Chromosome replication is an essential process for cell division. The mode of chromosome replication has important impacts on the structure of the chromosome and replication speed. Pseudoalteromonas, members of which consistently carry a chromosome and a chromid. We provide experimental and bioinformatic evidence that while chromids in a few strains replicate bidirectionally, most replicate unidirectionally. This is the first experimental demonstration of the unidirectional replication mode in bacterial chromids. Phylogenomic and comparative genomic analyses showed that the bidirectional replication evolved only once from a unidirectional ancestor and that this transition was associated with insertions of exogenous DNA and relocation of the replication terminus region (ter2) from near the origin site (ori2) to a position roughly opposite it. This process enables a plasmid-derived chromosome to increase its size and expand the bacterium’s metabolic versatility while keeping its replication synchronized with that of the main chromosome. A major implication of our study is that the uni- and bidirectionally replicating chromids may represent two stages on the evolutionary trajectory from unidirectionally replicating plasmids to bidirectionally replicating chromosomes in bacteria. Further bioinformatic analyses predicted unidirectionally replicating chromids in several unrelated bacterial phyla, suggesting that evolution from unidirectionally to bidirectionally replicating replicons occurred multiple times in bacteria.As typical bacterial replicons, circular chromosomes replicate bidirectionally and circular plasmids replicate either bidirectionally or unidirectionally. Whereas the finding of chromids (plasmid-derived chromosomes) in multiple bacterial lineages provides circumstantial evidence that chromosomes likely evolved from plasmids, all experimentally assayed chromids were shown to use bidirectional replication. Here, we employed a model system, the marine bacterial genus  Escherichia coli and the Gram-positive organism Bacillus subtilis, both of which have a single circular chromosome that replicates bidirectionally (–ori) site, after which two replication forks proceed in opposite directions and ultimately terminate in the terminus (ter) region, located roughly opposite the ori site on the circular chromosome , Bacillus subtilis (b), and Vibrio cholerae (c). Download FIG S1, PDF file, 0.5 MB.The chromosome structure for model bacteria Copyright © 2021 Xie et al.2021Xie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Vibrio cholerae, the causative agent of cholera, the secondary chromosomes have a nucleotide composition close to that of the main chromosome and frequently carry core genes that are found on the (main) chromosome in other species , and two replication forks proceed in the opposite directions  of the marine bacterium Pseudoalteromonas haloplanktis TAC125 does not show an otherwise expected GC skew, suggesting that it may be replicated unidirectionally  out of the total 26,371 replicons from 13,550 genomes. Among them, 2,304 were unpredictable and 13,722 were predicted to replicate bidirectionally and 77 unidirectionally (Proteobacteria (Gammaproteobacteria and Alphaproteobacteria), Firmicutes, and Bacteroidetes  was the major group that likely harbored a unidirectionally replicating secondary replicon  from NCBI RefSeq database based on GC skew. Download Copyright © 2021 Xie et al.2021Xie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Pseudoalteromonas, we collected type strains of the 26 species in this genus, which accounted for more than half of the currently recognized species of the genus. Using a combination of Illumina sequencing and PCR gap-closing, we obtained complete genome sequences for nine strains , helicase (dnaB), DNA polymerase III subunits , gyrase subunit B (gyrB), primase (dnaG), and the single-strand DNA-binding protein (ssb), all of which show high sequence similarities to those from E. coli  Replication origin sites (ori) and termination sites (dif) on the main chromosomes and chromids. Download Table S3, PDF file, 0.5 MB.(a) Genes involved in chromosome replication and their homologues in model organisms Copyright © 2021 Xie et al.2021Xie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Pseudoalteromonas spp. have two chromosomes, two chromosome partitioning systems were identified in their genomes: parA1B1 on Chr1 and parA2B2 on Chr2. The parA2B2 genes were located adjacent to the putative ori2 site in a conserved gene cluster, which also contained the tus gene, the putative replication initiator protein gene repA, the possible DNA helicase gene ywqA, and the DNA recombination protein gene rmuC . As in Pseudoalteromonas, in V. cholerae the initiation of chromid replication is also different from that of the main chromosome, which uses DnaA to initiate the main chromosome replication and RctB to initiate the chromid replication  was located at a position roughly opposite ori (ori1) .Similar to the chromid of the strain TAC125, most romosome , suggestr region , 33, 34.i (ori1) . Howeverchromids , dif2 waori site) should have an additional copy compared to the region that has not been replicated  (35–ori than those closer to ter (ori to dif on both sides of dif (or on both replichores)  , 35–37. r to ter . Our datichores)  and b, i chromid . Similar chromid . This isPseudoalteromonas species sequenced, P. spongiae harbored a chromid that showed a GC skew similar to that of the E. coli, B. subtilis, and V. cholerae chromosomes  and chromid  based on different alignment trimming strategies and the removal of compositionally heterogeneous genes. (g) Topology tests on two different topologies for the chromid trees. Download Copyright © 2021 Xie et al.2021Xie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02745-20.3FIG S3FIG S3, PDF file, 0.9 MB.Determination of the minimal replicon for the chromids. Download Copyright © 2021 Xie et al.2021Xie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02745-20.4FIG S4Pseudoalteromonas chromids revealed by GC skew analyses. Download FIG S4, PDF file, 1.1 MB.Unidirectional (a) and bidirectional (b) replication of Copyright © 2021 Xie et al.2021Xie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02745-20.5FIG S5FIG S5, PDF file, 0.5 MB.(a) Unidirectional replication revealed by deep sequencing at the exponential phase for seven species. (b) Bidirectional replication revealed by deep sequencing at the exponential phase for the strain SAO4-4. Download Copyright © 2021 Xie et al.2021Xie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Pseudoalteromonas genomes comprised two monophyletic groups, each consisting of two clades (clades I and II versus clades III and IV), with clade I consisting exclusively of the three strains carrying the bidirectionally replicating chromids. With this robust species tree, we applied the parsimonious rule of evolution to infer the evolutionary history of the chromid replication mode. Because a unidirectional replication mode of the chromid at the last common ancestor (LCA) of all Pseudoalteromonas requires only one evolutionary change , whereas a bidirectional mode at the LCA necessitates a minimum of two independent changes  to explain the phylogenetic distribution of the chromid replication mode of the extant species, the former was favored   . Clade Ito 9 MYA .10.1128/mBio.02745-20.6FIG S6Pseudoalteromonas. Download FIG S6, PDF file, 0.6 MB.Estimated divergence time of Copyright © 2021 Xie et al.2021Xie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ter2 relative to ori2 in the genus can be explained by gene insertion events between them, resulting in a shift of ter2 away from ori2. Consistently, genome annotation revealed the presence of a number of insertion sequences and integrons in chromids  Homologs of dif2 sites on chromids (Chr2) of P. spongiae JCM12884T, Pseudoalteromonas sp. SAO4-4, P. piratica OCN003T, and P. prydzensis DSM14232T predicted by BLASTN searches . Download Table S4, PDF file, 0.7 MB.(a) Integrons, insertion sequences, and prophages predicted on Copyright © 2021 Xie et al.2021Xie et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. tunicata DSM14096T with P. spongiae JCM 12884T to investigate the coordination modes used by unidirectionally versus bidirectionally replicating chromids. Again using deep-sequencing data, we analyzed the sequencing coverage for the main chromosome ori and dif sites (ori1 and dif1) and the chromid ori and dif sites (ori2 and dif2). As expected, during the stationary growth phase, a period lacking extensive replication, the sequencing coverage for each ori and dif site was nearly equal in both species  of their chromids.The appropriate timing of replication is essential for successful cell division . Unlike  species . At the were 1.0 , indicatPseudoalteromonas chromids was that the bidirectionally replicating chromids were larger (1.60 to 1.67 Mb versus 0.64 to 1.39 Mb). The coordination of the replication of multiple chromosomes implies that the time required for the complete replication of the main chromosome sets an upper time limit for the chromid replication  and other bacteria (50.4 ± 25.2% versus 18.8 ± 8.7%)  . It was romosome , though Pseudoalteromonas chromids replicate by the theta mechanism. Consistent with this, no such proteins in chromids from other bacteria have been reported. Considering the different replication factors involved in different mechanisms, a parsimony assumption is that chromids likely evolved from plasmids that also used the theta-type replication. Unlike in chromosomes, however, theta-type replication in plasmids is mostly unidirectional  may evolve into a large and bidirectionally replicating one (more chromosome-like) was raised. Deep genome sequencing and phylogenomic analysis revealed that the last common ancestor of Pseudoalteromonas chromids replicated unidirectionally and the unidirectional replication evolved into bidirectional replication only once, occurring at one of the basal clades of the Pseudoalteromonas phylogeny. Therefore, profiling of Pseudoalteromonas from marine environments ultimately revealed a basic biological mechanism that deepens our understanding of the early evolutionary history of bacterial chromosomes.Though chromids have been observed in different bacteria, only bidirectionally replicating chromids have been experimentally verified so far . Previoutionally . Here, tE. coli, the replication fork is blocked by complexes formed by binding of the Tus protein to the ter regions   genes to the chromosome while exerting little influence on the replication physiology of host bacteria. This is because replication initiation, elongation, and termination of chromids are not changed in spite of the fact that hundreds of genes are acquired on the two replichores of a chromid. Provided that the cell harboring the new bidirectionally replicating chromid adopted termination synchrony to coordinate replication, each DNA replication complex would have to replicate only half as much DNA in the allotted replication time period. Thus, there would be more than adequate time for the replication of additional genetic material that was inserted into the now bidirectionally replicating chromid, explaining the trend observed among the Pseudoalteromonas genomes that the bidirectionally replicating chromids are larger. While bidirectional replication of the chromid may benefit the organism, the reality is that unidirectional replication is more widespread within the genus. This suggests that other factors may also control the replication mode in this genus, which is a topic of interest for future studies.The switch from uni- to bidirectional replication of the V. cholerae as a model. The V. cholerae chromid replication is regulated by the initiator RctB encoded by the chromid  triggers the initiation of chromid replication . Similarly, there were more genes on the leading strand for the unidirectionally replicating chromids  and for the right replichores of the bidirectionally replicating chromids . However, such unequal distribution of genes on the two strands was not apparent on the left replichore of the bidirectionally replicating chromids , consistent with the hypothesis that the left replichore was newly generated from exogenous DNA insertion between the ori2 and dif2 sites. Replication also has a global impact on the mutation rate. Generally, there are higher mutation rates in late-replicating regions  may have occurred multiple times in bacteria.With the marine bacterial genus Pseudoalteromonas species were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures  and the Japan Collection of Microorganisms . The complete list of strains is presented in Type strains for 26 http://hannonlab.cshl.edu/fastx_toolkit/). The resultant clean reads were assembled using SOAPdenovo version 1.05 (http://soap.genomics.org.cn/soapdenovo.html). The remaining intra- and interscaffold gaps were closed using PCR. For the intrascaffold gaps, PCR primer pairs were designed on the flanking contig ends. To design primer pairs for closing the interscaffold gaps, the linkage information between scaffolds was predicted by comparison with genomes of strains TAC125 and SM9913 as well as other species sequenced in this study, using MUMmer version 3.23  according to the manufacturer’s instructions. Multiple Illumina paired-end DNA libraries of different insert sizes  were prepared for each strain . Genome ion 1.05 , with reion 1.05 . A few iion 3.23 . SequencOpen reading frames were predicted using Glimmer version 3.02 . Gene fuThe GC skew is calculated as (number of Gs – number of Cs)/(number of Gs + number of Cs), with a sliding window. The cumulative GC skew was calculated by summing up the GC skew values for all windows from the start window to the current window along the replicon. The circular representation of GC skew was produced using DNAPlotter .E. coli, B. subtilis, and V. cholerae were used as standards for bidirectional replication. The chromid of P. haloplanktis TAC125 was used as a standard for unidirectional replication.To explore the replication direction of bacterial chromosomes, all complete bacterial genomes in the NCBI RefSeq database (May 2019 version) were downloaded. Then, GC skew for all large replicons (>200 kb) was calculated using a self-written Perl script, with a window size of 1 kb and a step size of 1 kb. Lastly, the GC skew and cumulative GC skew were manually inspected. The chromosomes of ori sites were annotated based on the sequence similarities with the reference sequences, the locus of the neighboring gene dnaA (for ori1) or repA (for ori2), and the internal structures, including DnaA boxes and Dam methylation sites (ori sites (ori1 and ori2) of TAC125, V. cholerae, and E. coli as references, genome sequences were searched using BLASTN with an E value cutoff of 1e−5, a score cutoff of 60, a coverage cutoff of 50% (for both query and target), and an identity cutoff of 30%. The 28-bp dif sites were searched with dif sites of TAC125, V. cholerae, and E. coli as references using BLASTN with an E value cutoff of 1 and a query coverage cutoff of 40%. The obtained candidate dif sites were further confirmed based on the following criteria: located in noncoding sequences and roughly opposite or near the ori site and possessing a palindromic structure and a highly conserved XerD binding site  gene, while pOriT-4CM contains a chloramphenicol resistance (Cmr) gene  to obtain a series of recombinant plasmids . These plasmids were transformed into E. coli DH5α cells  and then were isolated and verified by Sanger DNA sequencing. The verified plasmids were then introduced into conjugation donor strain E. coli WM3064. Next, the plasmids were transferred into P. spongiae JCM 12884T or P. tunicata DSM14096T by intergeneric conjugations, as described previously  at 25°C to screen single colonies that contain the recombinant plasmids. Each single colony was then grown in marine LB broth supplemented with 100 μg/ml ampicillin and 35 μg/ml chloramphenicol, and the recombinant plasmids were extracted and verified by restriction enzyme digestion. The bacterial strains and plasmids used in this study are listed in The minimal replicon for the chromid was characterized by testing the ability of the eromonas . The onlP. tunicata and P. spongiae, the potential bias in the exponential-phase coverage was removed by correction using the stationary-phase coverage. First, based on the stationary-phase data, a correction factor for each bin was calculated as the coverage of the bin divided by the average coverage of the replicon. Next, the exponential-phase coverage of each bin was corrected after division by the corresponding correction factor. Besides, outliers were removed from the coverage profiles by comparing the coverage of each bin with its 50 neighboring bins and removing bins with a coverage difference that exceeded twice the difference between the first quartile and the third quartile of coverages of neighboring bins.For each strain, synchronized cells were obtained by starving in artificial seawater at 25°C for 24 h as described previously . These cAlgicola sagamiensis DSM 14643, a close relative of Pseudoalteromonas, was used as the outgroup. For the chromid tree, the tree was rooted at the midpoint, since the close relatives of the genus Pseudoalteromonas have no chromids and therefore cannot be used as the outgroup. Comparisons between different topologies were performed using the seven tests implemented in IQ-Tree  (Insertion sequences were annotated using the ISsaga server with default parameters (dex.php) . Integrodex.php) . Prophagdex.php)  and the dex.php) .CP011011, CP011012, CP011025 to CP011042, CP011924, CP011925, AHBZ00000000, AHCB00000000, AHCD00000000, AHCF00000000, AQGU00000000 to AQGW00000000, AQGY00000000, AQHA00000000 to AQHC00000000, AQHE00000000 to AQHH00000000, and AQHJ00000000. The alignments, phylogenies, and scripts (https://figshare.com/s/2145896d75319c896d58.Genome sequences obtained in this study have been deposited in GenBank/DDBJ under accession numbers  scripts  are avai


Antimicrobial resistance (AMR) is one of the greatest healthcare threats worldwide, with the number of deaths globally attributable to AMR estimated to reach 10 million per year by 2050 unless action is taken [The Japanese government published a National Action Plan on AMR in 2016 that also focuses on enhancing awareness among the general public. In April 2017, the AMR Clinical Reference Center (AMRCRC) was established in Tokyo to implement the National Action Plan. One of the missions of the AMRCRC is to enhance AMR awareness among both medical personnel and the general public. The AMRCRC has conducted various campaigns via websites, social networking services, media exposure, and public events.People’s knowledge about antibiotics and infectious diseases is not always accurate. According to a 2017 internet survey in Japan , nearly Tsuzuki et al. reported on the factors associated with sufficient knowledge of antibiotics and AMR . The useConstructing an attractive message is crucial for public health authorities to ensure successful AMR awareness campaigns. However, it is difficult to evaluate such messages because national campaigns are conducted simultaneously using various measures. Roope et al. reported some interesting findings about the kinds of messages that are effective for enhancing public awareness . They coRoope et al.’s research was conducted in the UK where general practitioners work as gatekeepers in the medical system, but the results are informative for other countries, especially those where antibiotics are prescription drugs, to raise public awareness of AMR. For more effective campaigns to combat AMR, we should continue to share the experiences of different countries in tackling the problem.


Organic acid is an important factor that accelerates the aging of cellulosic insulation materials. In this study, the interactions between cellulose and five acids, representative of what may be found in an aging transformer, were studied using molecular dynamics. The adsorption process of the five acids onto the surface of crystalline cellulose shows that the three low molecular acids are more readily adsorbed onto cellulose than the two high molecular acids. The deformation and adsorption energies of the acids all increase with an increase in molecular weight when they are stably interacting with cellulose. However, the differences between adsorption energies and deformation energies are positive for the three low molecular acids, whereas they are negative for the two high molecular acids. This indicates that the attachments onto cellulose of low molecular acids are considerably more stabilized than those of the high molecular acids. This is consistent with the experimental results. Furthermore, based on the calculated solubility parameters of acids, the experimental result that the three low molecular acids are to a large degree absorbed onto the cellulose, whereas the two high molecular acids remain in the oil, was theoretically elucidated using the theory of similarity and intermiscibility. Cellulosic paper is a major component of the transformer insulation system. It deteriorates with age, leaving the insulation vulnerable to stresses generated by thermal, mechanical and electrical transients ,2,3. WatLundgaard et al. found that carboxylic acids are aging products of oil-paper insulation materials. Low molecular acids  are formed by aging of cellulose, and high molecular acids (naphtenic acid and stearic acid) are formed by aging of insulating oils. Low molecular acids and water have a clear synergy in accelerating the aging of insulating paper, whereas high molecular acids do not influence paper aging significantly. Low molecular acids are readily absorbed by cellulose paper, contrary to high molecular acids that remain in oil . N. AzisAlthough the effects of different organic acids on the aging of cellulose have been widely studied, most relevant studies have been confined to laboratory experiments due to the complexity of the physiochemical process of the aging of cellulose. Investigations on the micro-mechanism underlying the influence of acids on the aging of cellulose remain at the exploratory stage. Therefore, the study of the different effects of different acids on the aging of cellulosic paper at the microscopic level can help shed light on the aging mechanism of the oil-paper insulation of transformers.In recent years, with the improvements in computer technology and computational methods, molecular simulation has developed into an effective means of analyzing complex systems at the molecular level ,12,13. MmoleD) and the centroids of the carboxyl groups (carbD) from the surface change with the simulation time in the final 100 ps of the MD. As shown by the graphs, the splits between the curves of the two centroids become increasingly obvious with an increase in molecular weight of the acid.There is one carboxyl group in each of the five acids, which is a typical polar group. Because cellulose is polar as well, the adsorption potential of the carboxylic group with cellulose is definitely higher than those of other non-polar groups with cellulose. Moreover, the active group for all the five organic acids is the carboxyl group; hence, the sites where the carboxyl groups were adsorbed are very significant. moleD increased with an increase in its molecular weight, as well as the difference between the distances of the two centroids (diffD = moleD − carbD). This indicates that the carboxyl group is the stable adsorbed point, whereas other parts of the acid molecule were adsorbed unstably. The greater the molecular weight of the organic acid, the looser its adsorption onto cellulose.Levulinic acid has an additional carbonyl group aside from the carboxyl group compared with the four other organic acids, as shown in carbonylD) is smaller than that between the centroid of the levulinic acid molecule from the surface (moleD) most of the time. From As shown in Ebind and Efree are the energies of the bound state and free state of the acid molecule, respectively.The deformation energy represents the change in the bound and free state energy of an atom, ion, or molecule during the deformation process. Apart from the levulinic acid molecule, the other four organic acid molecules were deformed when they were adsorbed onto the surface of cellulose. The degree of deformation can be characterized by the deformation energy as follows:As shown in E12 is the total energy of the system, E1 is the energy of acid molecules, and E2 is the energy of cellulose.The adsorption energy represents the decreasing energy produced by the bonding of two materials during the adsorption process of atoms, ions, or molecules attached to a substance surface. This energy is mainly used to calculate the chemical engineering properties and explore the adsorption mechanism. The adsorption energy of the acid molecule by cellulose can be calculated using the following formula:Eads/V. The calculated and the experimental volumes [E = Eads − Edef, for the five organic acids. As shown in  volumes  of the aCED represents cohesive energy density, which refers to the energy needed by 1 mol condensate per unit volume to overcome the gasification of intermolecular forces, and V represents molar volume.The solubility parameter, proposed by Hildebrand et al.  in the mThe solubility parameter has been widely applied in polymer engineering and related fields as an important parameter for measuring the compatibility of the insulation materials.According to the theory of similarity and intermiscibility, a solute can only be dissolved or swollen in a solvent that is of similar solubility to the solute. As shown in the table, the calculated solubility parameters deviate from their experimental values in the low molecular acids, whereas they agree roughly with the experimental values in the high molecular acids. The solubility parameters of the three low molecular acids are approximate to the solubility parameter of cellulose in contrast to those of the two high molecular acids, which are more similar to that of oil. The result explains the experimental phenomenon that the low molecular acids are to a large degree absorbed by cellulose, contrary to the high molecular acids that tend to dissolve in the oil. For the Coulomb component of the solubility parameter, either its absolute value or proportion decreases with an increase in the molecular weight. Contrarily for the van der Waals component, although its absolute value decreases with an increase in the molecular weight, its occupied proportion indeed increases. The proportion of the polar group in the acid molecule again determines this change.The five organic acids generated by the aging of oil-paper composites are shown in Cellulose, a linear polymer of glucose units linked to one another in a special manner, is the essential component of cellulosic paper ,21. TherMany studies have found that different surfaces of the Iβ crystal exhibit different properties ,24. Atomhttp://accelrys.com/products/materials-studio/index.html).Structural optimization was performed for the five organic acids, including annealing and energy optimization, to obtain their minimum energy conformations. Five models, with the same (1-10) surface and different acid molecules, were constructed. Only one acid molecule was added into the cell and placed at approximately 15 Å in front of the crystal surface for each model. Then it was let free to evolve under the lone influence of the cellulose. All cellulose chains were rigidly constrained to their initial positions except for the chains in the top layer. In fact, releasing all constraints would require a larger model that would entail more time for the simulation. Structural optimization and molecular dynamics (MD) were carried out using the second-generation polymer consistent force field (PCFF) ,31,32,33The adsorption behaviors of the five organic acids in transformer oil-paper insulation materials were studied using molecular dynamics. The main conclusions are as follows.The changes in distances of centroids of the five organic acid molecules from the surface of cellulose indicate that the three low molecular acids are more readily adsorbed onto cellulose than the two high molecular acids. The conformations of the acid molecules are greatly related to their polarities when adsorbed onto cellulose. The adsorption of acid molecules onto cellulose becomes increasingly unstable with an increase in the molecular weight of the acid molecules although all their carboxyl groups are stably adsorbed onto the cellulose.All five acid molecules deformed when they were adsorbed onto the cellulose, with an increase in the molecular weight; both the deformation energy of the acid molecule and the absorption energy of cellulose increased. However, the adsorption energy density of each acid molecule decreased. The difference in adsorption energy and deformation energy shows that the three low molecular acids can be stably adsorbed onto cellulose, whereas the two high molecular acids cannot. This is consistent with the experimental results.The solubility parameters of the three low molecular acids are approximate to that of cellulose, whereas the solubility parameters of the two high molecular acids are close to that of transformer oil. The result to some extent contributes to theoretically explain the experimental phenomenon that the low molecular acids are to a large degree absorbed by cellulose, contrary to the high molecular acids that tend to remain in the oil using the theory of similarity and intermiscibility.


Urothelial cancer (UC) includes carcinomas of the bladder, ureters, and renal pelvis. New treatments and biomarkers of UC emerged in this decade. To identify the key information in a vast amount of literature can be challenging. In this study, we use text mining to explore UC publications to identify important information that may lead to new research directions.We used topic modeling to analyze the titles and abstracts of 29,883 articles of UC from Pubmed, Web of Science, and Embase in Mar 2020. We applied latent Dirichlet allocation modeling to extract 15 topics and conducted trend analysis. Gene ontology term enrichment analysis and Kyoto encyclopedia of genes and genomes pathway analysis were performed to identify UC related pathways.There was a growing trend regarding UC treatment especially immune checkpoint therapy but not the staging of UC. The risk factors of UC carried in different countries such as cigarette smoking in the United State and aristolochic acid in Taiwan and China. GMCSF, IL-5, Syndecan-1, ErbB receptor, integrin, c-Met, and TRAIL signaling pathways are the most relevant biological pathway associated with UC.The risk factors of UC may be dependent on the countries and GMCSF, IL-5, Syndecan-1, ErbB receptor, integrin, c-Met, and TRAIL signaling pathways are the most relevant biological pathway associated with UC. These findings may provide further UC research directions. Urothelial carcinoma (UC) also known as transitional cell carcinoma includes carcinomas of the bladder, ureters, renal pelvis. UC is the fourth common cancer in men . Risk faThere is a large volume of publications on UC. Traditional ways of literature review tend to be time-consuming and labor-intensive. Machine-learning-based literature mining may analyze large collections of documents, identifies patterns in a dataset using statistical and computational methods, make predictions based on the discovered patterns, and minimizes human interventions. Machine learning has been used in biomedical informatics research and early prediction of treatment outcomes. Literature mining using machine learning is useful in summarizing key research themes and trends . A topicWe used the keyword “urothelial cancer” to search abstract from PubMed, Web of science, and Embase in Mar 2020. Fourteen thousand four hundred forty-three abstracts were obtained from Pubmed, 14,390 from Web of Science, and 24,110 from Embase. A total of 29,883 abstracts were analyzed after the removal of the duplicated ones. The title and abstract of each article were extracted and then combined into a single string. The keywords assigned by authors were not included [Latent Dirichlet Allocation (LDA) is a type of topic modeling. Lda2vec is an extension of word2vec and learns word, document, and topic vectors. LDA learns the powerful word representations in word2vec and constructs a human-interpretable LDA document. The LDA document is obtained by modifying the skip-gram variant. In the original skip-gram method, the model is trained to predict context words based on a pivot word. Lda2vec goes one step beyond the paragraph approach by working with document-sized text fragments and decomposing the document into two different components - a document weight vector and a topic matrix. The document weight vector represents the percentage of the different topics and the topic matrix consists of different topic vectors. A context vector is constructed by combining the different topic vectors in a document .Lda2vec is an unsupervised text mining method and to determine the optimal number of topics is critical. There is no best way of choosing the optimal number of topics . The perP (word|topic).For visualization of the content of topics, the most probable words to convey a topic meaning were listed with the RGB color model, an additive color model in which red (R), green (G), and blue (B) light are added together in various parameters to reproduce a broad spectrum of colors. The parameters of R, G, and B are all inversely proportional to the normalized probability of words, and the color is shaded in greyscale from black to white. The higher color depth indicates a higher probability. The RGB color model was plotted with python (wordcloud package version 1.6.0). The word clouds were also plotted to demonstrate the distribution of vocabularies over each topic. To make the visualization clear, we combined the singular and the plural forms of a word as one word if both forms were listed in the top 20 probable words for a given topic. The topics were individually presented as an unstructured set of word clouds, and the word size is proportional to the probability of the word within a topic, p-value of < 0.05 was considered significant [To investigate a comprehensive set of functional annotations of the hub gene. Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by using the “FunRich” . FunRichnificant .We explored the top 10, 15, 30 keys words and selected the top 15 keywords. LDA discovered separate and relative definite issues, the location of UC , gene (T7), treatment , and severity . Some of the topics are related. For example, the gene expression (T7) and tumor grade (T12) are associated with the decision of chemotherapy (T9), surgery , and survival (T3). The keywords in each topic are shown in Table There was an association between risk factors of UC and countries in the analysis of 13,725 abstracts -medicated signal events, interleukin (IL) 5-mediated signaling events, ErbB receptor signaling network, Syndecan-1-mediated signaling events, TNF-related apoptosis-inducing ligand (TRAIL) signaling pathway, Signaling events mediated by Hepatocyte Growth Factor Receptor (c-Met), Glypican pathway, Proteoglycan syndecan-mediated signaling events, Beta1 integrin cell-surface interactions, and Integrin family cell surface interactions Fig. . The perIn this text mining assisted literature review of UC, we found an increasing trend of publications regarding treatment, survival, and gene. A decreasing trend of publications regarding upper urinary tract UC, radical cystectomy, and lymph node metastasis was also observed. Immune checkpoint therapy is the hottest topic in the UC treatment. The majority of the publications are from the United States, China, Japan, Taiwan, Germany, Italy, and France. Cigarette smoking and aromatic amines are commonly reported risk factors , 20, folThe top ten pathways identified may help to explore new treatment for UC. One of the examples is Mycobacterium bovis bacillus Calmette-Guérin. Mycobacterium bovis bacillus Calmette-Guérin has been used as an effective treatment for UC because it activates the TRAIL signaling pathway that leads to tumor necrosis through the immune response . GMCSF iThere are some limitations to this study. First, only results from Pubmed were analyzed and the language is limited to English. This may lead to selection bias. Second, the analysis was conducted based on the extracted abstracts but not the full texts. More information may be obtained if we apply analysis on full texts. Third, we used LDA to extract articles. LDA was the concept of “bag of words” rather than the order of words. When a sentence was divided into separate words, it became meaningless or lost the original meaning. Forth, the frequency of words was presented but the frequency of the words may not necessarily stand for their significance.In this paper, we have presented an empirical study by utilizing LDA modeling to discover major research topics of UC. We analyzed the dynamics and intellectual structure of topics. We found growing researches on the treatment but not cancer staging. Cigarette smoking and arsenic are the most commonly reported risk factors worldwide and there is an association between UC risk factors and countries. GMCSF, IL-5, Syndecan-1, ErbB receptor, integrin, c-Met, and TRAIL signaling pathways are the top biological pathways associated with UC. The study provides a better understanding of the trends of UC research and potential future research directions.


Kaposi's sarcoma (KS) is a vascular tumor that originates from the endothelial and immune cells. Lesions usually appear on the skin and oral mucosa, but they may also extend to involve lymph nodes and visceral organs. Patients typically present with multiple painless purplish spots on the face, oral mucosa, and genitalia.We report a case of cutaneous KS in a 31-year-old male with an unknown positive HIV status. Clinical presentation and investigations were both toward KS. Therefore, our patient was treated immediately after diagnosis but could not tolerate the antiretroviral therapy and had unfortunate consequences. Kaposi's sarcoma (KS) is a vasoformative spindle-cell tumor associated with human herpesvirus-8  infection. There are four main clinical epidemiological types: epidemic-acquired immunodeficiency syndrome (AIDS)-related KS, classic sporadic KS, iatrogenic KS, and endemic KS . The mosA 31-year-old male presented to the dermatology clinic with complaints of multiple non-pruritic lesions over the scalp, face, trunk, and arms that started six months ago and gradually disseminated. Otherwise, the patient’s past medical and drug history were unremarkable. On physical examination, there were multifocal violaceous nodules and plaques on the auricle, scalp, face, trunk, and arms Figures . ConsequKS was firstly described in 1872 as an idiopathic, multi-pigmented skin lesion . There iAlthough KS can be sometimes diagnosed clinically by the appearance of lesion, biopsy should be taken to confirm the diagnosis. KS has been observed in almost all visceral sites, and therefore any patient with KS should be evaluated for any visceral involvement . TreatmeIrrespective of CD4 cell count, initiation of antiretroviral therapy (ART) is highly recommended for all HIV-infected patients. ART is the cornerstone in the treatment of HIV-related KS . A markeIn HIV patients not receiving ART, KS is a progressive disease with a considerable mortality rate. Nevertheless, diagnostic delay can lead to more opportunistic infections, which increase the risk of developing IRIS. Early cutaneous KS lesions can be easily mistaken as hematomas, purpura, angiomas, or nevi. Hence, our case emphasizes the importance of considering HIV-associated cutaneous KS as a differential diagnosis of any multiple painless violaceous skin lesions.


The interaction of strong laser fields with matter intrinsically provides a powerful tool for imaging transient dynamics with an extremely high spatiotemporal resolution. Here, we study strong-field ionisation of laser-aligned molecules, and show a full real-time picture of the photoelectron dynamics in the combined action of the laser field and the molecular interaction. We demonstrate that the molecule has a dramatic impact on the overall strong-field dynamics: it sets the clock for the emission of electrons with a given rescattering kinetic energy. This result represents a benchmark for the seminal statements of molecular-frame strong-field physics and has strong impact on the interpretation of self-diffraction experiments. Furthermore, the resulting encoding of the time-energy relation in molecular-frame photoelectron momentum distributions shows the way of probing the molecular potential in real-time, and accessing a deeper understanding of electron transport during strong-field interactions. Interaction of strong laser fields with matter provides powerful tools to image transient dynamics with high spatiotemporal resolution. The authors investigate strong-field ionisation of laser-aligned molecules showing the effect of molecular alignment on the photoelectron dynamics and the resulting influence of the molecular frame in imaging experiments. The radiative recombination results in the emission of high-energy photons by high-harmonic generation1, and this is a powerful tool to investigate the electronic structure with attosecond temporal resolution5. Alternatively, the rescattered portion of this electron wavepacket is exploited in laser-induced electron diffraction (LIED)6 experiments as a coherent diffraction pattern of the molecular target, potentially providing time-dependent images of the molecule at sub-femtosecond and few-picometer resolution. Recently, corresponding experimental results for the structure or dynamics of small or highly symmetric molecules were obtained12. At the same time, the initial conditions of the strong-field interaction have attracted much attention for capturing the intrinsic nature of strong-field physics.In the prototypical strong-field interaction, an intense driving field extracts a valence electron from the target through tunnel ionisation, accelerates the free electron in vacuum and eventually drives it back to the parent ion, predominantly resulting in rescattering or radiative recombination14, these initial conditions are still generally considered a weak perturbation in strong-field physics. All the results obtained in LIED experiments, for example, are interpreted in the framework of the strong-field approximation, where the electron is considered to be born in the continuum with a negligible initial momentum, and to propagate as a plane wave15. Furthermore, the post-ionisation dynamics before rescattering are assumed to be fully driven by the laser field, by neglecting, for example, the Coulomb interaction with the ionised molecule.While pioneering attosecond experiments and molecular-frame measurements revealed non-trivial spatiotemporal features in electron tunnelling15, where angular dependence in the final photoelectron wavepacket is introduced solely through rescattering. Within this approach, diffraction patterns were analysed utilising the angular8 or radial16 photoelectron distribution. However, the relevance of the ionised molecular orbital in the rescattered photoelectrons is still under discussion17. So far, this was included by an overall weighting factor in the rescattering probability19, or as a spatial phase or an angular feature in the rescattering electron wavepacket20. Recently, the influence of molecular alignment on molecular structure retrieval was discussed21. However, general predictions are still extremely challenging with new models appearing23.Common strategies to analyse photoelectron-momentum distributions rely on the quantitative rescattering theory (QRS)Here, we experimentally and computationally study molecular-frame photoelectron spectroscopy from strongly aligned molecules in order to investigate the relation between the molecular frame and the strong-field-induced ultrafast electron dynamics. We demonstrate that and how the molecular frame governs the rescattering time for the photoelectron and, consequently, its final kinetic energy.24 was adiabatically aligned in the laboratory frame, with  cos2θ2D = 0.9, by using a linearly polarised, 500 ps laser pulse, centred at 800 nm26, with a peak intensity I = 3 × 1011 W cm−2. The molecules were aligned in two different configurations, shown in Fig. Y and Z axes, named parallel and perpendicular alignment, respectively. A second laser pulse, centred at 1300 nm, with a duration of 65 fs, and a peak intensity I = 8 × 1013 W cm−2, was used to singly ionise the OCS molecules. For this intensity, the ponderomotive energy of the laser field is  Up ≈ 13 eV and the ionisation occurred in the tunnelling regime. The electric field of the ionising laser pulse, EL in Fig. Y axis (ellipticity ϵ = IZ/IY < 0.005). The produced molecular-frame angle-resolved photoelectron spectra (MF-ARPES) were recorded in a velocity map-imaging (VMI) spectrometer27 with its detector parallel to the XY plane. It is important to note that the de Broglie wavelength of rescattering electrons in the experiment was larger than 200 pm. In this regime no diffraction feature is expected to appear in the photoelectron distributions16. Figure pX < 0.5 a.u. (atomic units), while the spectrum for perpendicular alignment shows a number of angular features for transverse momenta pX between 0.5 and 1 a.u. These angular structures, which are much weaker in the spectrum for parallel alignment, could be identified as forward-rescattering features28. Focussing the attention on large longitudinal momenta pY, the counts for parallel alignment drop around 2.5 a.u. In the case of perpendicular alignment, however, the spectrum extends to larger momenta, showing an appreciable amount of counts at pY = 3 a.u. Following the strong-field approximation, the hard cut-off of photoelectron momentum is expected to only depend on the properties of the laser field29. Experimentally, this quantity is hard to measure. Thus, the turning-point of the signal drop, i.e., the minimum of the first derivative, at large longitudinal momenta is used instead. In the following, we use the term cut-off in the latter sense. Surprisingly, in the current study we found a clear dependence of the cut-off on the molecular frame. Figure pX and pY, between 0 and 4 a.u. Here, the spectra were split along the Y axis and the spectrum from parallel alignment is shown on the left and the one from perpendicular alignment on the right. Now, the differences at small momenta as well as at the cut-off are even more evident. To perform a quantitative analysis of the cutoffs, the momentum distributions were angularly integrated within a cone of  ±20° with respect to the longitudinal axis (Y) and converted to an energy scale. In Fig. Up. The perpendicular/parallel ratio of the two area-normalised spectra (green) shows a predominance of photoelectrons for perpendicular alignment in the energy range between 2 and 10 Up, where the distribution is dominated by rescattered electrons30. Furthermore, the ratio increases with energy, reaching the maximum around the cut-off. To evaluate the cutoffs, the first derivative of the energy distributions are shown in Fig. 30 and it was around 2 Up for both alignment cases. This excluded any significant alignment-dependent direct-electron-cut-off enhancement31. Surprisingly, the second minimum behaves differently for the two alignments. While it is located around 10 Up for perpendicular alignment, as expected from the well-established above-threshold ionisation theory29, the cut-off is shifted down to a value around 8.5 Up for parallel alignment. Figure 32 and a semi-classical molecular trajectory simulation set-up. Using TDDFT, the MF-ARPES probability was calculated by simulating the complete dynamics of the many-body ionisation process in real-time and real-space with the tSURFF method34, see Supplementary Note To unravel the experimental observations, state-of-the-art calculations were performed using both, time-dependent density-functional theory (TDDFT)35 in conjunction with a simple man propagation (SM)37 were conducted in order to track the molecular-frame electron dynamics during the strong-field interaction38, see Supplementary Note Up of the ratio of the two alignment cases  tunnelling theoryses Fig. h, this s40. Therefore, when the molecular axis was aligned along the polarisation axis of the strong field, the electron preferentially acquired an initial transverse momentum pX0 that was much larger than in the case of perpendicular alignment, shown by the red vertical ridges in Fig. To obtain a more comprehensive picture of the alignment-dependent photoelectron dynamics and, in particular, a glimpse at the initial electron wavepacket, we performed a differential analysis by subtracting the photoelectron distributions of the two alignment cases from each other. Figure 43. In general, they are defined by the tunnel-exit position as well as by the temporal phase and the momentum acquired during the ionisation with respect to the external field. Here, we demonstrate that the molecular potential, i.e., the combination of the electrostatic potential and the electronic structure of the molecule, has in fact not only a primary role in setting the initial conditions for electron emission, but that it also drives the whole photoelectron dynamics: it defines the cut-off of rescattered electrons and it shapes the time–energy relation for electron recollision. To investigate this, we exploited the ADK-SM calculations to analyse the final absolute momentum acquired by the electron after photoionisation as a function of the recollision phase. In the following discussion we will refer to revisit to describe passages of the electron nearby the cation on a relatively large spatial scale, where momentum transfer is relatively small and soft, whereas recollision, or rescattering, refers to the close approach of the electron to a nucleus, with an associated large-momentum transfer, e.g., in back scattering. While multiple revisits may occur during the interaction of the electron with its cation, a recollision event will drive the electron irreversibly away from the molecule. The resulting momentum distributions are reported in Fig. k + 1/2)π, k = 1, 2, 3…, for which the electron collides with the molecular cation when the laser field’s vector potential is maximum. The first recollision event, i.e., the first peak in Fig. π/2, allows the electron to reach the largest momentum as expected in the classical theory29. This is close to the maximum asymptotic kinetic energy, i.e., the 10 Up cut-off. The peaks appearing later correspond to electrons that have initially missed and then revisited the ion at later times. These subsequent rescattering events are expected to lead to lower photoelectron energies29. At the same time, these multiple revisits are possible only due to the Coulomb attraction of the ionised molecule29. Since the current understanding and analysis of strong-field self-diffraction experiments only consider the photoelectron recollision on the first revisit12, the relevance of Coulomb attraction is usually neglected. However, our results demonstrate that it is a crucial ingredient to correctly understand molecular-frame electron rescattering. Note that a small yield at large momentum (>3 a.u.) is visible for both alignments  revisit order(s) could be selected in the experiment with near-single-cycle (few-cycle) laser pulses.Furthermore, the photoelectron cut-off in the molecular frame carries crucial time information: While the cut-off for perpendicular alignment is strongly shaped by electrons recolliding 3/4 of an optical cycle after ionisation, as usually assumed, this is not true for parallel alignment: the cut-off is dominated by electrons revisiting the molecule much later, namely one or multiple optical cycles later. For a wavelength of 1300 nm, this corresponds, at least, to a delay of ~4.3 fs and it linearly increases with the wavelength. From Fig. We demonstrated, experimentally and computationally, that the molecular frame determines the momentum distribution of high-energy rescattered electrons in strong-field ionisation. The basic concept of molecular-frame strong-field ionisation is captured by considering the initial conditions imposed by the molecular potential in the dynamics of the photoelectron. Furthermore, from the analysis of the rescattering trajectories it is evident that the molecular interaction plays a crucial role in setting a clock for the emission and the dynamics of high-energy electrons. It highlights that the molecular frame has a strong impact on the relation between the photoelectron energy and the rescattering time. This finding redefines the delay step of time-resolved self-diffraction experiments and opens up a perspective on time-resolved diffraction experiments. These conclusions hold similarly for other observables related to electron recollision .12. We note that in such experiments mid-infrared lasers (λ ≈ 3 μm) are typically employed. Since the electron’s excursion length increases with increasing laser wavelengths, we expect our findings to be even more relevant for actual LIED experiments.Our result represents an important benchmark for any self-diffraction measurement and represents a breakdown of the usual interpretation of LIED experiments4. Furthermore, the earliest moments of a strong-field interaction are intrinsically imprinted in the initial conditions of the photoelectron and in the final energy distribution. Thus, molecular-frame strong-field-ionisation experiments, in principle, allow one to achieve a deeper understanding of electron tunnelling, for instance, regarding the tunnelling time, and to track the molecular potential in real-time.Our study highlights the molecular-frame conditions as a crucial ingredient of self-diffraction experiments. This framework is general and can, in principle, be extended to any molecular system. Furthermore, the molecular-frame strong-field interaction was quantitatively modelled here by a fully interacting-electron TDDFT calculation and, in conjunction, by a semi-classical single-active-electron theory. We exploited TDDFT to evaluate the contribution of different molecular orbitals to the ionisation-rescattering dynamics as a benchmark for the applicability of the semi-classical approach. We expect this double-sided theoretical framework to become more and more important with increasing molecular complexity, where modelling the photoelectron dynamics may go beyond the capabilities of a single-orbital picture. In general, this also opens the perspective to investigate electron-correlation-driven phenomena in molecular strong-field physicsSupplementary InformationPeer Review File


The global concern over antimicrobial resistance (AMR) is gathering pace. Low- and middle-income countries (LMICs) are at the epicentre of this growing public health threat and governmental and healthcare organizations are at different stages of implementing action plans to tackle AMR. The South Indian state of Kerala was one of the first in India to implement strategies and prioritize activities to address this public health threat.Through a committed and collaborative effort from all healthcare related disciplines and its professional societies from both public and private sector, the Kerala Public Private Partnership (PPP) has been able to deliver a state-wide strategy to tackle AMR A multilevel strategic leadership model and a multilevel implementation approach that included developing state-wide antibiotic clinical guidelines, a revision of post-graduate and undergraduate medical curriculum, and a training program covering all general practitioners within the state the PPP proved to be a successful model for ensuring state-wide implementation of an AMR action plan. Collaborative work of multi-professional groups ensured co-design and development of disease based clinical treatment guidelines and state-wide infection prevention policy. Knowledge exchange though international and national platforms in the form of workshops for sharing of best practices is critical to success. Capacity building at both public and private institutions included addressing practical and local solutions to the barriers e.g. good antibiotic prescription practices from primary to tertiary care facility and infection prevention at all levels.Through 7 years of stakeholder engagement, lobbying with government, and driving change through co-development and implementation, the PPP successfully delivered an antimicrobial stewardship plan across the state. The roadmap for the implementation of the Kerala PPP strategic AMR plan can provide learning for other states and countries aiming to implement action plans for AMR. The growing threat of antimicrobial resistance (AMR) is a major public health challenge, leading to complications in infectious disease management and contributing to high patient morbidity, mortality and cost of care . Deaths Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., as well as a rise in resistance to commonly used antibiotics for treating Salmonella, Shigella, Vibrio cholerae, Neisseria gonorrhoeae and Mycobacterium tuberculosis . The. The5]. rculosis , 7. AMR rculosis . Mortalirculosis  and overrculosis . A recenrculosis . Pneumonrculosis .Table 1AIn addition to this overwhelming AMR burden, the country leads in antimicrobial consumption with high use of broad spectrum agents, including penicillins which have a Drug Resistance Index (DRI) of 71.6 . DRI is th five-year plan in 2011. The Chennai declaration of 2012 had a positive impact in shaping national AMR strategies by engaging medical associations and societies for AMR containment. The Indian Council of Medical Research established AMR surveillance centres throughout the country [In India, inappropriate antibiotic use and over the counter sale of drugs are one of the key drivers of AMR, in addition to lack of universal access to water, sanitation and hygiene (WASH) . Several country , 16. In  country . The lau country . Taking  country . A Natio country . As partAntimicrobial Stewardship (AMS), is recognised as a key strategic intervention to reduce AMR in India’s NAP-AMR with demonstrated success in curbing AMR rates and reducing economic costs , 18. TheThe healthcare system in Kerala has been a culmination of resource redistribution policies and several decades long social investments in primary healthcare and universal education along with empowerment of women –23. WithThe heightened awareness and health seeking behaviours set in motion by high education levels provides a conducive environment for public healthcare endeavours at all levels of care. Various global reports on AMR policies have stressed the lack of collaborative efforts and emphasize the importance for a co-ordinated action in a political context to drive strategies to combat AMR –34. IdenThe increasing burden of AMR and antibiotic prescribing was the driving force for developing a state-wide PPP for AMR. Learning form lessons of other infectious disease public health interventions, and through its PPP, the State of Kerala was the first in the country to formulate the Kerala Antimicrobial Resistance Strategic Action Plan (KARSAP) for combating AMR in 2018 incorporating One Health. Like the Nipah public health campaign the KARSAP was overseen by the State Government, supported by the professional bodies, led by the PPP, and delivered through grassroots initiative involving champions, nodal officers and PPP representatives  across all primary, secondary and tertiary care institutions within the State , 42. TheThe impetus for change at state level was accelerated following a government led survey among general practitioners to understand the baseline antibiotic prescription practices. The survey identified key gaps including lack of knowledge amongst the respondents about the mechanism of action of antibiotics, and the pressure by patients to be prescribed antibiotics. These results led to the formation of a State Wide Task Force along with a formation of a Core Committee at the State level. A State Core Committee was convened with representation from key stakeholders to identify the steps in addressing AMR. The Committee interacted with all the major stakeholders and supervised action items decided at State Task Force meetings. In line with the scale of the program, it was decided to engage with all State chapter of professional societies. The senior office bearers of 14 professional bodies were contacted and they all agreed to participate under the leadership of IMA State chapter  sessions were rolled out for all physicians to sensitize them about the state antimicrobial policy and rational prescribing of antibiotics. As part of the TT sessions, a structured multiple choice questionnaire comprising of 30 questions addressing appropriate antibiotic use in general and accurate diagnosis in specific infectious diseases case scenarios was disseminated before and after the TT session among the physicians. AMR surveillance was established in PPP mode for 6 pathogens of AMR concern and for major foci of infections Fig. .The PPP identified state level action plan including specific strategies to combat AMR which were adopted across the healthcare organisations Box . The actIn collaboration with the Kerala University of Health Sciences (KUHS), the PPP developed recommendations for revising the curriculum of undergraduate, house surgeons and post graduate trainees at government and private medical colleges. These recommendations were presented to the respective Board of Studies and Universities Academic Council. The aim of these revisions was to offer uniform training to provide academic and practical knowledge and training on rational antibiotic prescribing and Infection Prevention at all levels. The objective of recommendations was to enable clinicians to adopt strong and robust evidence based infection control behaviours and to independently care for patients. The new curriculum included four hours of additional training for undergraduates during their third year placement training, ten hours of training during the induction program for trainee surgeons, and 14 h of department specific training during the induction program of post-graduates. The outline of the course is provided in Box All trainees had to pass an examination with a pass rate of 80%. Similar strategies have now been introduced to the curriculum in nursing, pharmacy and dental schools. A pilot is being conducted at one of the private medical colleges to provide learning for state-wide implementation in collaboration with the respective professional councils.Additionally, 102 clinicians from different specialties representing hospitals across the state were invited to participate in a train-the-trainer initiative on a voluntary basis . The aimIn this overview, we provided information on the strategy, advocacy and policy level efforts that were undertaken to establish a state and tertiary care level PPP for AMS. Multilevel strategies are essential to the successful implementation of AMS programmes. Working on developing an AMR containment strategy, exposed the magnitude of the AMR problem across the State. During the course of co-designing such strategies, a balance must be achieved in addressing the priorities across Human Health, Animal Health and the Environment. At Kerala, we worked on developing strategies across the One Health spectrum with equal emphasis. Attempts were made to collectively address the barriers of over the counter pharmacy shops, noncompliance of H1 prescription law, unwarranted antibiotic use in animal husbandry, making antibiotic prescription a legislative issue, poor continuous medical education, non-accredited microbiology laboratories and poor infrastructure, and sub-optimal infection control practices. The goal of having a strong and robust policy document along with an all-inclusive implementation plan was never lost. In order to do this, we had to cast the net wide, and this was only possible through the PPP approach.Key strengths of the PPP were in the collaborative work of 18 professional medical societies to formulate Clinical Guidelines on Antibiotic Prescription; strong political will and leadership within the State; participation from human, animal and environmental departments; engagement of civil societies; and the constitution of a task force to monitor the ongoing work.Turning the PPP initiative from a concept to implementation faced key challenges, which included: the effort to bring various groups (public and private) together; initial criticism of the initiative as there was no data to support the cause; changes in political leadership changing the priorities and delaying ownership of the programme; change in goal posts viz human health vis a vis One Health Approach; no dedicated funding available; bringing all stakeholders at one platform and agreeing on priorities / methodologies; lack of regulatory approach towards stewardship programme implementation; and lack of dedicated staff in public or private sectors to work on this initiative.Strong leadership pays dividends. In Kerala, the support from the government officials in the ministries of health was critical. There was a close coordination among all concerned public service departments and State Health Services, Medical Education, Drug Controller and Medical Services Corporation Ltd. The State Medical Association which maximized the private healthcare professionals’ participation. All professional societies were deeply invested into the strategic planning and drafting of the policies and developing the strategies. Collaborative work of multi-professional groups through regular meetings helped in co-design and development of disease based clinical treatment guidelines and state-wide infection prevention policy. Knowledge exchange though international and national platforms in the form of workshops for sharing of best practices is critical to success. Capacity building at both public and private tertiary care institutions included addressing practical and local solutions to the barriers e.g. good antibiotic prescription practices and infection prevention at all levels. Fig. .Fig. 2LeDespite limited financial support, the drive and will of each stakeholder motivated the teams and led to creation of history in being the first State in drafting clinical guidelines for antibiotic prescription, revising curriculum in medical schools, training of faculty and staff at public and private institutions and establishing a surveillance network.This manuscript describes the strategic, policy, and advocacy that was required to initiate a PPP for AMS in the state of Kerala, which remains an atypical state for health standards in comparison to the rest of India. As such the experiences and processes described here may not be applicable to other states. The lack structural and process measures to tackle AMS meant that it was not possible to measure practices effectively. Now that the structures and mechanisms for the PPP for AMS have been established, the next phase is to evaluate this initiative. This is the first phase of the PPP initiative and was initiated in started in public and private tertiary care settings because the problem is greatest in tertiary care, and the resources available for such an initiative were more readily accessible. Furthermore, the problem is not yet quantified in secondary and primary care. If sustained success is demonstrated in tertiary care, the initiative will be expanded to other settings.The State of Kerala was the first in India to have developed a strategic approach to AMR including empirical clinical guidelines for infection management, implementation of an education programme for under- and post-graduate medical trainees as well as a robust State-wide training program covering 50,000 + general practitioners. In LMICs, where resources may be limited, the private and public sectors can work together successfully to develop strategies for addressing public health, including AMR. The Kerala PPI model AMR containment and IPC strategic plan has proved to be a successful model which has led to sustainable and multidisciplinary change. The model has delivered a State-level Antimicrobial Resistance Strategic Action Plan, laboratory based surveillance system and a healthcare associated infection based surveillance program to assess the performance of IPC and AMR containment activities. The lessons learnt from Kerala AMR strategy can provide learning for other States and countries aiming to implement action plans for AMR.


The functionalized CNTs were well dispersed within the nanofibers and aligned along the direction of nanofibers. Therefore, beads formation on nanofibers decreased. The impedance of the nanofibers was found to decrease with the PPy content and CNT addition. These nanofibers had a great potential to be used as an electrochemical actuator or a tissue engineering scaffold.In this study, carbon nanotubes (CNTs) added polyacrylonitrile/polypyrrole (PAN/PPy) electrospun nanofibers were produced. Average diameters of the nanofibers were measured as 268 and 153 nm for 10 and 25 wt% of PPy contents, respectively. A relatively higher strain to failure values (23.3%) were observed for the low PPy content. When as-grown CNTs (1 and 4 wt%) were added into the PAN/PPy blends, disordered nanofibers were observed to form within the microstructure. To improve the interfacial properties of CNTs/PAN/PPy composites, CNTs were functionalized with H There is a great research and industrial interest to use carbon nanotubes (CNTs) as nanofillers to obtain polymer composites with improved mechanical, electrical and thermal properties [1–5]. Because of their superior mechanical properties such as modulus, strength and thermal and electrical conductivity with their low weight, they have been attractive candidate for composite systems . The main problem for using CNTs as filler materials is their poor dispersion in matrix. Dispersion and alignment of CNTs influences directly the mechanical properties of final composite. Agglomerated CNTs cause defect sites, porous regions, and crack initiation points, etc. which deteriorate the mechanical properties of composite. There are several techniques to achieve a good dispersion of CNTs in a polymer matrix such as, high speed mixing [8], in situ polymerization [9], chemical functionalization [10–12] and high shear mixing [13]. Surface functionalization of CNTs has been found be an effective method to obtain a good CNT dispersion and enhance the bonding between the CNTs and polymer matrix.In addition to their excellent electrical and mechanical properties [14], CNTs show electromechanical actuation behavior. This electromechanical behavior makes CNTs a good candidate for application such as sensors and actuators [15]. By incorporating CNTs, actuation response of the polymer is significantly improved and actuation occurs at lower voltages as compared to those of without CNTs [16]. It was also revealed that CNTs added into conducting polymers may increase actuation strain and conductivity, therefore, electrochemical efficiency in the actuation is provided [17]. Courty et al. [18] reported substantial dielectric anisotropy by aligning CNTs in the polymer and the actuation response was due to the large anisotropy in nanotube polarizability. Zheng et al. [19] fabricated polypyrrole (PPy)/CNT laminates by electrochemically polymerizing PPy on a glassy carbon substrate. Incorporation of the CNT sheets resulted in an increase of strength for both transverse and longitudinal directions and enabled actuation at much higher loads, as compared to those for electrolyte-contacting neat PPy. Electrical conductivity was also improved with the CNTs content.Park et al. [20] added flouro group bonded functional CNTs slurry to the PAN solution. An enhancement in mechanical properties of the carbon fibers was observed and it was attributed to the reduction of thermal damage on the surface of PAN during the stabilization process. The DSC results indicated that the cyclization enthalpy and temperature of F-Ph-CNT/PAN composite were much lower than those for homo PAN. The integration of SWCNTs into polyaniline fibers (PANi) in wet spinning was investigated by Mottaghitalab et al. [21] The addition of CNT in N,N’-Dimethyl propylene urea (DMPU) to PANi was found to limit aggregation of CNTs. By incorporating 2 wt% CNTs to PANi, its yield stress, tensile stress, and Young’s modulus were improved. Chitosan/PANi/SWCNT fibers were fabricated by wet spinning method by Spinks et al. [22] The conductivity of the synthesized material (10−4 S/cm) was 3 to 4 order of magnitude higher than those of the PANi/chitosan composite film. The mechanical properties were improved and the actuation strains (0.3%) were reduced due to the stiffening effect of the nanotubes. Tensile strength of chitosan/PANi/SWNT fibers was 2 fold of pure chitosan, and elongation at break were increased by 60%.PPy is one of the commonly studied conducting polymers due to its low actuation voltage, high conductivity and biocompatibility . Its actuation voltage is lower than 2 V, reach up to 30 MPa active stress and 39% maximum strain [25–27]. It reaches to the largest stroke value among all the conducting polymers for an electrochemical actuation [28]. PPy actuators have been synthesized by a variety of methods. Electrodeposition [29], drop casting [19] and electrospinning [30] are the most common methods. Electrospinning method provides extremely rapid formation of the nanofiber structure, high material elongation rate and a cross-sectional area reduction [31]. Because of its poor solubility, pure PPy cannot be electrospun, therefore PPy is used generally with a co-polymer such as poly ethylene oxide (PEO) [32], PAN [33], polyvinylidene difluoride (PVDF) [34] and poly strene (PS) [35]. In general, PPy based coatings containing nanofibers were investigated to obtain linear actuators. Ketpang et al. [34] synthesized PVDF/CNT electrospun nanofibers, then they coated those nanofibers by vapor-phase polymerization of PPy. Ju et al. [36] synthesized PAN/CNT electrospun nanofibers and coated with PPy by in situ chemical polymerization.In the present study, the preparation of CNT incorporated PAN/PPy electrospun nanofibers was investigated. As-grown and functionalized CNTs with various amounts were added to PAN/PPy blends. Their morphological and electrochemical properties were characterized. These composite nanofibers are expected to find use in tissue engineering as scaffold for different cell types with their 3-dimensional structure and they can be utilized as actuators through the actuation effect of PPy and CNTs.2SO4 (95-97%), HNO3 (≥65%), HCl (≥32%) and NH4OH were also purchased from Sigma-Aldrich Chemie GmbH and applied for CNT functionalization. All these reagents were used without further purification.To synthesize PAN/PPy nanofibers, PAN (MW = 150000), PPy (conductivity >0.005 S/cm) and solvent N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich Chemie GmbH . H4 gas as a carbon source was fed to the system to initiate CNT growth and the growth lasted for 40 min. At the end of growth time, CH4 flow was terminated and the system was left for cooling under H2 flow. Growth was performed at atmospheric pressure.CNTs were produced on Co-Mo/MgO catalyst as reported in our previous study [37]. Before CNT growth, Co-Mo/MgO catalyst was pretreated under 200 sccm H2 flow for 1 h at 850 °C. Then, at CNT growth temperature of 1000 °C, CH3 at 60 °C for 30 min to remove MgO and the other catalyst components. Then, CNTs were washed with distilled water several times until the solution became neutral and dried at 100 °C. For functionalization, purified CNTs were sonicated in a mixture of H2SO4 and HNO3 with volume ratio of 3:1 (30 mL/10 mL) for 2 h, and then kept in this solution for 24 h. Subsequently, 20 mL HCl was added to the solution and it was sonicated again for 2 h. Finally, ammonium hydroxide was added to the solution in a controlled manner to neutralize the solution. After neutralization, CNTs were filtered and washed with deionized water until the pH was 5.5 [38].As-grown CNT weighing 200 mg was purified by mixing it in 30 ml HNOAs-grown and functionalized CNT samples were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Raman spectroscopy and Fourier transformed infrared spectroscopy (FTIR).PAN/PPy nanofibers were synthesized by electrospinning method. PPy was taken as a conducting polymer. It is difficult to synthesize pure PPy nanofibers via electrospinning method because of its poor solubility and high 30. To enhance its poor solubility and mechanical properties, PAN was used as a host polymer in the present study.The electrospinning solution was prepared by dissolving PAN in DMF (8 wt%) until a clear solution was obtained by mechanical stirring. To this solution, PPy amounting 10 and 25 wt% of the total polymer mass was added, and then it was stirred for 3 days at 60 °C. For CNT containing fibers preparation, different contents of as-grown CNT  and functionalized CNTs (1 and 4 wt%) were added into the PAN/PPy solution. They were dispersed in ultrasonic bath for 24 h.The resultant solution was filled into a 20 mL syringe connected to a high voltage for electrospinning. For all samples, 15 kV voltage was applied and flow rate of solution was kept at 1.5 mL/h. The distance between the syringe and collector was kept as 30 cm and the fibers were collected on an Al foil.The morphology and diameter of PAN/PPy and PAN/PPy/CNT nanofibers were characterized by SEM (FEI QUANTA 250 FEG) with an acceleration voltage of 5 kV and a secondary-electron detector. For this purpose, the samples of dried nanofibers were fixed onto metallic stubs with double-sided carbon tape. Micro structure and defects determination of CNT embedded nanofibers were determined by TEM. Tensile tests of nanofibers were performed by using a TA.XT plus texture analyzer with a 5 kg load cell and 0.1 mm/s test speed. For tensile tests, sample dimensions were kept as 8 ×60 mm.Electrochemical impedance spectroscopy (EIS) measurements of the nanofibers were performed by using a three-electrode electrochemical cell: working electrode was graphite electrode covered with our nanofibers during electrospinning process, reference electrode was Ag/AgCl and counter electrode was a Pt wire. The electrolyte solution was Fe[CN6]3-/4 solution.-1) and graphitization-derived G-band (1574 cm-1). The high intensity of the G band with respect to D band of as-grown CNTs indicated low defect density and high crystallinity. After acid treatment, these characteristic peaks of CNTs were still present, which indicated that acid treatment did not damage the structure of CNTs. However, after functionalization ID /IG ratio was increased. This might be due to breakage of some bonds of CNTs and chemical groups bonded to these groups after H2SO4/HNO3/HCl treatments. These chemical groups could be interpreted as defects on the structure, which lead to increase in D band intensity. SEM images of the as-grown and functionalized CNT samples displayed that as-grown CNTs were straight, nontangled and individual. However, functionalized CNTs were observed to be in the form of CNT groups instead of individual CNTs.Raman spectra of the CNT samples given in Figure 1 displays the characteristic peaks of CNTs; defect-derived D-band . The high symmetrical structure of CNTs gave very weak infrared signals because charge state between carbon atoms do not show significant difference and this small difference of charge state causes very small induced electric dipole [38]. The peaks between 2800 and 3500 cmPPy is a conducting polymer and it increases electrospinning solution conductivity. Solution conductivity is a significant parameter for morphology of electrospun nanofibers. When a polymer solution with high electrical conductivity is utilized, an electrical force which elongates the polymer jet emerging from Taylor cone is created and it provides formation of uniform fibers with smaller diameter. However, if the solution is very conductive, it will be unstable in electrical field and causes significant bending of the polymer jet at Taylor cone and fiber diameter will become larger [39]. Radius of the polymer jet is inversely proportional with the cube root of the conductivity of the polymer solution [40]. In literature Chronakis et al. studied PPy/PEO electrospun fibers [30]. They indicated that a crytical amount of PEO should be inserted into PPy solution to obtain a fibrous structure. Ji et al. [41] studied PPy with PAN and PAN/PPy bicomponent nanofibers exhibited increasing disordered structure with the increase of PPy concentration and intermolecular interactions were formed between PAN and PPy during nanofiber formation. It was reported ordered and beadless structure of PAN/PPy nanofibers with 10 and 25wt% of PPy contents previously [42]. Increasing PPy content resulted in an increase in solution conductivity and a decrease in solution viscosity, therefore producing nanofibers with smaller diameters .PPy has very poor mechanical properties and exhibits a brittle behavior. Therefore, in this study, PAN was used as a host polymer for PPy to improve its mechanical properties. Tensile tests were performed to measure the mechanical properties of PAN/PPy nanofibers. Table 2 shows the tensile properties of the PAN/PPy nanofibers containing 10 and 25 wt% of PPy. The tensile strain at failure of the nanofibers containing 10 wt% of PPy was much higher than those for 25 wt% of PPy. This was attributed to the brittle nature of PPy. However, the tensile strength of nanofibers were not significantly affected by PPy content.PPy is a hydrophobic material. However, as a copolymer PAN affected the surface properties and electrospun PAN/PPy nanofibers exhibited a hydrophilic property in water, as observed on contact angle measurements given in Table 2. As increasing PPy amount from 10 to 25%, contact angle value increased about 7o .The polymer solution viscosity also has a significant influence for uniformity and diameter of obtained fibers. The viscosity values of the solutions measured are given in Table 2. The viscosity of prepared solution containing 10wt% PPy was measured to be about 2 times higher than those of the solution containing 25wt% PPy. Increasing PPy amount in solution decreased the solution viscosity. So, higher solution viscosity resulted with larger PAN/PPy nanofibers in diameter [42] because it is more difficult to overcome surface tension for electrical forces at higher viscosity values. It was observed that a high viscosity solution prevented continuous and ordered fiber formations.Two different amounts of CNTs (1 and 5 wt%) were added into the PAN/PPy solutions containing 25% PPy and then the fibers were electrospun from the prepared solutions. Nanofiber formation was affected by the addition of CNTs in two different ways. The first effect of CNT incorporation was increament in the solution conductivity, the second effect is CNT addition broke the solution homogenity. As a result, some nanofibers were achieved with 1 wt% of CNTs addition, although they contained many beads and irregularities. On the other hand, almost no fiber formation was observed for 5 wt% CNT content. Only discrete beads were formed in this case which might be due to high solution viscosity and agglomeration of CNTs. To obtain better nanofibers with CNTs, PPy content was decreased to 10 wt% and different amount of as-grown CNTs were added . SEM micrographs of these samples are shown in Figure 3. For 1 wt% CNT content, obtained electrospun nanofibers were smooth and almost no bead formation was observed. However, increasing CNT content terminated continuous spinning and as a result, number of beads increased dramatically. Besides, with increasing CNT content average dimeter of nanofibers were obtained as 95.27, 85.33 nm, 81.77 nm and 69.94 nm for the CNT amount of 1, 2, 3, and 4 wt%, respectively. It was indicated that CNT incorporation decreased the average dimeter of nanofibers. When the bead diameter was analysed, for 2 wt% CNT content the average bead dimeter was 345.76, for 4 wt% CNT amount, the average bead diameter desreased to 217.74 nm. As a result, with the increasing amount of CNT, number of beads increased, however, fiber and bead diameter decreased. In literature Kaur et al studied CNT incorporated PAN nanofibers and they also reported nanofiber diameter decrease with CNT addition [45]. It was observed that nanofibers with 4 wt% CNT sample contained a high number of beads and disordered features on their surfaces. TEM images of this sample showed that some of the CNTs were not fully embedded into the nanofibers and they agglomerated either forming beads or simply stuck to the surface of the fibers .To further improve nanofiber formation, functional CNTs (1 and 4 wt%) were added to 10 wt% PAN/PPy mixture. The SEM micrographs of the PAN/PPy/CNT nanofibers prepared with functional CNTs are given in Figure 5. For 1 wt% of functional CNTs, the amount of nanofibers produced were higher and the numbers of beads were lower, as compared to the electrospun nanofibers prepared with as-grown CNTs. Similarly, a better nanofiber formation was observed for 4 wt% functional CNTs compared to as grown CNTs. When the average diameter of nanofibers containing functional CNTs were examined; for 1 wt% CNT amount, average diameter was 92.16 nm and very little increase was observed compared to nanofibers containing 1 wt% as-grown CNTs. For 4 wt% CNT amount, the average diameter was 73.84 nm. Here, a little decrease was observed in average dimater compared to the sample containing as-grown CNTs. Functionalization of CNTs was not significantly influenced fiber diameter. It was revealed in this study that functionalization of CNTs improved electrospun nanofiber formation significantly.-1 and 2354 cm-1 are the characteristic peaks of PAN and the peaks at 1270 cm-1 and 1654 cm-1 are induced by C-N and C=N stretching, respectively. The peak at 1454 cm-1 and 1068 cm-1 corresponded to the C=C and C-H. This data confirmed the PPy existence in PAN. Addition of as-grown CNTs did not change the FT-IR spectrum of PAN/PPy nanofibers due to the symmetrical structure of as-grown CNTs. However, sample including functionalized CNTs showed a broad peak at about 3400 cm-1 which was a characteristic of the O-H stretching of hydroxylic group. Peaks between 2800 and 3500 cm-1 were characteristic peaks of C-H and O-H bonds which could be related to carboxylic and hydroxylic groups.Figure 6 shows the FT-IR spectra of CNT embedded PAN/PPy nanofibers. Peaks observed at 1454 cm-1 and 1590 cm-1, respectively. Nitrile group (-CN) peak was observed at 2240 cm−1 . ID/IG ratio was significantly low if as-grown CNTs were utilized due to highly graphitized CNTs. ID/IG value increased with the addition of functional CNTs. Graphitization decreased with functionalized CNT addition because of broken bonds after acid treatment.Raman spectra of the PAN/PPy/CNT electrospun nanofibers containing 1 wt% CNT is displayed in Figure 7. D and G peak were observed at about 1300 cmThe concentric tubular structure of functional CNTs could be clearly observed in TEM images of the nanofibers . They were dispersed uniformly and aligned along the direction of nanofibers.EIS data give information about the nature of electrochemical process occurring at the electrode/electrolyte interface. Analysis of the interficial properties such as resistance and capacitance of the sample are combined by EIS by applying a very small amplitude sinusoidal voltage. During EIS measurements electrospun nanofibers displayed electroactivity, they remained stable and no deformation or decomposition was observed in their structures. The Nyquist graphs of PAN/PPy nanofibers were given in the frequency range of 0.01 Hz to 100 kHz . Nyquist plots of PAN/PPy nanofibers exhibited semicircles. The diameter of the semicircle represents the charge-transfer resistance (Rct) of PAN/PPy nanofibers .[46]. Rct of nanofibers decreased with inceasing PPy amount which depends on the change in charge of the surface of PAN/PPy nanofibers. With 25 wt% PPy content Rct decreased significantly. CNT addition in PAN/PPy nanofibers also decreased Rct value of nanofibers as given in Table 3. The impedance value of nanofibers calculated from Nyquist plots decreased with increasing PPy amounts as given in Table 3. While PAN nanofibers showed a 2603 ohm Rct value, Rct values were 1688 and 1066 ohm for PPy ratios of 10 and 25 respectively. It means the highest PPy content showed the highest electrical conductivity. CNT addition also decreased the Rct value from 1688 to 1481 ohm. As a result, increasing PPy and CNT addition decreased Rct and improved electrical properties of nanofibers. PPy as a conducting polymer and CNT as a conducting nanomaterial enhanced the conductivity of nanofibers, therefore the decrease in Rct values was observed with the addition of these materials as expected.PAN/PPy and CNT added PAN/PPy nanofibers were prepared by electrospinning method. The diameter of the nanofiber decreased by increasing PPy content in PAN/PPy. The nanofibers with low PPy amount were found to be more ductile. The nanofibers with beads and agglomerated CNTs were observed when as-grown CNT was added to PAN/PPy. With increasing CNT content, extensive bead formation and disordered sites were observed on nanofibers. The formation of electrospun nanofibers became more difficult at high PPy and CNT contents. This was probably due to agglomeration of CNTs, high solution viscosity and conductivity. Functionalization of CNTs enhanced the dispersion of CNTs within the nanofibers, and hence CNT alignment along the direction of nanofibers under high electrical field. In order to obtain more homogen nanofibers with higher tensile strain and higher hydrophilicty, PPy should be utilized low contents in PAN solution in DMF and CNT incorporation also should be kept at low contents and functionalized CNTs shold be used. The EIS results indicated that CNT addition and increasing PPy amount in nanofibers decreased the resistance value and enhanced electrical properties of nanofibers.