactams, e.g. cfxA6 or blaACI were more often found in pigs and were seen at comparable levels in both tested poultry species e.g. cfxA, blaOXA-347. It is worth emphasizing that blaOXA-347 prevailed in foxes and a few other beta-lactam genes encoding AmpC type β-lactamases, e.g. blaCFE, blaDHA, blaCMY (the blaCMY–blaBIL–blaLAT cluster) were unique to this species  gene conferring cross-resistance to phenicols, lincosamides, pleuromutilins, streptogramin A and oxazolidinones was only found in farm animals -Ia, aph′-III, anies Figs . cfr(C)  animals . AMR tow)-Ia, aph′-III, anals Figs .Dendrogram for genes was clustered on Pearson correlation coefficients, whereas for samples it was based on the Bray-Curtis dissimilarity indexes. Complete linkage clustering was applied for dendrograms. Heat map presents the 50 most abundant determinants but all resistance genes found in samples were included in computations.The highest entire plasmid content were observed in wild boars and foxes  in comparison to other animals tested. Lower levels were found in poultry species (over 590 FPKM in chickens and 480 FPKM in turkeys) and in rodents (over 360 FPKM). Interestingly, pigs had the least observed number of reads mapped to the plasmid database with less than 250 FPKM . EvidencBacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria were the most abundant, but discrepancies in specific phyla contribution were observed in different animals. The highest level of Bacteroidetes was found in farm animals   . The rodReads assigned to plant, vertebrate and invertebrate genomic material represented 0.02%, 20.22% and 0.03% of all reads respectively . Poultryvia food of animal origin but also through direct contact with the animal or animal husbandry environments [Data collection on AMR determinants in animals is crucial to effectively combat resistant bacterial pathogens that may affect consumer health not only ronments . Thereforonments , 20.Not surprisingly, our findings revealed higher AMR level in domestic animals in comparison to wild. Tetracycline resistance dominated AMR profiles and this probably results from ongoing selective pressure in the environment, as tetracyclines have been the most widely applied antimicrobial class in veterinary medicine and horticulture over decades , 26. AccIn comparison to wild animals, resistomes of domestic species were characterized by greater gene diversity and richness. These results and the overall AMR level in domestic animals would clearly indicate the selective effect of antimicrobial usage (AMU) . High coCajanus cajan) was noted [Other aspects that may affect the result are indicated by analysis of the potential diet. We found the presence of DNA host material in all of the tested samples and expected diet composition. As predicted, evidence of fodder plants in farm animals, including imported ones, e.g. pigeon pea  gene in all tested animals except rodents and found it in highest abundance in poultry, particularly turkeys. The result is cause for concern, as the gene encodes resistance not only to tetracyclines but also to tigecycline, the last resort antimicrobial against multi‐drug resistant Enterobacteriaceae and methicillin‐resistant Staphylococcus aureus [tet(X) in pig and chicken samples were reported formerly [Importantly, our study shown the presence of s aureus . The abuformerly , 31.blaOXA-347 gene both in chickens and turkeys. We assume that this result might be an outcome of penicillins or cephalosporins use as carbapenems are prohibited in livestock treatment in Poland. Previously presence of blaOXA-347 was described in porcine gut microbiome as a result of amoxicillin treatment [cfiA gene encoding metallo-β-lactamase. Other studies confirmed the gene in Bacteroides associated with human infections [Traces of determinants encoding class D beta-lactamases were noted in poultry. Among them we found reatment . In factreatment . In chicfections , 46 and Suidae: wild boars and pigs. High levels of AMR towards tetracycline, beta-lactams, and macrolides in pigs comparing to wild boars indicate the selective effect of antimicrobial classes widely used in pig production sector [Anthropogenic impact on AMR spread might be highlighted by AMR profiles in two species belonging to n sector . Interesn sector , 49. AMRn sector . This gln sector . Glycopen sector . Recent n sector . It shoun sector . Hence, n sector .blaOXA-347 gene encoding class D beta-lactamase, the same as found in both poultry species, was previously noted in Capnocytophaga stomatis and C. cynodegmi, that constitute oral flora components of healthy cats and dogs [E.coli and enterococci isolated from red foxes [E. coli and Enterococcus spp. isolated from this species but without indicating the specific AMR genes [oqxA and oqxB genes. These pumps are disseminated on plasmids among Enterobacteriaceae and Enterococci [Salmonella spp. occurrence, these findings are cause for concern, even taking into account the limitations of the method in plasmid analysis [Our analysis revealed a significant AMR level including resistance to drugs highly important for public health in red foxes. Exposure to AMR derives from discarded food and agricultural waste near urban settlements visited by scavenging animals might to some extent explain the AMR level in this species. The results also indicate that apex predatorial red foxes may accumulate resistance determinants form wild birds, small mammals or reptiles of which traces were found in their potential diet . Numerouand dogs . Beta-laed foxes , 64, buted foxes . FindingMR genes . The AMRerococci –69. Giveanalysis , 71.E. coli derived from rats from Hong Kong revealed high rate of ESBL producers in the rodents living in underground sewers [The fact that we observed only traces of resistance in rodents compared to other animals should be underlined. The choice of this group of animals, collected at forest and meadow areas, was dictated by the fact that their probable contact with human settlements was occasional and therefore the animals had little contact with antimicrobials. Both the current and our previous results confirmed these assumptions . Althougd sewers . Recent d sewers .Insight into animal gut microbiomes and diet indicates that diverse dietary preferences result in differences in bacterial composition. A more varied diet seems to drive gut microbiome diversity, as in the case of wild animals. We could also expect higher diversity among resistance determinants found in wildlife. The contrary seems nevertheless to be the case; the variety of AMR genes appears to be greater in farm animals. The explanation for this might be significant exposure of those species to several drugs, disinfectants and metal ions e. g. copper used as feed additives . The uneTo the best of the authors’ knowledge, this is the first study on AMR in animals that examined the possible diet of selected animals in the context of AMR spread.Notwithstanding bias due to the limited number of samples could have been introduced in this study. The applied method has a limitation in plasmid analysis still, the obtained results were hard to ignore and the authors decided to include this data to indicate the considerations for further studies. Moreover, the overall quinolone and polymyxin resistance level described in this study might be underestimated due to the applied approach. The chromosomal mutations in the quinolone resistance determining region (QRDR) and mutations leading to colistin resistance might have been missed.Here, we applied metagenomics to investigate the epidemiology of AMR in various animal species. The study revealed higher AMR levels as well as higher resistome diversity and richness in domestic species, pointing to antimicrobial usage in the animal production sector as the main AMR driver. Our results also indicate that wildlife constitutes a reservoir of AMR determinants including those encoding resistance to antimicrobials highly important in human medicine. The potential of wildlife as AMR transmission vectors has been proven by plasmid profiles revealed in wild boars and red foxes. We also demonstrated that discrepancies between AMR found in the intestinal microbiomes of various animals probably resulted from different antimicrobial exposure, habitat, and diet. The overall results allowed us to highlight at least a few factors that may foster AMR spread in animals and clearly indicate anthropogenic impact on AMR dissemination.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Poultry production can function as an instrument for poverty alleviation and economic development. As low-income countries transition into higher incomes alongside growing urban populations, there will be an increasing demand for quality sources of animal products. Consequently, poultry production systems will continue to shift from subsidence agricultural practices to intensive food production that implies routine antimicrobial usage. Promotion of intensive poultry production could increase antimicrobial resistance (AMR) within resource-limited settings lacking in effective biosafety and biosecurity measures. Bacterial resistance lessens the portfolio of antimicrobials available in poultry husbandry and potentially human medicine. This issue requires a systems framework in order to evaluate the various social and biological factors driving the emergence of resistance within the context of intensive poultry production.The emergence, spread, and persistence of antimicrobial resistance (AMR) remain a pressing global health issue. Animal husbandry, in particular poultry, makes up a substantial portion of the global antimicrobial use. Despite the growing body of research evaluating the AMR within industrial farming systems, there is a gap in understanding the emergence of bacterial resistance originating from poultry within resource-limited environments. As countries continue to transition from low- to middle income countries (LMICs), there will be an increased demand for quality sources of animal protein. Further promotion of intensive poultry farming could address issues of food security, but it may also increase risks of AMR exposure to poultry, other domestic animals, wildlife, and human populations. Given that intensively raised poultry can function as animal reservoirs for AMR, surveillance is needed to evaluate the impacts on humans, other animals, and the environment. Here, we provide a comprehensive review of poultry production within low-resource settings in order to inform future small-scale poultry farming development. Future research is needed in order to understand the full extent of the epidemiology and ecology of AMR in poultry within low-resource settings. Antimicrobial resistance (AMR) remains a growing threat for human and animal health, lessening the ability to treat bacterial infections and furthering the risk associated with morbidity and mortality caused by resistant bacteria. Ensuring the effectiveness of antimicrobials to treat bacterial infections remains a pressing issue for both veterinary and human medicine ,2,3,4. TAnimal agriculture within low-resource settings is of high importance as many countries transition to more intensive animal farming practices, leading to greater AMU and thus an intensified risk of AMR exposure to animals and humans worldwide . As low-In LMICs, AMU increased by 65% from 2000 to 2015 . The mosGallus gallus domesticus) comprise of 90% of global poultry production, amounting for approximately 23 billion chickens [Poultry, one of the fastest per capita produced livestock ,36 Figu, will cochickens . Foundational reviews have suggested that there is not a global standard in biosecurity practices for small-scale poultry farming ,47. MoreThis review aims to provide a systems lens of the major drivers of AMR in poultry farming within the context of low-resource settings in order to inform future veterinarian and public health policy and implementation. Therefore, this paper will first provide introduction of AMR evolution and spread, followed by description of the origins of AMR in poultry, and then an overview of poultry production systems, thereafter, an evaluation of small-scale poultry development, and concluding with barriers to improved antimicrobial stewardship programs in low-resource settings. Our review provides a novel eco-epidemiological framework for assessing the impacts of intensive poultry farming within low-resource setting.Escherichia coli [AMR bacteria are naturally found in the environment because many antibiotics are produced by other organisms such as fungus  and soil bacteria  . In manyhia coli . Since ihia coli . Typicalhia coli ,59. In chia coli , making hia coli ,61,62,63Acquired bacterial resistance is caused by four general mechanisms including inactivation, target alteration, decreased permeability, and increased efflux . First, Two fundamental biological pathways that facilitate the evolution and dissemination of resistance include vertical gene transfer (VGT) and horizontal gene transfer (HGT) . First, The discovery that antimicrobials fed in subtherapeutic concentrations to poultry expedited their growth was accidental . In 1946Determining productivity gains of AGPs at a global scale remains extremely difficult due to the lack of availability of quality data outside of a few HICs . Bans onThe use of antimicrobials in intensive poultry production is becoming increasingly common at smaller scales within low-resource settings because of its high throughput of meat and egg products ,85,86,87It is important to highlight the varying risk factors associated with large-scale farms in relationship to smaller, family operated operations. Within the context of LMICs, AMR exposure from commercial farms is generally localized to occupational exposure or to animal fecal environmental contamination of AMR pollutants to surrounding soil or water runoff ,94,95. CFamily managed farming operations are found throughout the world and make up the majority of global poultry production  Figure . “FamilySmall-extensive and extensive scavenging poultry farming typically involve local breeds of poultry that can be characterized with a variety of terms ranging from “village”, “indigenous”, “backyard”, or “household” ,105. ForIn contrast to small-extensive and extensive scavenging poultry farming, semi-intensive and intensive family operations apply similar practices as intensive animal production systems at the household level . Small-sUN launched the 2030 Agenda for Sustainable Development to promote peace and prosperity for all people and the planet both present and in the future. This agenda established 17 Sustainable Development Goals (SDGs) for mitigating impacts of human pressure on the planet that has led to planetary crises such as biodiversity loss, climate change, environmental degradation, and negative impacts on health and nutrition. Studies have acknowledged the interrelatedness nature of the SDGs ,126,127.Although poultry development has existed for decades, the recent UN SDGs have stimulated increased development projects centered around poultry husbandry; many of the SDGs overlap within the context of small-scale poultry husbandry ,55 Tabl. These pThere have been numerous studies highlighting how small-scale poultry facilitates improved economic stability, increased food security, and gender equity ,43,55. FDespite the positive outcomes of poultry development, there are many constraints associated with local stakeholder poultry production that need to be acknowledged. These constraints include various predators, nutrition quality, genetic breeding, training and management, infrastructure and capital, farmer organization, governmental policies, and most relevant to public health are the associated biosafety and biosecurity risks . MoreoveUnfortunately, applying intensive poultry farming to low-resource settings can potentially exacerbate pre-existing health burdens . Small-sThe spreading of AMR and infectious diseases in small-scale productions is also related to live-animal markets where different animal species are sold to local farmers. The convergence of humans and animal species coming from several locations provides a unique opportunity for infectious agents to jump species and propagate. Poultry sell in wet markets usually come from intensive operations with a wide AMU. As a result, cases of 1-day old chickens harboring multidrug-resistant bacteria have been reported in LMICs ,140,141.Intensive small-scale poultry systems raise larger flock volumes, which can lead to potentially higher economic yields . Men of The increasing global burden of resistance challenges experts to design innovative interventions to disentangle the complex social and ecological dimensions that facilitate the evolution, spread, and persistence of AMR. This effort stems from Boulding’s Skeleton of Science , demonstEscherichia coli, non-typhoid Salmonella spp., and Campylobacter spp. because of their importance to veterinarian medicine and public health [We recognize that the presence of AMR determinants is an important driver in the dynamics of bacterial resistance in poultry production. It is important to acknowledge that selection for resistance is heavily influenced by variability in antimicrobial administration, which can include antimicrobial classification, duration of therapy, and Defined Daily Dose ,161,162.c health ,167,168.c health ,169. TheFurthermore, mechanisms facilitating the emergence of resistance within the avian microbiota vary by poultry farming practices ,172,173.E. coli in backyard chickens [E. coli resistance profiles of backyard chickens and children mirror changes of AMR profiles of intensively raised broiler chickens [There is a gap in the literature evaluating the epizoology of resistant bacterial populations spread from poultry to other animals. In particular, intensively raised poultry can serve as reservoir hosts for AMR bacteria ,160 Fig; Table 4chickens . Within chickens .E. coli reveals that chickens, pigs, and cattle demonstrate are very similar with respect to their AMR phenotype [Enterococcus spp. [E. coli isolates resistant to tilmicosin, tetracycline, ampicillin, amoxicillin with clavulanic acid, ticarcillin, cephalothin, and ARGs corresponding to tetracycline resistance [In addition to AMR exposure between breeds of chickens, intensive poultry farming has been linked to AMR transmission between various domestic and wild animal species . Bacterihenotype . Moreovecus spp. . In Costsistance . Similarsistance . Furthersistance ,201,202.blaCTX-M and mcr-1 in ESBL-producing E. coli of avian and human origin; further phylogenetic analysis revealed close relatedness, which suggests an active transmission of ESBL-producing E. coli and mcr-1 in both clinical medicine and veterinary medicine [E. coli from a rural community in Ecuador, found that livestock (including poultry), companion animals and humans shared similar AMR profile; however, genetic analysis revealed that ARGs were located on different plasmid structures and bacterial strains, revealing that HGT plays a significant challenge for understanding the movement of AMR in a community [AMU in poultry production contributes to the dissemination, selection, and persistence of AMR in human populations. Understanding the primary transmission routes of zoonotic bacterial resistance is critical for veterinary medicine and public health. Resistant bacteria of avian origin that have the potential to colonize human microbiota is not a novel concept . Studiesmedicine . Furtherommunity .In many regions of the world, small-scale poultry operations are connected to vast market networks centralized in urban live markets. These markets can pose an enormous threat for AMR and zoonoses emergence since poultry are housed and later slaughtered within the same setting of other domestic animals and wildlife ,229,230.3), phosphorus (3.4% P205), and potassium (1.7% K2O) for crop fertilizer and recognized as the best organic fertilizer collected from terrestrial food animals [Poultry production generates large volumes of excretion that comprise of solid waste and wastewater. Primarily, solid waste consists of litter , abattoir waste, and carcasses whereas wastewater usually comes from disinfecting and washing hatchery and abattoir environment ,234,235. animals . Through animals . The aqu animals . Despite animals ,94,238.  animals . FurtherWithin the context of LMICs, fecal contamination has largely been attributable to the emergence of pathogenic and AMR bacteria across all scales of poultry production ,240,241.mcr-1, originally detected in poultry production in China, is now globally present among livestock and humans resistomes [mcr-1 remains present in the environmental resistome [E. coli among dogs, humans, flies, commercial poultry operations, and farmers [In LMICs, poultry litter is typically locally disposed within the community landscape as either livestock feed or crop fertilizer ,249. Evesistomes ,257,258.esistome ,259,260. farmers . The envClostridium perfringens, Streptococcus pneumoniae, Enterococcus faecalis, and strains of Bacteroides [Burkholderia cepacia, Ochrobactrum intermedium, and Stenotrophomonas maltophilia) to function as reservoirs for AMR [Many mechanisms can facilitate the colonization of AMR bacteria or the transmission of ARGs into human microbiota through environmental resistome. Monitoring these dynamics within a low-resource setting is understudied ,261. Limteroides . Studies for AMR . Evidently, environment functions as a reservoir for active antibiotics, metabolites, and genetic material in the form of ARGs and MGSs ,267,268.AMR, facilitated by antibiotic consumption, remains a dire global public health issue. Antimicrobial stewardship programs (ASPs) are curtailed by a variety interactive factors ,271,272.In resource-limited settings, disease surveillance typically captures a marginal understanding of the system. Surveillance is heavily hindered by the lack of resources and political commitment in supporting AMS agendas ,280,281.In LMICS, misperceptions of AMU and AMR emergence is common among small-scale poultry farmers ,271. ThiQuality veterinary services are fundamental to mitigating global emergence of bacterial resistance. Recent analysis conducted by the World Organization for Animal Health (OIE) reported that the majority of national veterinary services are suboptimal . This fiThe majority of antimicrobial use (AMU) is for food animal production . PoultryThroughout the world, national veterinary service standards fall short of meeting international standards . Access Effective veterinary services need to work in partnership with human medical services within a broader public health; many scientists have recognized this effort as a ‘One Health’ framework ,293,294.The Global Action Plan on Antimicrobial Resistance endorsed by the member states of the WHO and affirmed at the high-level meeting on antimicrobial resistance during the 71st General Assembly of the UN , recommeIt has been widely accepted that LMICs require unique interventions compared to HICs due to their unique structural, cultural, and socioeconomic factors affecting AMR emergence . AMR emeThe arsenal of antimicrobials administered in raising food animals is rapidly declining, while remaining essential for animal health, agrarian livelihoods, and public health. Careful evaluation of antibiotic use surrounding intensive poultry development could prevent further dissemination of drug resistance. Veterinary medicine implementations should target existing regions where resistance is emerging. Adopting sustainable poultry husbandry practices could lessen the rise or resistance. There is an obligation of all countries to improve stewardship of antimicrobials as an effort to improve biosafety and biosecurity.


The enzyme soluble epoxide hydrolase (sEH) plays a central role in metabolism of bioactive lipid signaling molecules. The substrate-specific hydrolase activity of sEH converts epoxyeicosatrienoic acids (EETs) to less bioactive dihydroxyeicosatrienoic acids. EETs exhibit anti-inflammatory, analgesic, antihypertensive, cardio-protective and organ-protective properties. Accordingly, sEH inhibition is a promising therapeutic strategy for addressing a variety of diseases. In this review, we describe small molecule architectures that have been commonly deployed as sEH inhibitors with respect to angiogenesis, inflammation and cancer. We juxtapose commonly used synthetic scaffolds and natural products within the paradigm of a multitarget approach for addressing inflammation and inflammation induced carcinogenesis. Structural insights from the inhibitor complexes and novel strategies for development of sEH-based multitarget inhibitors are also presented. While sEH inhibition is likely to suppress inflammation-induced carcinogenesis, it can also lead to enhanced angiogenesis via increased EET concentrations. In this regard, sEH inhibitors in combination chemotherapy are described. Urea and amide-based architectures feature prominently across multitarget inhibition and combination chemotherapy applications of sEH inhibitors. Arachidonic acid (ARA) is an omega-6 polyunsaturated fatty acid (PUFAs) and metabolized by three major classes of enzymes including cyclooxygenases (COXs), lipoxygenases , resulting in the formation of various inflammatory mediators such as prostaglandins, prostacyclin, lipoxins and leukotrienes . ThroughThe CYP2C and CYP2J enzymes convert arachidonic acid to four EET regioisomers, namely 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET . EETs arEpoxide hydrolases have been detected in prokaryotes and eukaryotes ranging from plants to mammals ,12,13. IsEH is composed of two structurally and functionally independent domains with two distinct activities. The C-terminal domain is responsible for hydrolase activity, while the N-terminal domain shows phosphatase activity . While sThe C-terminus epoxide hydrolase motif of sEH transforms four regioisomers of EETs, namely, 5,6-, 8,9-, 11,12-, and 14,15-EETs, to the corresponding dihydroxyeicosatrienoic acids (DHETs), whereby the biological effects of EETs are diminished, eliminated or altered . SpectroOrganisms are routinely exposed to endogenous and exogenous epoxide-containing compounds such as EETs. An epoxide (or oxirane) possesses distinctive reactivity patterns owing to the highly polarized oxygen-carbon bonds in addition to a highly strained ring . ReactivEETs generate an anti-inflammatory effect on the endothelium by inhibiting cytokine-induced NF-κB transcription factor . The traThe sphingosine kinases (SphK1 and SphK2) are a distinctive group of lipid kinases responsible for ATP-dependent phosphorylation of sphingosine to produce Sphingosine-1-phosphate (S1P). SphK1 has emerged as a significant signaling enzyme because of its contribution to the growth, metastasis and chemoresistance of various human cancers ,33. SphiMaterials inflammation-driven cancer is based on the infiltration of white neutrophilic granulocytes, and involvement of cytokines and chemokines . SolubleSoluble epoxide hydrolase inhibitors (sEHIs) have been leveraged for addressing a variety of diseases including hypertension ,38,39, cTrans-3-phenylglycidols and chalcone oxides were first reported as sEHIs, and it was demonstrated that substituted chalcone oxides are more potent inhibitors of sEH enzyme than trans-3-phenylglycidols ,64,65. A50 values of less than 5 µM [50 values are depicted in Other moieties that have shown promising sEHI activity include aminobenzisoxazole, acyl hydrazones, 4-benzamidobenzoic acid hydrazide, adamantyl thioureas and sulphoxide derivatives ,96,97,98han 5 µM . A varieAlisma orientale and reported their potential sEH inhibitory activities [Research on natural product-based sEHIs development has sought to address the typical drawbacks of synthetic small molecule inhibitors. A plethora of natural products, including stilbenes, carbazole, anthraquinones, selaginellin, cycloartane triterpene, capsaicin, dihydrocapsaicin, anthocyanin derivatives and phenolic glycosides derivatives, possess sEHI activity  107,108,118,119.tivities . Naturaltivities . Similartivities . A numbetivities . Belyanstivities .Giancarlo and coworkers developed a distinctive dual inhibitory mechanism of the endogenous 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2) whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522 remote to the active site. This mode of inhibition expands the scope of development of new allosteric inhibitors of hsEH .Soluble epoxide hydrolase inhibitors have been studied in combination with other target-specific inhibitors as potential multitarget therapies. The consideration of sEH in anti-inflammatory drug development is notable in this regard. Nonsteroidal anti-inflammatory drugs (NSAIDs) and selective cyclooxygenase 2 (COX-2) inhibitors (coxibs), which are capable to block COX-2 mediated conversion of ARA to prostaglandin E2 (PGE2), are widely used to treat inflammation and pain . NSAIDs 50 of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay. It was demonstrated that LTA4H specifically converts the instable epoxide LTA4 toward the proinflammatory LTB4. Thus, dual inhibition of LTA4H and sEH is likely to have superior effects in resolving inflammation due to accumulation of the anti-inflammatory EETs and blockage of the chemoattractant LTB4. Hiesinger et al. developed amide derivatives with a benzothiazole core as a dual inhibitor of sEH and LTA4 hydrolase, thereby demonstrating another future route for development of anti-inflammatory agents [sEHIs have been suggested as being capable of synergistic reduction of the side effects of NSAIDs ,130,131.y agents . Hefke ey agents . Other sy agents . Substity agents . A hybriy agents . Other sy agents . Meirer y agents . Dual iny agents ,145 Dualy agents . On a siy agents . Recently agents . Inceogly agents . Furthery agents .Chemotherapy promotes tumorigenesis, angiogenesis and metastasis via apoptotic tumor cell-induced macrophage chemotaxis and proinflammatory cytokines ,152,153.While cisplatin is among the most prominent chemotherapeutic agents, cisplatin-based therapy is highly toxic, underlying the need for improvement in efficacy of cisplatin-therapies . The modPancreatic cancer is one of the most lethal malignant neoplasms. Chronic pancreatitis and mutant Kirsten rat sarcoma viral oncogene homolog KRAS gene  are the most common entities involved in pancreatic carcinogenesis, with more than 90% of human pancreatic carcinomas carrying the KRAS gene mutation ,161. Mutω-3 epoxy fatty acid metabolites derived from ω-3 polyunsaturated fatty acids (PUFAs) metabolism by cytochrome P450 epoxygenases have been suggested to play a crucial role in inhibiting pancreatic cancer growth. Several studies have suggested the importance of ω-3 epoxide as highly potent metabolites against inflammation and carcinogenesis, particularly via targeting inflammatory signals ,166,167.The use of Bleomycin (BLM) in cancer chemotherapy is constrained by its pulmonary toxicity. A recent study showed that AUDA can reduce pulmonary toxicity in the BLM-induced lung fibrosis in mice model . t-AUCB The ARA pathway and EETs are deeply interlinked with a variety of physiological outcomes ranging from vasodilation, angiogenesis and inflammation. sEH is a crucial component of the ARA pathway, and sEH inhibition forms a classical strategy for blocking the infiltration of inflammatory mediators. Due to the interconnections between sEH activity, angiogenesis and inflammation-induced carcinogenesis, multitarget therapies involving sEHIs hold promise for treatment of inflammation and a variety of cancers. While urea, carbamate and amide derivatives have been widely reported as sEHIs, these scaffolds have also been successfully leveraged for developing dual inhibitors towards sEH and suitable ARA pathway targets such as FLAP, LTA4 hydrolase and 5-LO. Further, the combination of sEHIs and NSAIDs is capable of synergistically suppressing primary tumor growth and metastasis with reduced toxicity. Multikinase inhibitors in combination with sEHIs offer a promising treatment strategy for hepatocellular and renal cell carcinoma. The use of polyunsaturated fatty acids in combination with sEH inhibition, as well as the dual inhibition of sEH and c-RAF, are potent approaches for preventing pancreatic cancer. Thus, small molecule sEHIs can be effectively leveraged as dual inhibitors and as components of combination chemotherapy. The widespread tissue and cytosolic distribution of sEH emphasizes the need for sEHIs possessing superior solubility and metabolic stability that can facilitate sEH inhibition-based multi-target therapies to achieve better results.


This article advances the thesis that disciplinary proceedings may constitute a tool for breaking the rule of law in Poland. In 2017, as part of a package of legal changes to the judiciary, a disciplinary system was created in Poland to ensure that judges were subservient to the political will of the authorities. From the beginning, new disciplinary officers appointed by the Minister of Justice  have targeted judges who disagree with unconstitutional changes to the judiciary. Disciplinary proceedings are by no means repressions that affect judges who demand that other authorities respect the rule of law in Poland. The article discusses, on a step by step basis, the practical mechanisms taken by the political authorities to break the rule of law in Poland. Particular attention is paid to the measures which have been taken concerning the judiciary. The article discusses the judgment of the CJEU on 19 November 2019 in combined cases C-585/18, C-624/18, and C-625/18 and the implementing resolution of the combined Civil, Criminal and Labour and Social Insurance Chambers of the Supreme Court on 23 January 2020 as well as the collapse of the rule of law in Poland from a practical perspective. The analysis of the recent events shows that after the so-called Muzzle Law  on 20 December 2019.) came into force, the application of the resolution of the combined Civil, Criminal and Labour and Social Insurance Chambers of the Supreme Court on 23 January 2020 implementing the CJEU judgment in the joined cases C-585/18, C-624/18, and C-625/18 of 19 November 2019 can be and, in fact, is penalized by further disciplinary proceedings, which constitutes a real threat to the already weakened rule of law. Institutions and, above all, judges who are safeguarding the rule of law are being destroyed. This article puts forward the notion that a proper form of disciplinary proceedings is a necessary element of independent and impartial courts. Independent and impartial courts in our opinion are active guardians of the rule of law. Furthermore, courts in democratic countries exercise judicial authority so that their main function is to ensure the right of access to a court under Art. 451) of the Constitution of the Republic of Poland or more widely under Art. 6(1) of the Convention for the Protection of Human Rights and Fundamental Freedoms of the C4The concept of the rule of law is certainly not new. The concept has a strong presence in legal theory,9In what follows we deal with the rule of law as a practical ideal. Needless to say, this maxim is not meant to be taken literally. Rather, the decisions made by legal actors—legislators, judges, and other officials—should be governed by certain ideals and standards.The rule of law will be operationalized by pointing to various institutional and extra-institutional aspects, taking into account the separation of powers. In recent years a new element has appeared in the discussion on the rule of law, the separation of powersIn this context it is beyond any doubt that one of the elements for ensuring the courts’ independence is a proper form of disciplinary proceedings. Furthermore, there can be no doubt that an improper form of disciplinary proceedings may create a tool for limiting the independence of judges. In other words, the CJEU transformed a case that initially seemed to be about the judicial review of an austerity measure into a decision on the organization of the European judicial system.30This judgment created a new dimension in the significant and active role of the courts in protecting the rule of law in the EU legal order. For a long time Article 2 TEU seemed to be nothing more than a mere proclamation of values with only limited implications (Article 7 TEU). It is generally argued that a violation of Article 2 TEU cannot itself be a ground for judicial action.32Of course, the aspect of the separation of powers and the rule of law is very important, but we are in full agreement with Martin Krygier on the issue of the essence of the matter in question. What about institutional and extra-institutional aspects of the rule of law in Poland? Do we really have the rule of law in Poland? Does the law still really matter?Respect for the rule of law has always played an important role in the EU, particularly in the case law of the CJEU. References have been made to the principles of legality, legal certainty, the prohibition of arbitrariness in executive powers, the right to a fair trial before independent and impartial courts, the separation of powers and equality before the law.Constitutional Tribunal. In transgressing the law as it stands , the rulings of the Tribunal in 2016 were not published until two years later (on 5 June 2018), when Poland thus made concessions in its dispute with the European Commission. This was done with a caveat that the rulings were issued in violation of the provision of the Act of 25 June 2015 on the Constitutional Tribunal, and that they were with reference to “a normative act that had lost its binding power”.Sejm (the lower chamber of the Polish Parliament) elected other judges in their place – referred to as stand-ins (doubles). They themselves said that they represented the government.nemo iudex in causa sua. The president of the Constitutional Tribunal expressed her own opinions on the constitutionality of the Act on the public television service. The Acts enacted in 2015 and 2016 regulate the Constitutional Tribunal in such a way that it violates the Constitution. This all resulted in constitutional bodies such as the Disciplinary Officer, the National Council of the Judiciary and the First President of the Supreme Court withdrawing their constitutional complaints to the Constitutional Tribunal as the Tribunal had become nothing more than a facade institution. At present, nobody in their right mind would see the Constitutional Tribunal as a guardian of the Constitution but rather as a highly politicised body.45The first safeguard that was eliminated by politicians was the The government’s next plans were as follows: to target how judges and prosecutors are trained; how the ordinary courts operate; and how judges are promoted, disciplined and dismissed. The focus shifted to the National School of Judges and Prosecutors, the Polish National Council of the Judiciary (NCJ), the ordinary courts and finally the Supreme Court. All of these goals have been achieved. The laws that have been enacted and which amend the laws on the Supreme CourtA series of laws have been enacted, including as from January 2018 a law concerning the Polish Council for the Judiciary (KRS). The above-mentioned laws stipulate that it is the Sejm which will elect judges as members of the KRS. This is in violation of the constitutional principles of the independence and separateness of the judiciary. Taking into consideration the fundamental defectiveness of the proposed mechanism for appointing judges to the KRS, it does not matter whether Parliament elects by a simple or a qualified (three-fifths) majority. It should be stressed that the KRS is meant to ensure the independence of the courts and the impartiality of judges (Art. 186 Polish Constitution). The composition of the KRS cannot depend solely on the decision of the remaining two branches of power. This is why the Constitution clearly limits the number of KRS members elected by representatives of the executive (the President) and the legislative branch. The Minister of Justice  is also a member of the Council.shortening the terms of office of the presidents of the courts or other functions, and to appoint people of their own choice in their place. All of the authority to appoint presidents of the courts at all levels has now been granted to the Minister of Justice, simultaneously stripping the organs of the professional self-governing bodies of the judiciary of any competences in this respect (Arts. 23–25 of the Act: Law on the organisation of the common courts51). The new way of appointing the presidents of the courts is a departure from the standards as presented in the jurisprudence of the Constitutional Tribunal and a violation of the constitutional principle of the separateness and autonomy of the judicial authority (Art. 178 para. 1 Polish Constitution).The politicians presently governing Poland have introduced changes which more or less amount to increased political oversight of the courts, with a significant limitation of the role of the professional self-governing corporation of judges. This is just as it was in communist Poland when the Ministry of Justice had special rights to dismiss judges without restriction by The next step was a bill amending the Act on the Organization of the Ordinary Courts, the Act on the Supreme Court and the Act on the National Council of the Judiciary which was submitted on 12 December 2019, and then voted on by parliamentary majority in the lower house of the Polish Parliament (Sejm) on 20 December 2019. It gained the well-deserved nickname of being a ‘muzzle law’, as it seems to be the greatest ‘achievement’ of the executive and the parliamentary majority on the path leading to the political subordination of the polish judiciary. This new so-called muzzle law introduces new types of disciplinary torts for judges, politicizes new disciplinary proceedings against judges even more so, deprives the bodies of judicial self-government of any significance , imposes an obligation on judges to disclose their affiliation to judicial associations (information on this will be posted on the Internet), gives the President the right to correct any defectiveness in the nomination procedure for a judge and enables the ruling party to take over the position of the First President of the Supreme Court by empowering each member of the General Assembly to nominate its own candidate, thereby reducing the quorum necessary for the election and enabling the President to nominate the temporary president of the Supreme Court for the election period.After the subordination of the Public Prosecution Office, the Constitutional Court and the National Council of the Judiciary to the political authority, the Supreme Court became the last standing independent body for legal protection in Poland. Firstly, they have changed the method for the allocation of cases between judges of the Supreme Court. Contrary to the ordinary courts, where the cases are allocated at random by an electronic system, in the Supreme Court they are allocated by the President of the Court. The next change was the creation of two entirely new Chambers, the members of which are chosen by the new politicized National Council of the Judiciary : they are the Disciplinary Chamber and Chamber of Extraordinary Claims and Public Affairs. The President of the Supreme Court, Prof. Malgorzata Gersdorf, the last key institution in the judicial system not fully controlled by the PiS government, has been replaced by a person affiliated to the PiS. Professor Małgorzata Manowska was appointed judge of the Supreme Court in a procedure that was grossly in breach of the Constitution.Furthermore, the disciplinary proceedings were changed to limit the procedural guarantees of judges. What especially raises doubts is the change in appointing disciplinary officers. The Minister of Justice was given the arbitrary right to appoint the deputy disciplinary officer for judges of the common courts and his two deputies.Granting the Minister of Justice the right to lodge an objection leads to the situation where his position will be highly privileged with respect to other entities entitled to submit a request to instigate an investigation by the disciplinary officer. The said objection as indicated above is binding as far as the disciplinary officer is concerned, as well as the recommendations concerning further proceedings. This means that the disciplinary officer may be obliged to instigate or continue disciplinary proceedings even though there are no grounds for this in his opinion. What is more, this right is completely arbitrary, and the possibility of lodging an objection on an unlimited number of occasions in the same case may - in extreme cases - lead to a constant prolongation of a situation in which a given judge will be faced with disciplinary accusations. Such a solution has to be evaluated also in the context of another provision under which the disciplinary limitation period does not run during the disciplinary proceedings until the day when the final and binding decision ending disciplinary proceedings is taken. This means that the Justice Minister has gained the possibility of having a significant influence on the course of disciplinary proceedings, meaning that certain judges are left with permanent accusations against them. Such a solution not only undermines the independence of the judiciary but also contradicts the essence of the statute of limitations. It means that, in practice, with such a system of disciplinary proceedings, the Minister of Justice may not only instigate disciplinary proceedings against a specific judge but may also choose the disciplinary officers in each case, as well as personally appointing his own disciplinary officer to proceed against a given judge, or may leave him or her with Kafkaesque permanent accusations against him/her. He also has an influence on the appointment of disciplinary court members at first instance. The Minister of Justice may also challenge a decision to discontinue proceedings in cases in which he has submitted the petition for instigating the disciplinary proceedings and refer the case back to the disciplinary court. The clear conclusion is that this is an inquisitorial model of proceedings.Attention should also be paid to the so-called “Panel for the Justice Minister’s measures taken in the disciplinary procedure against judges and assistant judges”, created at the Justice Ministry whose aim is to “carry out an analysis and present the Justice Minister’s recommendations” within the scope of the “disciplinary procedure against judges”. In practice, according to media reports the members of this panel have spread untrue and defamatory information about judges. As a result of the disclosure of the defamatory attacksLay judges have been added to the panels at the Supreme Court’s Disciplinary Chamber. These lay judges are elected by the Senate (the upper chamber of the Polish Parliament). This in effect means that disciplinary courts have become political. It should be underlined that active politicians will choose the lay judges so a social element decides on the disciplinary liability of judges, including their removal from office.A separate issue also involves the Disciplinary Chamber of the Supreme Court,The President of the Disciplinary Chamber not only occupies an autonomous position with respect to the First President of the Supreme Court but also has an influence on the administrative functioning of disciplinary courts at first instance. The President of the Disciplinary Chamber of the Supreme Court not only appoints the presidents of the disciplinary courts for a 3-year term of office but may also dismiss them during their term in office based on general and unspecific grounds such as “a gross or persistent failure to meet professional obligations” or if “the further performance of the function cannot be combined for other reasons with the administration of justice”. He also may inspect the operations of the disciplinary courts at first instance. The President of the Disciplinary Chamber of the Supreme Court determines, thereby arbitrarily ignoring the random choice provisions (S. 110§ 3 LCCS), the disciplinary court that is competent to hear the case at first instance and this holds true for all forms of disciplinary misconduct. The Disciplinary Chamber is comprised of persons appointed by the new National Council of the Judiciary which is considered to be a body that is dependent on the political authorities. Half of them are made up of former prosecutors, including a prosecutor who interrogated the suspect during an earlier stage of the proceedings63In order to complement the disciplinary system so-called “Muzzle Law” was passed. This new so-called “Muzzle Law” has introduced new types of disciplinary torts for judges and it has politicized new disciplinary proceedings against judges even more so . It has introduced further changes in S. 107, 109, 110, 112, 114a, 128 LCCS with respect to disciplinary proceedings. The changes contained therein with respect to disciplinary proceedings are aimed at the situation where Polish judges do not respect the decisions of the CJEU and SC, do not protest against a breach of the Constitution by the legislative and executive authorities and do not carry out their obligations to maintain law and order. The changes provide for new types of misconduct and constitute grounds for holding a judge accountable for disciplinary reasons, where unclear and sweeping criteria are used which do not meet the constitutional requirement of acts prohibited by the law having been determined. It is even more striking that, in principle, these forms of misconduct (which do not constitute a crime) result in the judge in question being removed from the profession or transferred to another court.In particular, S. 107 § 1 (3) LCCS provides for the liability of a judge for “actions questioning the existence of the professional relation of the judge, the effectiveness of the appointment of the judge”, “or the empowerment of the constitutional body of the Republic of Poland”. It leads to the judges in question being prohibited from sitting on the “neo-NCJ”, exercising the prerogative of the President or being elected as representatives of the NCJ. The sanction provided in this case is dismissal from office. Next in S. 107 §(4) LCCS the Sejm has introduced a disciplinary tort consisting of engaging in a public activity which cannot be reconciled with the principles of the independence of the courts and the sovereignty of judges. The entry into force of this regulation means that, contrary to European standards, judges will not have a public right to oppose changes that affect law and order. The changes introduced in S. 110 of LCCS lead in fact to depriving the disciplinary courts of competences concerning the waiver of a judge’s immunity, permission to hold him criminally accountable and to arrest him temporarily and then to refer the case to the Disciplinary Chamber of the Supreme Court. So, the basic sanction for verifying the correctness of the way judges were appointed and their jurisdictional authority involves dismissal from office. It means that when applying European law Polish judges will be penalized with at least the loss of their job. Finally, the system is meant to create a situation where only judges who do not criticize other authorities, and do not question the correctness of the measures taken by the other two authorities, will be left in Poland.What is especially dangerous is that the most important disciplinary cases will be heard in both instances by the same body  and in such a case the penalty will in principle be dismissal from office or a transfer to another court. Such a body cannot be objective, is certainly not independent from other authorities and is not sovereign if it adjudicates in cases in which it is a priori interested in a specific decision, due to the way it is formed and its structure.The Act has changed the principles for appointing deputy disciplinary officers, entrusting this competence to the disciplinary officers of the common courts without indicating any criteria or requirements for obtaining permission, making the appointment decision completely arbitrary. Simultaneously the self-governing judiciary and judges have been deprived of having any influence on the choice of local officers. This means, above all, that a system has been created where officers will be appointed by the disciplinary officers of the common courts who, in turn, have been appointed by the Justice Minister who is a politician. This change will increase the influence of the political authorities on the system of the disciplinary liability of judges.The already existing system which has been changed as a result of the so-called Muzzle Law does not meet any constitutional, EU or international standards.65In 2017 a disciplinary system was established under the Minister of Justice, Zbigniew Ziobro, to ensure that judges were subservient to the political will of the government. The disciplinary officer Piotr Schab and his deputies, Przemysław W. Radzik and Michał Lasota, have targeted judges who oppose unconstitutional changes to the judiciary, a judge who wore a T-shirt with the slogan “Constitution”, judges who have submitted preliminary questions to the EU Court of Justice, and judges who have delivered a verdict that is not in line with the preferences of the political authorities. There are currently 34 judges suffering formal persecution in the form of disciplinary charges after having opposed recent legislative measures because they undermine the rule of law in Poland, or because they have applied EU Court of Justice rulings. The charges may lead to suspension, a 40% loss of salary, and ultimately dismissal. On 4 February 2020 Judge Paweł Juszczyszyn was suspended and deprived of 40% of his salary after being the first judge to implement the verdict of the EU Court of Justice (of 19 November 2019) establishing criteria for independent and impartial courts; 26 judges have suffered informal persecution in the form of an unjustified change of duties or a change to their place work, threats of disciplinary charges, anonymous public harassment by representatives of the legislative or executive authorities and there has been one case of a judge being criminally charged. Judge Igor Tuleya may still have his immunity waived and be tried under the so-called Muzzle Law established in February 2020, to be applied retroactively for his ruling on 18 December 2017 allowing the media to be present as he ordered an investigation into a Law and Justice Party vote in Parliament.Let us take the case of Judge Paweł Juszczyszyn as an example.Judge Igor Tuleya has repeatedly spoken in public about the state of the rule of law in Poland and in his statements he has always boldly defended the independence of the courts, the independence of judges and the principle of a democratic state subject to the rule of law, thereby openly criticising the unconstitutional changes introduced in the area of justice by those currently in power. Judges regularly meet with citizens in meetings on the rule of law, judicial independence, the independence of judges, the principle of the democratic rule of law and human rights. Judge Igor Tuley is also the instigator of a preliminary question to the Court of Justice of the European Union concerning the compatibility of Polish law with European law. As in the case of Judge Ewa Maciejewska from Łódź, the deputy disciplinary prosecutor for judges of the common courts, Michał Lasota, called on Judge Igor Tuleya to make a written statement concerning possible “judicial excessiveness”. In the opinion of the disciplinary prosecutor, the Polish court’s request to the Court of Justice of the European Union for a preliminary ruling on the compatibility of Polish law in the area of justice with European Union law may constitute grounds for initiating disciplinary proceedings. Judge Igor Tuleya’s educational and civic activities and judicial activity as a judge were met with a systemic response from the disciplinary prosecutor.72Judge Waldemar Żurek has also repeatedly spoken in public on the state of the rule of law in Poland, and in his statements he has always boldly defended the independence of the courts, the independence of judges and the principle of a democratic state subject to the rule of law, thereby openly criticising the unconstitutional changes introduced in the area of justice by those currently in power. In January 2018, Judge Waldemar Żurek was dismissed from his position as the spokesman of the Regional Court in Kraków with regard to civil cases. In July 2018, Judge Waldemar Żurek was transferred from the 2nd Civil Appeal Division to the 1st Civil Division (1st instance), which was criticized by “Themis” (the Association of Judges) and “Iustitia” (the Association of Polish Judges), which described this decision as being politically motivated harassment of this judge and as an attempt to intimidate judges who openly act against actions aimed at political subordination to justice. Judge Waldemar Żurek had taken part in meetings with citizens, where current changes concerning the justice system, including the independence of the courts judges, were discussed. Judge Waldemar Zurek’s activities were met by a reaction from the disciplinary officer.73Another example is the situation of the Judges of the Court of Appeal in Katowice, Aleksandra Janas and Irena Piotrowska. Judges Aleksandra Janas and Irena Piotrowska adjudicate appeal cases at this Court of Appeal. While examining an appeal against the judgment of the Gliwice regional court in a divorce case, the judges decided to examine the legal status of the judge sitting in the court who had issued the decision at first instance. To this end, on 11 December 2019, judges Aleksandra Janas and Irena Piotrowska decided to submit a legal question to the Supreme Court. The judges requested an answer to the question of whether it could have been be considered as a duly composed court if the adjudicating judge had been appointed by the new, politicized National Council of the Judiciary. Deciding on the status of a judge issuing a ruling is crucial to resolving a case. If the composition of the adjudicating court is contrary to provisions of law, this results in the invalidity of the proceedings. The decision issued by judges Aleksandra Janas and Irena Piotrowska aimed to assess, among other things, the legal status of the body performing the function of the National Council of the Judiciary in terms of meeting the criteria set out in the judgment of the CJEU of 19 November 2019 in joint cases AK (C-585/18), CP (C-624/18) and DO (C-625/18). Thus, judges Aleksandra Janas and Irena Piotrowska, by submitting a legal question to the Supreme Court, had acted on the basis and within the limits of the law, thereby applying the judgment of the CJEU of 19 November 2019, which they were obliged to do so. Aleksandra Janas and Irena Piotrowska together with judge Paweł Juszczyszyn are all judges who have taken responsibility for the implementation of the CJEU judgment of 19 November 2019, and this was met with the immediate reaction of a closed disciplinary/clerical system created by the politicians in power in Poland, which has always had one goal – to take control of the courts.As a result of doubts which appeared with regard to disciplinary proceedings, applications for a preliminary ruling on their interpretation were submitted by the Supreme Court. In the application for a preliminary ruling on interpretation in the case C-585/18 and in the second and third applications in cases C-624/18 and C-625/18, the court wanted to determine whether Arts. 2 and 19(1) (par. 2) TEU, Art. 267 TFEU and Art. 47 of the Charter of Fundamental Rights should be interpreted in such a way that the chamber of the highest court of a member state, such as the Disciplinary Chamber, which is to adjudicate in matters that are subject to EU law, meets, with respect to the conditions on which it was established and on which its members were appointed, the requirements of independence and impartiality required on the basis of EU law. If this is not the case, the court asked whether the principle of EU primacy should be interpreted in such a way that it requires the court to waive the application of national provisions reserving the competence for hearing such cases to the said court chamber. The basic doubts concerned whether in the light of the national provisions concerning the formation of a specific body, such as the Disciplinary Chamber, and defining in particular the competence held by it, its composition and conditions and principles for appointing judges sitting in the chamber, as well as the conditions and principles on which they were appointed, such a body and its members meet the requirements of independence and impartiality which a court must meet under Art. 47 of the Charter of Fundamental Rights if it adjudicates in a dispute in which the individual alleges a breach of EU law, as in this case. In this respect the CJEU is some kind of supreme court, assuming that the national courts co-share adjudication with the CJEU, being peculiar “common courts with respect to CJEU”, due to the decentralization of the application of EU law. The national courts play a leading role in maintaining the rule of law in the EU in cooperation with the Court of Justice, acting under national laws but also under European law in order to ensure that individuals have the effective rights to which they are entitled.As a result of the CJEU judgment the following judgments of the Supreme Court were delivered: 5 December 2019,83To begin with, a few formal issues should be underlined. The issue to be determined was not the issue of presidential prerogatives, the issue of the legislative competence of the Supreme Court or the constitutional understanding of the principle of the separation of powers.The resolution has a significant meaning, however, because, as a legal principle, it is binding on the adjudicating panels of the Supreme Court. Above all it is aimed at the harmonization of case law and to dispel all doubts concerning adjudication, and what is more significant, it aims to provide stability for the legal order. The resolution is balanced and is assumed to have long-term consequences.So, do we have a rule of law in Poland? In our opinion Martin Krygier90The first question is that of the efficacy of the law. The inefficiency of the machinery of justice is a great obstacle to establishing a State of Law in Poland.Secondly, the rule of law is linked with Polish democracy and its fate, and so major threats to the rule of law amount to threats to Polish democracy in general.94Thirdly, as the courts are the guardians of the rule of law, there is no rule of law without the right of access to a court. After the destruction of the institutions, the time has come to eliminate the judges, who are unfavourable to the populists. Disciplinary proceedings may create an instrument for breaking the rule of law in Poland. In 2017, as part of a package of legal changes to the judiciary, a disciplinary system was created in Poland to ensure that judges are subservient to the political will of the authorities. New disciplinary officers appointed by the Minister of Justice have targeted judges who oppose unconstitutional changes within the judiciary. Disciplinary proceedings are by no means the only forms of repression that affect judges who demand that other authorities respect the rule of law in Poland. The ongoing large-scale use of disciplinary proceedings against Polish judges who apply EU law will inevitably lead to a chilling effect and weaken the effectiveness of the law in the legal order. The idea of the EU as the community of law will be compromised. As no Polish legislation may prevent judges from other Member States applying the CJEU ruling of 19 November 2019, legal chaos undermining the stability of the EU legal order is an inevitable scenario. The system of EU law based on mutual trust and cooperation between the courts from all the Member States will be under a severe threat. Despite the judgment of the Supreme Court, the Disciplinary Chamber is still functioning. An analysis of the recent events shows that the application of the resolution by the combined Civil, Criminal and Labour and Social Insurance Chambers of the Supreme Court dated 23 January 2020 implementing the CJEU judgment in joined cases dated 19 November 2019  can be and is penalized by further disciplinary proceedings, which constitutes a real threat to the already weakened rule of law.The situation of the rule of law in Poland has been dramatically aggravated over the last few months. The Polish government has refused to respect the recent judgment by the CJEU regarding judicial independence in Poland  and has enacted a law aimed at preventing its implementation. Polish judges already face disciplinary and criminal charges for submitting preliminary questions to the CJEU and for following its recent judgment. The Commission filed a complaint in October 2019, and in January 2020 – being especially concerned about the “Muzzle Law” before the Polish Parliament, which further tightened the disciplinary liability system – it applied for interim measures. On 8 April 2020, as a response to this complaint by the European Commission, the CJUE ruled that: “The Republic of Poland shall be required, immediately and pending the judgment in Case C 791/19, to suspend the application of the provisions of Article 3(5), Article 27 and Article 73(1) of the Supreme Court Act of 8 December 2017 , as amended, which constitute the basis for the jurisdiction of the Disciplinary Chamber of the Supreme Court in both the first and second instance, in disciplinary cases of judges; refrain from referring cases pending before the Disciplinary Chamber of the Supreme Court for consideration by a panel which does not meet the requirements for independence indicated in particular in the judgment of 19 November 2019, A.K. and others (Independence of the Disciplinary Chamber of the Supreme Court) .” A new chapter in the conflict concerning the rule of law has begun: The PiS has openly rejected these Commission and CJEU decisions and appeals. What is at stake is no longer the conformity of the Polish judicial system with European standards but also respect for legally binding decisions by EU institutions. Most importantly, on 9 June 2020 in Warsaw, the meeting of the Disciplinary Chamber on waiving judge Igor Tuleya’s immunity took place against the decision of the CJEU. The Supreme Court Disciplinary Chamber has not yet revoked Judge Igor Tuleya’s immunity,