Electrospun polymer nanofibers (EPNF) constitute one of the most important nanomaterials with diverse applications. An overall review of EPNF is presented here, starting with an introduction to the most attractive features of these materials, which include the high aspect ratio and area to volume ratio as well as excellent processability through various production techniques. A review of these techniques is featured with a focus on electrospinning, which is the most widely used, with a detailed description and different types of the process. Polymers used in electrospinning are also reviewed with the solvent effect highlighted, followed by a discussion of the parameters of the electrospinning process. The mechanical properties of EPNF are discussed in detail with a focus on tests and techniques used for determining them, followed by a section for other properties including electrical, chemical, and optical properties. The final section is dedicated to the most important applications for EPNF, which constitute the driver for the relentless pursuit of their continuous development and improvement. These applications include biomedical application such as tissue engineering, wound healing and dressing, and drug delivery systems. In addition, sensors and biosensors applications, air filtration, defense applications, and energy devices are reviewed. A brief conclusion is presented at the end with the most important findings and directions for future research. Poly L 182]..182].The superior properties of electrospun nanofibers have contributed to the large interest in processing them and their utilization in industry. The relative simplicity of the electrospinning process made this interest increase. The great effect of the high ratio of surface to volume due to the nano-scale presence gives rise to improved strength, better surface reactivity, larger surface energy, and higher electrical and thermal conductivity. Therefore, seeking to mathematically model the spinning process and resulting properties became of great interest. In what follows, we briefly review some of the most important models in the literature.τ viscous force, One-dimensional steady state mathematical model for an electrospinning polymer jet ,184,185:The mathematical model for a steady state polymer jet in the electrospinning process was developed by Spivak and colleagues ,187,188.The mass balance equation results in:Linear momentum balance:Electrical charge balance:The right-hand side momentum balance equation is the sum of viscus and electrical forces.The Wan–Guo–Pan mathematical model considers the combined impacts of heat, electricity, modified Navier–Stroke equation, and hydrodynamics considered by this model ,190. TheThere are three different current constituents: (a) Ohmic bulk conduction current I” represents the current, R the resistance, E the voltage, and According to Ohm’s law, current flows to the low voltage gradient in proportion to the resistance of circuit. It can be shown that: follows :(19)g∼rαIn the case when α = 2, specimens behave like a metal conductor, and the modified ohm is used for bulk conduction.In the case when α = 1, there are no free ions or electrons in the bulk, therefore, the current is produced by surface charge, divided along the surface which is in motion. Therefore, surface convection current:Thus, the value of α will lie between 1 and 2 as conduction of synthesized charged jet falls between Ohmic bulk conduction and surface convection. We can alternatively consider the conductance and polymer concentration scale with each other in the following ways ,190:(22)One of the most significant applications of polymer nanofibers is in the biomedical sector, particularly in the domains of medication delivery and tissue engineering. Given that biological molecules and nanoscale fibers have similar sizes, the latter are poised to perform well in simulating biological environments and natural extracellular matrices. Nanofibrous meshes exhibit enhanced biological activities, such as increased cell adhesion, differentiation, and proliferation, due to high porosity, large surface area to volume ratio, and interconnectivity of porous matrices comparable to macromolecular ones. Additionally, it is also conceivable for biological molecules to load for nutrients and wastes to exchange through pores ,192. TheElectrospun polymer nanofibers utilized as scaffolds in tissue engineering for different tissues such as nerve, bone, blood vessels, cartilage, and skin, etc. have attracted much attention . RestoriIn wound rehabilitation pursuit, the large porosity of electrospun fibers may provide additional structural space for accommodation of transplanted cells, promote cell migration and proliferation, and increase oxygen exchange and waste outflow. The tiny pore size of nanofibrous scaffolds can prevent dehydration and wound infection throughout the healing process. Additionally, tunable mechanical properties of electrospun nanofibers can maintain mechanical consistency between tissue engineering grafts and parent tissue and prevent the wound from wrinkling or shrinkage during implantation.For skin tissue engineering, various natural and synthetic polymers are electrospun to nanofibrous scaffolds. Natural polymers such as collagen, fibrinogen, chitosan, silk, etc. have been processed for wound healing. Collagen is a principal element of the human skin extracellular matrix; it develops a three-dimensional fibrillar network structure with diameters in the range of 50–500 nm to normalize attachment, differentiation, and proliferation in skin texture. Electrospun cellulose gelatin/acetate nanofibers have resemblance to the extracellular matrix composition of skin. It was studied that 75/25, gelatin/cellulose acetate nanofibers revealed distinctive adhesive features and improved fibroblast proliferation to human skin. However, utilizing natural polymers for tissue engineering revealed poor mechanical properties and less resistivity to enzymatic degradation, which are among major challenges facing this promising technology. A variety of synthetic biodegradable polymers such as polyglycolic acid, polylactic acid, polycaprolactone, and copolymers are generally used for skin tissue engineering because of their advantageous mechanical and biodegradable characteristics. For example, electrospinning polylactic-co-glycolic acid with glycolide/lactide having a molar ratio of , respectively, can accomplish the required biodegradable scaffolds for the replacement of damaged skin. The usage of synthetic polymers is, however, constrained by their hydrophobic surface and limitations of cell-detection signals .Portable Electrospinning for Wound DressingAn important promising development is in portable electrospinning technology. Portable electrospinning works by directly producing healing nanofibers on the wound to customize the dressing, relieve the pain, and increase the correspondence to wound bed. These features give it the advantage over using pre-prepared nanofibers in different types of wounds such as cut skin, irregular skin wounds, burned skin, liver cuts, dural repairs, etc. Furthermore, it also leads the way for personal wound dressing. Practically, in situ wound dressing is possible by a portable electrospinning setup that is user friendly and safe to operate. A portable electrospinning machine was utilized for in situ spinning of poly (lactic-co-glycolic acid) fibers in the wound dressing process, where they were deposited precisely on the injured areas .For in situ electrospinning, the versatility of portable devices depends upon biological collector and modified components of devices such as spinneret and voltage supply systems. The portability of devices depends upon the requirements and nature of modifications. Portable devices have different sources of power supply, which are summarized in Various drug delivery systems such as polymer micelles, liposomes, and nanofibers are studied to diminish the toxicity of dosage and increase therapeutic efficacy ,200,201.Nano-based sensors and biosensors are utilized according to combined chemical and physical principles based on the two main components of the receptor and the transducer. The receptor reacts chemically to the change and creates a form of energy that the transducer measures and signals . SignifiResponding to air pollution challenges, which stem basically from especially fine particle in the environment, has become a pressing priority, whether in industrial sites or in urban areas. With the increasing pace of industrialization and energy consumption and mining, the urgency is even greater. Volatile organic compounds and microbes in air can seriously harm human life . VariousPolymer nanofibers are considered as exceptional membrane materials for the defense industry in smart textiles for the detection of biological and chemical warfare agents with high sensitivity. Their high sensitivity to biological and chemical pollutants at concentrations of parts per billion make them attractive candidates for sensing interfaces for warfare agents . In one Renewable energy resources are becoming more dominant to ensure economic growth due to the diminution of fossil fuels and huge increase in demand for energy. It was reported that nanofibers perform better than typical materials in devices for energy storage, harvesting, and conversion, offering good alternative materials for use in energy devices such as lithium-ion batteries, nanogenerators, and solar cells . NanofibTo conclude this section on applications of polymer nanofibers, the mechanisms by which different polymer nanofibers act to achieve the needed outcome in different applications are summarized in The versatile applications for electrospinning of polymer nanofibers stem from the quite diverse sets of technologies employed, polymers and solvents used, conditions of processing … etc. In all applications, there are the driving forces of market volume, demand, and how critical the application is. The medical field is a strong market, but there are also other critical applications such as water treatment and air filtration, in addition to textile and other industries. Due to their unique characteristics, ease of production, and diversity of combinations, in addition to superior properties, electrospun polymer nanofibers (EPNF) are poised to play a larger role in the development of many technologies that span different areas of human demand in different sectors. In this paper, we presented an encompassing review of polymer nanofibers’ attractive features, production techniques, and, especially, electrospinning, including a detailed description of the process. The polymers that are used in electrospinning were covered. Special coverage of the most important electrospinning process parameters was included so that the processes’ different variations can be understood through manipulation of such parameters. EPNF mechanical and other properties of EPNF were reviewed, with special attention to mechanical tests and techniques. Due to the importance of models in predicting the features and behavior during processing and in service, a brief review of important mathematical models was also presented. The increasing and widening applications of EPNF were included in the final section, covering various fields from biomedical applications, sensing, and biosensing technologies to defense and energy applications.The field is ripe for more advances in research and industrial endeavors, due to the interesting findings in both diversification of processing technologies and techniques and also in the lateral and vertical expansion of applications. More efforts should be made in the experimental, computational, and theoretical directions. With the wide choices for the direction of experimental research, the need is great for the development of theoretical and computational models to streamline choices of experimentation, whether in the choice of pristine polymers or processing techniques and parameters. The ability to characterize features and behavior, even with some degree of idealization and simplifications, will offer better informed choices for development directions in the experimentation to improve electrospinning processes’ efficiency and precision and directions in which to expand and improve utilization throughout the wide spectrum of applications.


The monitoring of antimicrobial resistance (AMR) in bacterial pathogens of animals is not currently coordinated at European level. To fill this gap, experts of the European Union Joint Action on Antimicrobial Resistance and Healthcare Associated Infections (EU-JAMRAI) recommended building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet). In this study, we (i) identified national monitoring systems for AMR in bacterial pathogens of animals (both companion and food-producing) among 27 countries affiliated to EU-JAMRAI, (ii) described their structures and operations, and (iii) analyzed their respective strengths, weaknesses, opportunities and threats (SWOT). Twelve countries reported having at least one national monitoring system in place, representing an opportunity to launch EARS-Vet, but highlighting important gaps in AMR data generation in Europe. In total, 15 national monitoring systems from 11 countries were described and analyzed. They displayed diverse structures and operations, but most of them shared common weaknesses  and common threats , which could be addressed collectively under EARS-Vet. This work generated useful information to countries planning to build or improve their system, by learning from others’ experience. It also enabled to advance on a pragmatic harmonization strategy: EARS-Vet shall follow the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards, collect quantitative data and interpret AMR data using epidemiological cut-off values. To fill this gap, experts of the European Union Joint Action on AMR and Healthcare Associated Infections (EU-JAMRAI),ckle AMR , recommeckle AMR . The objIn the One Health approach, EARS-Vet should be designed to complement and integrate with existing European monitoring systems for AMR, i.e., the European Antimicrobial Resistance Surveillance Network (EARS-Net)  and the Escherichia coli, Klebsiella pneumoniae, Mannheimia haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus hyicus, Streptococcus uberis, Streptococcus dysgalactiae, and Streptococcus suis); and relevant antimicrobials for their treatment , complemented with antimicrobials of more specific public health interest . Although reviews of AMR monitoring systems in the animal sector have already been published  identify existing national monitoring systems for AMR in bacterial pathogens of animals among 27 countries affiliated to EU-JAMRAI , (ii) describe their structures and operations, and (iii) analyze their respective strengths, weaknesses, opportunities and threats (SWOT).EU-JAMRAI stakeholders from 27 countries of the EU/European Economic Area (EEA) were contacted in 2018–2020 and asked if a national monitoring system for AMR in bacterial pathogens of animals was in place in their country: Austria, Belgium, Croatia, Cyprus, the Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Ireland, Latvia, Lithuania, Malta, the Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, and the United Kingdom. A national monitoring system for AMR in bacterial pathogens of animals was defined as any system collecting and regularly analyzing AST results produced on bacterial isolates from clinical samples of animals that can be considered as having a national coverage. However, no criteria were established on geographic data representativeness or scope of bacterial and animal species.Stakeholders in individual countries who reported having a national monitoring system for AMR in bacterial pathogens of animals were invited to have their system described and performance analyzed, using a common methodology across participating countries.The description of existing national AMR monitoring systems was done through a questionnaire covering the following key areas: (i) political and financial support, (ii) monitoring objectives, (iii) central institutional organization, (iv) laboratory network, (v) monitoring procedures, (vi) laboratory techniques, (vii) monitoring data, (viii) communication and (ix) evaluation .The performance analysis of national monitoring systems was done using SWOT  analyses . In couna posteriori and used to describe the main strengths, weaknesses, threats and opportunities of national monitoring systems. After completing the questionnaires and SWOT analyses, further clarifications were obtained via email exchanges on an ad hoc basis between the expert from ANSES and the coordinators of the national monitoring systems. Questions considered of lower relevance or not leading to factual answers were excluded from the data analysis.Both questionnaires and SWOT analyses were completed in 2019–2020 during 1-day physical meetings, except for one country (Estonia), where two virtual meetings were organized due to travel restrictions linked to the COVID-19 pandemic. Each physical or virtual meeting was jointly organized by the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) and the EU-JAMRAI partner institution(s) of the visited country. Participants were relevant national stakeholders, including the coordinator(s) and key experts of the national AMR monitoring system, as well as an expert from ANSES, who asked the questionnaire and facilitated the completion of the SWOT analyses. Frequently reported topics in SWOT analyses were identified The following 12 countries reported having a national monitoring system for AMR in bacterial pathogens of animals: the Czech Republic, Denmark, Estonia, Finland, France, Ireland, Germany, the Netherlands, Norway, Spain, Sweden, and the United Kingdom. Stakeholders from Denmark, the Netherlands and Sweden reported more than one monitoring system in their countries . Ten couThe questionnaire and SWOT analysis were completed for all countries identified with a national monitoring system for AMR in diseased animals, except the United Kingdom, where the country visit got canceled due to the COVID-19 pandemic and could not be rescheduled virtually within the project time frame. In total, 15 monitoring systems from 11 countries were described and analyzed .Results are presented per each key area, as defined in section “Materials and Methods.”All countries under investigation had a National Action Plan (NAP) to tackle AMR. All NAPs, except in the Netherlands and Estonia, specify the need to perform AMR monitoring in diseased animals and often request specific organizations to perform this monitoring. However, as shown in Even if not regulated or not integrated in NAPs, national monitoring systems can still benefit from governmental support such as in the Netherlands, where AMR monitoring is funded by the Dutch government  or by both the government and producer/interbranch organizations (for livestock). Still, some national monitoring systems remain independent initiatives such as the monitoring of the University of Copenhagen (UC) in companion animals, without dedicated governmental or specific financial support  .When dedicated national funding is allocated, it can consist of a subsidy (i) to decrease the AST costs for farmers (in the Czech Republic and Spain) or (ii) to do more pathological examinations on farm animals, which are, ultimately, an important source of AST data . In this way, subsidies support two goals: collecting more monitoring data and supporting veterinary antimicrobial stewardship. In some countries (Norway and Germany), a budget has been allocated to re-test isolates at a central laboratory. This ensures AST harmonization, irrespective of isolate origin, in terms of method, antimicrobials tested, and interpretive criteria applied. It also enables to test antimicrobials which are not tested by field veterinary diagnostic laboratories, but which may be of interest in a public health perspective . Alternatively, funding can also be intended to complementary monitoring programs to fill gaps in the regular monitoring in bacterial pathogens of animals . Other costs of AMR monitoring, such as human resources of the coordination team, may either be supported by the regular budget of the coordinating institution or by dedicated funding  .Monitoring objectives are summarized in Nearly all systems aim to monitor AMR trends (14/15) and detect emerging AMR (13/15) for antimicrobials of interest to veterinary medicine. Although 13 monitoring systems aim to inform veterinarians on AMR levels to help them in their antimicrobial treatment decisions, only six systems produce AMR data with the specific aim to develop antimicrobial therapy guidelines intended to veterinarians. It is worth noting that the Czech National Monitoring of Target Pathogens’ Antimicrobial Resistance (CZ NMTP) was established primarily to support antimicrobial stewardship by incentivizing the use of AST and evidence-based antimicrobial therapy by veterinarians.Most systems (12/15) also aim to monitor AMR trends and detect emerging AMR in a public health perspective, demonstrating the frequent adoption of the One Health approach. Furthermore, in seven systems such AMR data are intended to be used to assess the risk of AMR transmission between animals and humans via non-food related routes, as well as (in six systems) to better understand AMR epidemiological links between the animal and human sectors.Political and Financial Support), only seven and five systems produce AMR data with the specific objectives to advise policy makers on interventions to mitigate AMR and to monitor the impact of the NAP, respectively.Although most NAPs include AMR monitoring in diseased animals . As shown in Sistema Español de Vigilancia de Animales Enfermos—SEVAE), the French surveillance network for antimicrobial resistance in diseased animals (RESAPATH), the GD Animal Health Surveillance System and the national monitoring system coordinated by the Technical University of Denmark and the Danish Veterinary and Food Administration (DTU/VFA). They show various compositions, ranging from representatives of a single organization to representatives of diverse institutions from both public and private sectors .A steering committee was defined as a committee defining the vision and objectives of a monitoring system, as well as approving monitoring procedures , are also very different across systems . They maSweden, Denmark, and the Netherlands have more than one monitoring system for AMR in clinical animal isolates, but these are only integrated in Sweden. However, collaboration between systems usually exists, such as in Denmark, where SEGES provides AST data from swine to DTU/VFA.RM-Vet) and SEVAE, which operate through 71, 30, and 22 laboratories, respectively. Networked laboratories either belong to the same institution , or comprise independent laboratories, belonging to both private and public sectors  or with a joint public/private status (CZ NMTP). The National Reference Laboratory (NRL) for AMR in the animal sector is involved in six systems.As shown in All laboratory networks, except in SEVAE, include one or two central laboratories. In RESAPATH, the two central laboratories (both belonging to ANSES) are responsible for AMR monitoring in different animal species. Central laboratories may have diverse missions, depending on the system: analysis of diagnostic specimens, re-testing isolates of specific interest from field laboratories, re-testing and performing complementary molecular analyses on isolates with specific phenotypes and organizing proficiency testing (PT) on AST as part of reference activities .All systems are primarily based on a passive monitoring procedure, i.e., AST results come from samples, that are routinely submitted by field veterinarians. However, two of them also include an active monitoring component (SvarmPat and DTU/VFA system), where diseased animals are sampled specifically for the purpose of AMR monitoring (and would likely not be sampled otherwise). These active monitoring components aim to fill knowledge gaps and generate more representative AMR data than by passive monitoring. They remain project-based and monitoring priorities can change over time.Laboratory techniques and standards of the 15 monitoring systems are described in Regarding bacterial identification, the most commonly used method was the Matrix Assisted Laser Desorption Ionization—Time of Flight (MALDI-TOF) (by 14/15 systems), followed by Analytical Profile Index (API) galleries (by 3/15 systems). Among national monitoring systems based on a laboratory network and which do not re-identify all bacterial species at a central laboratory, SEVAE, CZ NMTP, and the DAFM system have developed agreements or standard operating procedures, so that all member laboratories use the same bacterial identification method. In CZ NMTP and the DAFM system, PT is also in place to ensure the performance of participating laboratories in bacterial identification. Conversely, no standard or recommendation is provided to field laboratories in RESAPATH and the Finnish Veterinary Antimicrobial Resistance Monitoring and Consumption of Antimicrobial Agents (FINRES-Vet).Regarding AST, broth microdilution is the reference method in 12 systems, followed by disk diffusion for four systems. Both methods are used as part of FINRES-Vet, depending on the animal species. Broth microdilution is also used in the DAFM system for confirmatory testing and in the Estonian system, coordinated by the Veterinary and Food Laboratory and the University of Life Sciences (VFL/ULS), for testing colistin resistance. Eight countries follow the standards of the Clinical and Laboratory Standards Institute (CLSI) and interpret results according to its veterinary clinical breakpoints (when available). Different alternatives are used by countries when veterinary breakpoints are not available (a situation reported as frequent): epidemiological cut-off values (ECOFFs) of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), epidemiological cutoff values (ECVs) of CLSI, human clinical breakpoints of EUCAST or CLSI, ECOFFs of the veterinary guidelines of the Antibiogram Committee of the French Society of Microbiology (CA-SFM), clinical breakpoints suggested by pharmaceutical companies, minimum inhibitory concentration (MIC) 90%, or internal ECOFFs . On the other hand, NORM-Vet and Svarm/SvarmPat follow the EUCAST guidelines and its ECOFFs (when available), while RESAPATH follows the AST technique and ECOFFs (when available) recommended in the veterinary guidelines of CA-SFM. In case of missing ECOFFs from their respective standards, RESAPATH does not provide any interpretation, NORM-Vet and Svarm/SvarmPat use internal ECOFFs (when possible) or CLSI ECVs (when available), while Svarm/SvarmPat may also use CLSI clinical breakpoints.®, proposed by the Animal and Plant Health Agency in the United Kingdom. Other quality control measures like the use of reference strains with a defined MIC range are also in place, hence all systems have some level of quality control.For the six systems that operate on a laboratory network without systematically re-testing all isolates centrally , a single AST standard is used. To guarantee the quality of AST results , laboratories are either accredited on the AST technique and/or participate in PT in 13 systems. PT can be organized by the NRL, the central laboratory, or an institution from a different country, such as the VETQASMonitoring data are presented in As shown in Outil d’Analyse des Systèmes d’Information en Santé (OASIS) , using the  (OASIS) . For the (OASIS) . Only RE (OASIS) , such asEleven SWOT analyses were produced (one per country), available in Flexibility and Utility of the system were reported as a major strength in nine countries out of 11. All of them highlighted their system had a broad scope, making it possible to monitor a diverse range of animal species, bacterial species and antimicrobial combinations, of relevance to the local epidemiological situation. Utility for veterinary practice  was reported as a strength by three countries. Another frequently reported strength (n = 10) was the level of Harmonization of the AST analyses performed within the monitoring system, with most participating laboratories being accredited for AST. Geographical coverage was reported as good (n = 5) or unknown (n = 1).The n = 9) reported that a strength of their system was the very good collaboration they had with other AMR monitoring stakeholders or partners, including those in the human sector (n = 5) and in healthy animals (n = 3), ministries of agriculture (n = 3), veterinary research institutes (n = 2), or private industry (n = 1) (Collaboration and Integrated monitoring). Five countries also reported their system relied on a small team, thereby facilitating internal communication and coordination.Most participating countries , while three countries reported poor data quality, such as incomplete or invalid metadata . Capacity for storing isolates collected through the monitoring system was reported as an issue in only one country.Representativeness, i.e., the ability to provide a reliable picture of AMR through sufficient and representative data for each combination of animal species/bacteria, which was reported as low (n = 9) or unknown (n = 2) in most countries. In most systems, this was related to the use of passive sample collections to monitor AMR.Another weakness shared across systems was the n = 8) considered the societal context, in terms of Public awareness and Policies, as an opportunity for their monitoring system. Six countries mentioned an increasing level of public awareness on AMR, and five reported AMR was a policy priority in their country. Three countries claimed their system was supported by national or EU Action plans on AMR, and four countries had national legislation or programs in place to enforce or encourage the use of AST in the veterinary sector, thus contributing to an increase in the volume of data submitted to the monitoring system.Most participating countries ((Collaboration and Integrated monitoring). Six countries also reported EARS-Vet as an opportunity to learn about practices and solutions from other countries that could be applicable to their own system.While collaboration between sectors was perceived as a strength in most participating countries, four countries reported there was an opportunity to improve the One Health integration of their system n = 9) reported the economic vulnerability as the main threat to their monitoring system, with a regular decrease in available financial and human resources (Sustainability and Resources). Three countries reported that lack of funding prevented them from performing more advanced data analyses or improving data representativeness. Of note, two countries also reported a lack of skilled AMR experts in animal health in their country.Almost all participating countries , or the use of alternative techniques  preventing access to more conventional AST results (n = 2). The development of large and competitive private laboratory companies was perceived as a threat in five countries, although three countries had good collaborations with these laboratories, and perceived them as an opportunity to further expand their laboratory network (Collaboration and Integrated monitoring).Various threats to To our knowledge, this is the first review of national monitoring systems for AMR in bacterial pathogens of animals describing system structures and operations and analyzing their performance with a standardized approach. We identified 12 countries with at least one national monitoring system, among 27. This led to a description and analysis of 15 systems in 11 European countries thanks to a questionnaire and SWOT analyses.A strength of our approach was to include in the study almost all countries of the EEA . This was facilitated by the EU-JAMRAI network of partner institutions and collaborating stakeholders, which facilitated contact with AMR experts in 27 countries. It enabled the identification of monitoring systems, which may not be easily identified through online searches, when no report is publicly available or only available in local language . However, no information could be retrieved for Latvia, Lithuania, Malta, Slovakia and Slovenia, despite several attempts to establish collaboration through the network. In addition, the questionnaire and SWOT analysis were not completed for the United Kingdom.The survey questionnaire allowed the provision of a broad overview of the structure and operations of national monitoring systems, by covering areas, which were not limited to microbiology, but also funding, regulation, institutional organization, procedures, laboratory networks, communication, and evaluation. Various methods exist to assess the performance of monitoring systems , but a sThe review clarified gray areas reported in a previous literature review , by confOn the one hand, many Southern and Eastern European countries do not have any national monitoring system for AMR in bacterial pathogens of animals , emphasiOn the other hand, the 12 countries with one or more national monitoring systems represent an opportunity to launch EARS-Vet.Our survey showed the diversity of organizations and operations of monitoring systems and may be particularly valuable to countries planning to establish or improve their system, by learning from the experience of other countries. As all systems had their own strengths and weaknesses, a lesson to learn may be that there is no single best way to monitor AMR in bacterial pathogens of animals and that each system needs to be adapted to its national context, capacities and objectives.Despite their differences, many systems shared common weaknesses, e.g., in data management and representativeness, and common threats, such as economic vulnerability and data access. Moreover, only two monitoring systems underwent evaluations, although this is an important practice to allow more transparent interpretation of outputs, more objective decision-making and resource allocation, as well as improvements in system design and enhanced acceptance of system outputs by stakeholders . HoweverOur review also contributed to the consolidation of a preliminary EARS-Vet network, thanks to numerous interactions with key experts from national monitoring systems. Such a network represents an opportunity to address frequent challenges of monitoring systems collectively and in a coordinated way.This review generated key information to advise the development of AMR monitoring in clinical animal isolates in Europe and more specifically to advance on the definition of the EARS-Vet framework. Indeed, EARS-Vet would need to collect accurate, representative and harmonized AST results produced and communicated by national monitoring systems.First, we showed that coordinating institutions own their AST data in all but two countries. This should facilitate the development of an EARS-Vet data sharing agreement. Moreover, all systems collect quantitative AST results, namely MICs for broth microdilution and inhibition zone diameters for disk diffusion, which would facilitate the re-calculation of proportions of non-wild type (or resistant) isolates, should interpretation criteria change over time.Second, most national monitoring systems are based on laboratories that are accredited on the AST technique and/or participate in PT and all of them implement quality assurance. Therefore, the capacity of national systems to produce accurate AST results is considered high. Still, the development of a European PT, as done in the framework of EARS-Net, should be considered.Staphylococcus pseudintermedius , EARS-Vet should accept only MIC data. Regarding the interpretation of AST results, EU-JAMRAI experts suggested using EUCAST ECOFFs, although more countries currently use CLSI veterinary clinical breakpoints. The reason for this is that for many drug-bug combinations, animal- and infection-specific clinical breakpoints are missing and will likely not be established in the short term. Moreover, EUCAST ECOFFs enable early detection of changing AMR trends and would facilitate the integration of EARS-Vet with the EFSA monitoring in zoonotic and indicator bacteria from healthy food-producing animals. Although many EUCAST ECOFFs are currently missing for the combinations to be monitored in EARS-Vet , ECOFFs Thus, the current landscape of AMR monitoring in the EEA showed many gaps but also an opportunity to launch EARS-Vet, as 15 national monitoring systems were already in place in 2020 with strong capacity in AST. Thanks to a thorough understanding of their practices, a pragmatic harmonization strategy could be proposed for AST. This work, combined with the definition of the EARS-Vet objectives  and scopThe original contributions presented in the study are included in the article/Evelyne Jouvin-Marche, Marie-Cécile Ploy, Sadika Bernard, Yohann Lacotte, Céline Pulcini, Marielle Bouqueau, Anton Hlava, Vera Buhmann, Eline Vandael, Lieven De Raedt, Blazenka Hunjak, Bojana Raickovic, Barbora Macková, Helena Žemlièková, Ute Wolff Sönksen, Sissel Skovgaard, Jüri Ruut, Ljudmila Linnik, Arina Zanuzdana, Nadiya Oezcelik, Flora Kontopidou, Mariana Tsana, Alkiviadis Vatopoulos, Achilleas Gikas, Aimilia Magkanaraki, Alessandra Cozza, Domenico Martinelli, Rosa Prato, Annalisa Pantosti, Francesca Prestinaci, Luca Busani, Roberta Creti, Uga Dumpis, Asta Dambrauskiene, Astra Vitkauskiene, Silvija Kiveryte, Agniete Mazzella, Rolanda Valinteliene, Robertas Petraitis, Jasper Claessen, Rosa Perán, Elma Smeets, Pita Spruijt, Svein Høegh Henrichsen, Christine Årdal, Mari Molvik, Oliver Kacelnik, Anne Margrete Urdahl, Dorota Żabicka, Sérgio Gomes, Razvan Ciortea, Maja Šubelj, Nina Jemec, António López Navas, Cristina Muñoz Madero, Laura Alonso Irujo, María Santacreu García, Gloria Oliva, Marta Massanés, Elena Ferragut, Eusebi Castaño, Casimiro Jiménez Guillén, Marisol Fragoso, Germán Peñalva, José Miguel Cisneros, Milena Estevez, Sophie Monteau, María José González de Suso, Pilar Gallego Berciano, Daniele Alioto, Lotta Edman, Axana Haggar, Elisabet Lindal, Jakob Ottoson, Anna Nordenfelt, Björn Bengtsson, Patriq Fagerstedt, Birgitta Lytsy, Jean Yves Madec, Lucie Collineau, Rodolphe Mader, Anne Berger-Carbonne, Karl Pedersen, Monika Larsson, Annicka Reuss, Anne Swalue, Cristina Portugal, Hans Fredrik Wilhelmsen, Jesús Oteo-Iglesias, Janicke Fischer, Helga Katharina Haug, Maria Da Graca Freitas, and María del Pilar López Acuña.RM coordinated the overall study, led the questionnaire and SWOT analyses in the 11 countries, coordinated the group discussions to develop the initial EARS-Vet framework, and drafted the initial manuscript. LC analyzed the SWOT results and drafted the corresponding section. J-YM, CMM, and J-PA supervised the overall work. CMM, BA, CB, EMB, PC-R, PD, WF, AH, JH, HK, MN, KP, TP, LP, RS, CTJ, AMU, J-YM, and J-PA contributed to the physical and virtual meetings to answer the questionnaires and SWOT analyses. All authors have contributed to developing the methodology, and joined discussions, which led to initial agreements on the EARS-Vet framework, reviewed the draft for important intellectual content and approved the final version.The views expressed in this publication are those of the authors and do not necessarily reflect the opinions of consulted experts and organizations.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We further showed that pharmacological inhibition of A2AR with istradefylline partially normalised striatal GDNF and striatal and cortical dopamine levels in mice. Lastly, we found that GDNF levels are increased in the cerebrospinal fluid of first episode psychosis patients, and in post-mortem striatum of schizophrenia patients. Our results reveal a possible contributor for increased striatal dopamine signalling in a subgroup of schizophrenia patients and suggest that GDNF—A2AR crosstalk may regulate dopamine function in a therapeutically targetable manner.Presynaptic increase in striatal dopamine is the primary dopaminergic abnormality in schizophrenia, but the underlying mechanisms are not understood. Here, we hypothesized that increased expression of endogenous GDNF could induce dopaminergic abnormalities that resemble those seen in schizophrenia. To test the impact of GDNF elevation, without inducing adverse effects caused by ectopic overexpression, we developed a novel in vivo approach to conditionally increase endogenous GDNF expression. We found that a 2–3-fold increase in endogenous GDNF in the brain was sufficient to induce molecular, cellular, and functional changes in dopamine signalling in the striatum and prefrontal cortex, including increased striatal presynaptic dopamine levels and reduction of dopamine in prefrontal cortex. Mechanistically, we identified adenosine A2a receptor (A Schizophrenia is a debilitating neuropsychiatric disease affecting 1% of the global population. The clinical features of schizophrenia include positive, negative, and cognitive symptoms, and at least some of these clinical manifestations are thought to reflect neurochemical disturbances, such as abnormalities in dopamine, glutamate, serotonin, adenosine, and gamma-aminobutyric acid signalling . Among tGlial cell line-neurotrophic factor (GDNF) is a secreted protein expressed predominantly in striatal interneurons . GDNF isWe and others have shown that ectopic gene overexpression in highly structured tissues, including the brain, can induce side-effects and artefacts, yielding results that are difficult to interpret –40. Thes2AR as a possible therapeutic target and show that administration of specific A2AR antagonist istradefylline partially normalises striatal and cortical dopamine levels in mice. Lastly, we find that GDNF levels are increased in the cerebrospinal fluid (CSF) of first episode psychosis (FEP) patients and in post-mortem striata of schizophrenia patients. Thus, our work demonstrates that 2–3-fold elevation in endogenous GDNF expression increases striatal presynaptic dopaminergic signalling and induces hypodopaminergia in prefrontal cortex in mice and suggests that elevated GDNF could contribute to the disease in a subgroup of patients with elevated GDNF levels. Our data also suggests A2AR as a potential drug target in a subgroup of schizophrenia patients with elevated GDNF.Here, we generate a novel mouse model allowing us to conditionally upregulate endogenous GDNF. We find that increased levels of GDNF are sufficient to induce altered dopamine signalling, including striatal hyperdopaminergia and cortical hypodopaminergia, as well as behavioural deficits and gene expression alterations similar to individuals with schizophrenia. We then identify AGdnf conditional hypermorphic mice, Gdnf cHyper). To that end, we inserted a FLEx cassette [Gdnf gene  in an iene Fig. . Based oern Fig. .Fig. 1InGdnf cHyper mice, we first analysed the kidneys, where Gdnf expression pattern and the outcome of Gdnf gene level alterations are well characterised. Mice lacking GDNF have no kidneys [Gdnf cHyper mice to a Pgk1-Cre line, where Cre expression results in ubiquitous recombination [Gdnf cHyper mice to a tamoxifen-inducible Esr1-Cre line [Gdnf  [Gdnf cHyper;Nestin-Cre mice  . In the onwards)  that regonwards) . We founice Fig. , whereasice Fig. . We thenice Fig. . In addiice Fig. . The tisice Fig. , and theice Fig. . AltogetGdnf cHyper;Nestin-Cre mice. We found a marked reduction in PFC dopamine levels in both Gdnf wt/cHyper;Nestin-Cre and Gdnf cHyper/cHyper;Nestin-Cre mice compared with controls  test, which reflects the ability to successfully integrate and inhibit sensory information. PPI refers to a reduction in startle response in response to an intense stimulus if preceded by a lower intensity stimulus [Gdnf cHyper/cHyper;Nestin-Cre mice compared with controls  –72, we mnsponder . We founage Fig.  and incrage Fig. , suggestage Fig. , compulsage Fig. , learninage Fig. , motor iage Fig.  or problage Fig. .Gdnf cHyper/cHyper;Nestin-Cre mice are associated with specific deficits in sensorimotor gating, daily activity and drinking behaviour, but not with general locomotor activity, anxiety, compulsive behaviour or cognitive function.Together, our results suggest that striatal hyperdopaminergia and PFC hypodopaminergia in Gdnf cHyper;Nestin-Cre mice at 2 months of age. We identified 311 differentially expressed (DE) genes in Gdnf wt/cHyper;Nestin-Cre mice and 214 genes in Gdnf cHyper/cHyper;Nestin-Cre mice compared to controls  overlapped with DE genes in either of these two studies, including Adora2a, Arc, Btg2 and Inf2  of 2A receptor (A2AR) is a G-protein coupled receptor that regulates the balance of adenosine/dopamine signalling through its interactions with D2R [2R, while A2AR antagonists, such as caffeine are believed to enhance dopamine signalling through D2R [Gdnf cHyper;Nestin-Cre mice  from first episode psychosis (FEP) patients. CSF was collected from 70 FEP patients, who were either fully naive to antipsychotic drugs as per interview and available medical records or had been prescribed an antipsychotic drug for less than 2 months. For comparison, we used CSF from healthy controls (HCs). We found that FEP patients naive to antipsychotic drugs  for severity of illness (CGI-S) scores. This analysis indicated that CSF GDNF levels correlated with a more severe clinical profile Fig. , Table 2GDNF mRNA levels in post-mortem striatum in individuals with schizophrenia compared to controls  in Gdnf promoter [2AR antagonism acutely reduces GDNF-induced dopamine release in synaptosomes and striatal slices [2AR signalling could promote GDNF-triggered dopamine release. Thus, A2AR antagonism may alleviate schizophrenia symptoms by two complementary mechanisms: acutely via inhibition of enhanced GDNF-driven striatal dopamine release and chronically via downregulation of striatal GDNF expression . Demographics and clinical characteristics of the study participants are provided in Table Animal experiments were conducted according to the 3R principles of the European Union Directive 2010/63/EU and following local laws and regulations. Protocols were authorised by the national Animal Experiment Board of Finland.g for 10 min at 4 °C. Supernatant was divided into aliquots and stored at −80 °C. The panel ‘Inflammation’ from Olink Bioscience  was used to assess protein levels in the CSF.CSF sampling was performed between 7:45AM and 10:00AM. Samples were centrifuged at 1438 Gdnf cHyper allele .In human studies, research subjects correspond to individual patients or controls. In animal experiments, research subjects correspond to individual animals, except for FSCV experiments, where the experimental units are brain slices.Because the genotype of the animal determined the experimental group, randomization to treatment was not possible. To reduce possible bias, only litters including animals from different genotypes were included. In behavioural tests, animals were tested in a random order. Treatments were administered in a random order.Experiments were performed by investigators blinded to experimental groups.t-test or Welch’s t-test, where appropriate. Multiple comparisons were performed with one-way or two-way analysis of variance (ANOVA), followed by Tukey’s or Sidak’s post hoc test. Statistical significance was set at P < 0.05.Values are presented as mean ± standard error of the mean. Statistical comparison between two groups was performed using unpaired Student’s Supplementary TablesSupplementary FiguresSupplementary Methods


Gram staining is a classic but standard and essential procedure for the prompt selection of appropriate antibiotics in an emergency setting. Even in the era of sophisticated medicine with technically developed machinery, it is not uncommon that a classic procedure such as Gram staining is the most efficient for assisting physicians in making therapeutic decisions in a timely fashion.Vibrio sp., or Aeromonas sp. as a causative pathogen. A characteristic of his residency in an estuarine area is that raw seafood ingestion, as documented in this patient prior to the current admission, predisposes those who have a chronic liver disease to a life-threatening Vibrio vulnificus infection.A 65-year-old Asian man with alcoholic cirrhosis complicated by esophageal varices was brought to the emergency division of Saga Medical School Hospital in early August, complaining of severe pain, redness, swelling, and purpura of the lower extremities. On physical examination he appeared in a critically ill condition suggestive of deep-seated soft tissue infection, raising a pre-test probability of streptococci, staphylococci, V. vulnificus. After two sets of blood culture, intravenous meropenem and minocycline were immediately administered before the patient underwent emergency surgical debridement. The next day, both blood culture and wound culture retrieved Gram-negative rods, which were subsequently identified as V. vulnificus by mass spectrometry, matrix-assisted laser desorption/ionization. The antibiotics were switched to intravenous ceftriaxone and minocycline.Given the pathognomonic clinical features suggestive of necrotizing fasciitis, our immediate attempt was to narrow down the differential list of candidate pathogens by obtaining clinical specimens for microbiological investigation, thus inquiring about the post-test probability of the causative pathogen. The Gram stain of the small amount of discharge from the test incision of the affected lesion detected Gram-negative rods morphologically compatible with V. vulnificus infection was further validated by on-site Gram staining in the emergency division. This case report highlights the significance of a classic procedure.The pre-test probability of  Vibrio vulnificus is a disease with a high mortality rate , heart rate was 126 beats per minute (bpm), blood pressure 82/53 mmHg, temperature 39.5 ℃, respiratory rate 22/min, and SpO5], heartThe patient’s postsurgical course was uneventful being put on oral minocycline at 200 mg/day alone until day 30 when rupture of esophageal varices developed resulting in death 10 days later due to decompensated liver failure. Because his family did not wish to, an autopsy was not performed .V. vulnificus was instantly suspected by Gram staining, and definitive treatment with meropenem plus minocycline, could be started within 15 minutes after the patient’s arrival on the first day of admission. Gram staining is said to be important [V. vulnificus is a Gram-negative bacterium that causes fatal bacteremia, typically complicated by necrotizing soft tissue infections [V. vulnificus is brackish water and it proliferates with constant salinity and water temperature: V. vulnificus grows in estuaries where salinity has optimally decreased due to flooding [Vibrio infections may increase in higher latitudes due to recent increases in the sea water temperatures [We reported a case in which mportant , but thefections . The prefections . The natflooding , and it eratures . The patV. vulnificus necrotizing fasciitis serving as a high pre-test probability of this ominous infection, the on-site Gram-staining showing homogeneous Gram-negative rods significantly increased a post-test probability of this life-threatening infection.Given these clinical and regional epidemiological features, including temporal weather conditions of heavy rain, pointing to V. vulnificus infection, including minocycline and ceftriaxone, or fluoroquinolones [There have been many previous reports that have recommended appropriate antibiotic regimens for inolones . The impinolones .Rather than discussing target antimicrobial therapy following the identification of bacteria, trying to predict the most probable causative organism based on the theoretical assumption making use of fundamental clinical reasoning such as pre- or pro-test probability is not only fast and frugal, but crucially important for timely initiation of quasi-target therapy.Although the clinical outcome of this case was poor, this does not contradict the usefulness of on-site Gram-staining in semi-identifying micro-organisms, which should continually be encouraged among emergency care physicians. This strategy will also contribute to the judicious use of antibiotics in the era of antimicrobial resistance.V. vulnificus is fatal and requires early appropriate antimicrobial therapy. Estimation of the causative organism by Gram staining is of vital importance in the treatment strategy for this life-threatening disease.Necrotizing fasciitis caused by


The nanofibers were calcined at different temperatures. All CoFe2O4 nanofibers show excellent oxygen evolution reaction (OER) performance. The nanofibers calcined at 750 °C have a multi-particle nanochain structure. The nanochain exhibits excellent catalytic performance for OER in 1 M KOH (pH = 14) producing a current density of 10 mA cm−2 at an overpotential of 0.34 V, and the small onset potential of 1.32 V versus RHE, better than that of the commercial Ir/C (20%) catalyst. Furthermore, the stability of CoFe2O4 multi-particle nanochains toward the OER decreases by only 0.78% even after a long period of 80 000 s. Our finding suggests that CoFe2O4 nanofibers with a multi-particle nanochain structure could serve as a new group of OER electrocatalysts with excellent performance.One dimensional spinel CoFe 2O4 nanofibers were synthesized via the electrospinning technique.One dimensional spinel CoFe The mixed solution contain ethanol, N,N-dimethyl formamide (DMF), iron nitrate nonahydrate (Fe(NO3)3·9H2O) and cobalt nitrate hexahydrate (Co(NO3)2·6H2O). For the typical synthesis, 2 mmol Fe(NO3)3·9H2O and 1 mmol Co(NO3)2·6H2O were initially dissolved in a mixed solution of 5.0 mL ethanol and 5.0 mL DMF under rapid stirring at room temperature by a magnetic stirrer to obtain a homogeneous sol–gel. The electrospinning process was carried out by applying a DC voltage of 14 kV, with a 14 cm spacing between the needle tip and the aluminum foil at room temperature. After the electrospinning process, the PVP/[Co(NO3)2 + Fe(NO3)3] precursor nanofibers were deposited on the aluminum foil. The as-spun nanofibers were then dried for 4 h in a drying oven at 60 °C. The nanofibers were then divided into three parts. These three parts were denoted as sample S1, S2, and S3, and calcined in furnace under air atmosphere at 550 °C, 650 °C, 750 °C for 2 h with the heating rate of 1 °C min−1, respectively. After all, the samples were naturally cooled down to room temperature.CoFe2O4 nanofibers were performed using a field emission scanning electron microscope  and a high resolution transmission electron microscope  equipped with energy dispersive X-ray spectroscopy . X-ray diffraction was used to study the crystal structure of the samples . The chemical states of the elements were identified by the X-ray photoelectron spectroscopy .The morphology and chemistry characterization of the CoFe−1. The electrolyte was first cleaned by high purity N2 gas. The potentials were converted with reference to the reversible hydrogen electrode (RHE) through RHE calibration in 1 M KOH (pH = 14), E(RHE) = E(Ag/AgCl) + 197 mV + 0.059 × pH. The Nyquist plots were tested at 1.4 V versus RHE, and the impedance data was fitted to a simplified Randles circuit with frequency ranging from 100 kHz to 0.1 Hz to obtain the charge-transfer resistances.The electrocatalytic activity for OER was measured using an electrochemical analyzer  in a configuration of conventional three-electrode cell, where the modified glassy carbon coated with catalyst was used as the working electrode, a Pt/C and a Ag/AgCl foil served as the reference and counter electrode, respectively. Linear sweep voltammetry (LSV) was performed in 1 M KOH at the room temperature with the scan rate of 2 mV sxOy nanofibers is investigated by varying the calcination temperatures at 550 °C, 650 °C and 750 °C. xOy nanofibers (S1–S3). Clearly, by the SEM images  diffraction peak of the XRD line broadening using the Scherrer formula:λ is the wavelength of X-ray source, β is the line broadening at half of the maximum intensity, and θ is the Bragg angle. The nanofibers are formed with CoFe2O4 nanoparticles, with uniform sizes. The average grain sizes for S1, S2 and S3 calculated by 2O4 is increased with increasing of calcination temperatures from 550 to 750 °C.XRD analysis of the as-prepared CoFe2O4 nanofibers. After standardized by the C 1s peak (284.6 eV), as shown in 3/2) and 795.3 eV (Co 2p1/2) are observed for CoFe2O4, with a spin orbit separation of 15.3 eV. In addition, there are two shake-up satellite peaks at around 771.3 eV and 784.8 eV at the lower binding energy edge of the Co 2p1/2 and Co 2p3/2 peaks. The shake-up satellite peaks and main peaks indicate the valence of Co is 2+ state.33–35 The Fe 2p spectrum  were measured to obtain the valence state and chemical composition in CoFespectrum  show two2O4 nanofibers, the samples were first coated on a clean glass carbon electrode, which also provided the contact area for measurement . The mass loading of the uniform catalyst film was measured to be 0.32 mg cm−2. Linear sweep voltammetry (LSV) was performed in O2-saturated 1 M KOH with a pH of 14 at the room temperature. IR) correction. The IR losses caused by electrolyte resistance, were corrected by subtracting IR, and the value of R is low and consistent. Clearly, CoFe2O4 nanofibers exhibit considerably enhanced activity to OER with the smallest onset potential of 1.32 V versus RHE. The CoFe2O4 nanofibers even show a comparable onset potential with the conventional precious metal benchmark for OER , S2 (512 mV) and even Ir/C (381 mV).11 Our results confirm that the S3 own a higher electrocatalytic activity for OER. Next, the OER electrocatalytic kinetics of the above catalysts were investigated by Tafel plots, as displayed in −1, which is the smallest among the CoFe2O4 nanofibers catalysts. It is noted that, despite with a similar structure, the S3 have a further decrease in the slope value compared with S1 and S2. This suggests that the S3 have the fast charge transfer progress.To evaluate the electrocatalytic performance of the CoFe, 1.52 V ). In add2O4 nanofibers, we also tested the Nyquist electrochemical impedance spectroscopy (EIS) to study the electrode kinetics under OER condition. The result of EIS is used to investigate the charge transfer resistance of the samples during the OER process, as shown in Rct values of 142.5, 140.3, and 44.8 ohm for S1, S2, and S3 suggest that an obvious decrease of charge transfer resistance is related to the improved OER performance. The finding suggests that S3 has the fastest charge transfer process and consequently facile OER kinetics at the electrode/electrolyte interfaces, and results in outstanding OER activity. In addition, the double-layer capacitances (Cd1) were estimated of the CoFe2O4 nanofibers by using a cyclic voltammetry method. Cd1, which is linearly proportional to the effective electrochemically active surface areas (ECSA), can be measured to compare the effective ECSA of the electrocatalysts. As shown in Cd1 values are measured 1.8, 6.6, and 10.7 mF cm−2 for samples S1, S2, and S3, respectively. The highest Cd1 of S3 is about 6 times of S1, indicating that sample S3 has the largest active sites.To further measure the electrocatalytic efficiency in terms of OER for CoFe2O4 nanofibers for OER in alkaline electrolyte (pH = 14), continuous OER was performed at the samples' static overpotential. As shown in versus RHE negligibly decreases by only 0.78% even after 80 000 s. In contrast, the OER current density on S1 and S2 decrease 2.87% and 23.88% after 80 000 s, respectively. Results reveal that S3 possess superior operational stability for future applications.As is well-known, the stability of electrocatalysts is another critical parameter for practical utilization. To assess the durability study of the CoFe2O4 multi-particles nanochains can be applied as an efficient OER electrocatalyst. The improved OER catalytic activity of S3 can be attributed to several reasons: (1) S3 has the best charge transfer rate owing to the lowest Tafel slope and charge transfer resistance, which is confirmed by Tafel slope and EIS measurements. (2) Rough surface and small diameters of S3 are favorable to increase the specific surface area of the sample and lead to more active sites between the electrode and electrolyte. It can be seen from the Cd1 estimation. (3) The chemical synergistic effect of oxygen, such as more oxygen vacancies, also contributes to the enhanced activity, but it can be ignored owing to same XPS's results of three samples.From the above results and discussions, the as-prepared CoFe2O4 nanofibers have been synthesized by electrospinning technology followed via calcination under different temperature. The nanofibers were characterized by SEM, TEM, EDX, XRD, and electrochemical measurement in detail. Our results showed that increasing calcination temperature caused the morphology change of the CoFe2O4 nanofibers. Further, we show that the nanofiber calcined at 750 °C has a multi-particles nanochain structure, and act as an efficient OER catalyst. CoFe2O4 multi-particles nanochains exhibit enhanced OER catalytic activity compared with CoFe2O4 nanorods, with lower onset potential and overpotential. Notably, the stability of S3 shows negligible degradation in OER current density over 80 000 s of continuous operation. This work offers a new pathway for designing high efficient OER electrocatalysts using a simple electrospinning technique.In summary, CoFeThere are no conflicts to declare.


However, molecular generation for structural optimization is often powerless for new targets. DNA-encoded library (DEL) can generate systematic, target-specific activity data, including novel targets with few or unknown activity data. Therefore, this study aims to overcome the limitation of molecular generation in the structural optimization for the new target. Firstly, we generated molecules using the structure-affinity data (2.96 million samples) for 3C-like protease (3CLpro) from our own-built DEL platform to get rid of using public databases . Subsequently, to analyze the effect of transfer learning on the positive rate of the molecule generation model, molecular docking and affinity model based on DEL data were applied to explore the enhanced impact of transfer learning on molecule generation. In addition, the generated molecules are subjected to multiple filtering, including physicochemical properties, drug-like properties, and pharmacophore evaluation, molecular docking to determine the molecules for further study and verified by molecular dynamics simulation.Molecular generation (MG)  Drug structural optimization  is to deThe strategy for molecular generative models through deep learning can be divided into ligand-based and structure-based . Ligand-based molecule generation requires a set of experimentally validated active compounds. ML generates molecules by learning the common features of the active compounds . StructuDeep learning generative algorithms have been explored for aided drug design. Generally, standard inputs in generative models are linear input symbols like Simplified molecular input line entry specification (SMILES) and molecular graphs. Common generative model architectures include recurrent neural networks (RNNs) , autoencH1 and H2  values. We provide the information of DEL in the supplement material. We cut out the data with very low count, the remaining data is 3,702,672. Then it was divided into a training set and a test set (0.8/0.2), of which the training set and test set have 2,962,138 and 740,534 molecules, respectively. First, the training set is used to train the molecule generation model. Then, molecules 18,129) with higher count values in DEL were selected as the positive samples of transfer learning to fine-tune the pre-trained model.SMILES or molecular graphs are commonly used for molecular representation. SMILES is a text of molecular structures . MoleculRecurrent neural network (RNN)  are desiVariational autoencoder (VAE)  consistsAdversarial Autoencoder (AAE)  is similTransfer learning  is a finValidity is the proportion of valid molecules in the generated molecules.Uniqueness is the proportion of molecules not duplicated in the generated molecules.Novelty is the proportion of molecules that do not exist in the training set.Internal diversity (IntDiv)  is a metScaffold similarity (Scaff) represents the similarity between scaffolds in the generated set and reference dataset. The value range is . The higher the value of Scaff, the more similar the two are.Each model generated 10,000 molecules, which were evaluated using the evaluation metrics provided in Moses , includiThe number of molecules generated by each model is 1,000,000 molecular datasets. First, the dataset was filtered for validity and reproducibility, followed by drug-likeness: 250 ≤ MW ≤ 750, logP ≤ 5, HBD ≤ 5, HBA ≤ 10, RB < 10, and 0.5 < QED. The next step was improving drugability by applying Medicinal Chemistry filters (MCFs)  and Pan The remained SMILES after validity and repeatability filtration and the original 3,702,672 SMILES from the DEL dataset were transformed into Morgan fingerprints with 1024 dimensions and 2 radius . These fThe model for affinity prediction was established according to our previous study . First, The 3D molecular similarity was calculated through the shape and color similarity score (SC score), which represents the pharmacophoric feature similarity  and the The A-chain of the complex PDB:7L13 (resolution 2.17 Å) of 3CLpro protein was split as a docking template to obtain accurate docking results. Subsequently, the complex was preprocessed using the Protein Preparation Wizard module of the Maestro suite  with the default setting, including the addition of hydrogen and side chains, removal of water molecules, and calculation of partial charges and protonation states using the OPLS4 force field . Followe+ and Cl− ions to neutralize the charge. Electrostatic interactions are handled separately using the Particle Mesh Ewald (PME) and Verlet algorithms. The heavy atoms of the protein are constrained, and the energy minimization is carried out through 50,000 steps using the steepest descent method. The simulated system was equilibrated for 100 ps using a canonical ensemble (NVT) and an isothermal-isobaric ensemble (NPT). Both van der Waals and Coulomb interactions were calculated using a cutoff of 1.4 nm. Afterward, the system was run at constant temperature (300 K) and constant pressure (1 bar) for 100 ns molecular dynamics simulations with a time step of 2 fs and trajectory data saved every 5 ps. Finally, the ligand and protein complex’s root mean square deviation  at 100 ns was measured. By examining the interaction of the ligand with active site residues and the structural changes of the complex, the complexes were considered stable.Amolecular dynamics simulation was carried out to analyze further the dynamic interaction process between protein and ligand and the stability of binding status. Molecular dynamics simulation is a popular technique to study protein motion by tracking its conformational changes over time . MoleculWe evaluate each model’s validity, uniqueness, novelty, intDiv, and Scaff metrics , 2. The The results visualization using UMAP dimensionality reduction are shown in According to the calculated SC scores of all molecules in the dataset and native ligand, 5,000 ligands with the highest SC score were selected for the follow-up study.Molecular docking was utilized to analyze the 3D conformational and pharmacophore-based screens and to study the structural basis of the interaction between 3CLPro and ligands. First, the reliability of the glide docking algorithm  was confirmed by re-docking the native ligand to the receptor.The re-docked conformation was presented in supporting information. Subsequently, the selected 5000 molecules were preliminarily docked to the revealed binding site using standard precision mode. According to the docking evaluation score and molecular conformation, 500 different conformations were selected, and four times enhanced conformational sampling was used to generate the ligand-binding pose more accurately.In addition, the docking scores with and without transfer learning for pharmacophoric models were analyzed . ApplyinMoreover, the molecules filtered by the affinity model also performed molecular docking as a pharmacophoric model. This aims to explore the possibility of replacing an external pharmacophore model with an own-built affinity model. The docking scores of the filtered molecules were combined in N1-N8 were finally selected for follow-up research considering the above ADMET properties with results of binding energy calculation  shows that hydrophobic interaction is the main contributor to the ligand binding process. According to the molecular docking conformation, we selected N1 and N2 for subsequent research, in which the position and interaction of N1 and N2 on receptors are consistent with the previous report (From s report  (Zhang es report .N2 is believed to be more compatible with the receptor pocket (ligand conformation), and the calculation of MMGBSA binding free energy indicates that N2 has a stronger interaction with the receptor, molecular dynamics simulations of the complex formed by N2 were conducted.To further analyze the interaction-related atomic details between molecules and 3CLPro, GROMACS was applied for molecular dynamics simulations using the above docking results. 100 ns run time for MD simulation is considered sufficient for side chain rearrangement. The result will confirm whether or not the complex remains in the most stable association. Since N2 and the ligand N2 showed steady kinetics after 30 and 50 ns, respectively (N2 and receptor, and finally, the system reaches a steady state. The RMSF value showed minimal fluctuation, and it remained in the range of 0.05–0.2 nm throughout the simulation period for most residues, except that a peak in RMSF value was observed only at residue 1. The less fluctuating performance confirms the strong attachment of the ligand to the protein (RMSD values plotted over the simulation time revealed a stable kinetic equilibrium of the complex. In detail, the 3CLPro protein with ectively . By moni protein .As far as we know, this is the first study that DEL’s dataset has been used for the molecular generation, which will promote the development of the application field of DEL combined with AI. This study preliminarily found that it may be feasible to use DEL data instead of public databases for molecular generation. In particular, for the new target pipeline, molecular generation and affinity model establishment based on DEL data are expected to become a tool with dual functions of drug discovery and further structural optimization. This advantage would be difficult to achieve with public databases due to the scarcity of datasets.


On univariate analysis, increasing patient age negatively correlated with a larger number of granulomas (p = .011). Granuloma presence was associated with finding intact and motile sperm from the vasal stump intraoperatively (p = .001), and vasoepididymostomy anastomosis (p < .001). However, granuloma presence (and quantity) did not correlate with obstructive interval or maximum TMC. Tobacco use and body mass index (BMI) were not associated with granuloma presence. On multivariate analysis, granuloma quantity was not associated with TMC. Obstructive interval and vasovasostomy anastomosis were associated with higher TMC, while BMI was negatively associated with TMC. In conclusion, increasing age was negatively correlated with granuloma formation. Granuloma presence was associated with more favourable intraoperative fluid findings and anastomosis type, but not post‐VR TMC, suggesting men with and without granulomas undergoing skilled microsurgery will have similar patency rates. Heavier men should be encouraged for weight loss prior to vasectomy reversal as increasing BMI was associated with lower TMC.The objective of this study was to identify factors that predict for sperm granuloma formation and the impact of sperm granuloma presence and quantity on vasectomy reversal (VR) outcomes. A cross sectional retrospective review of prospectively collected data, on the impact of granuloma on VR outcomes from a single academic center was performed. The impact of age, obstructive interval, intraoperative vasal fluid findings, anastomosis type, body mass index, tobacco use and total motile count (TMC) was determined. A total of 1550 men underwent VR between January 2000 and August 2019. Granulomas were present unilaterally in 23.3% ( No difference in pregnancy rate with or without granulomas were seen  and tobacco use. We also seek to understand the impact of sperm granuloma presence and quantity on factors known to impact post‐VR total motile sperm count (TMC), including intraoperative vasal fluid composition, obstructive interval, male age and anastomosis type.2Institutional review board approval was obtained. A retrospective review of VRs performed between January 2000 and August 2019 by one of two high‐volume microsurgeons (SFM or PJB), at a single institution. All VR were being performed for fertility restoration. Men were advised to undergo post‐VR semen analysis (SA) within four months of VR. Men were excluded if they did not have postoperative SA testing or were using exogenous testosterone.All vasovasostomies (VVs) were performed using a mini‐incision two‐layer microsurgical technique, with six equally spaced 10.0 nylon sutures on the mucosa, and six equally spaced 9.0 nylon sutures on the muscularis. All vasoepididymostomies (VEs) were performed using the end‐to‐side intussusception technique, using four equally spaced 10.0 nylon sutures on the mucosa and four 9.0 nylon sutures on the muscularis. VV was performed if the vasal stump fluid was clear, thin, copious and if sperm or sperm parts were seen at intraoperative bench microscopic evaluation. VE was performed if the fluid obtained was pasty, thick and no sperm or sperm parts were seen.The impact of one or more sperm granulomas on post‐VR outcomes were analysed, including male age, anastomosis type , post‐VR TMC and the vasal obstructive interval. The impact of patient factors on granuloma presence and quantity was also analysed, including age, patient‐reported tobacco usage , and BMI.t‐test and one‐way ANOVA, or their non‐parametric equivalent, were used to test for differences in continuous variables and Pearson's chi‐squared or Fisher's Exact test were used to test for associations in categorical variables. Multivariable logistic and ordinal logistic regression were used to evaluate the associations between independent variables of interest with granuloma presence, while controlling for patient age. Multivariable negative binomial regression was performed to evaluate the association between independent variables of interest and maximum TMC, while controlling for age. All statistical analyses were performed using R version 4.0.2. All tests were two‐sided and a p‐value <.05 was considered statistically significant.Descriptive statistics were presented using median (IQR) or mean (SD), dependent upon distribution, for continuous variables, and frequency (percent) for categorical. Two‐sample 36 sperm.During the study period, 4817 men underwent VR, and after excluding those that did not meet criteria, 1550 were included in analysis. The mean ± SD male age was 39.9 ± 6.8 years. The mean ± SD male BMI was 27.9 ± 4.8. The mean obstructive interval was 8.6 ± 5.6 years. The majority, 1466 (94.6%) of men underwent VV/VV, 71 (4.6%) underwent VV/VE and 13 (0.8%) underwent VE/VE. The mean post‐VR TMC was 58.2 ± 79.0 × 10p‐.011). Patients with two granulomas were younger than patients with zero or one granuloma. Granulomas were present on one side in 23.3% (n = 361) and both sides in 14.2% (n = 220). Granuloma presence was associated with intraoperative vasal fluid findings, including the presence of both complete motile sperm (p = .001) and complete non‐motile sperm (p = .001). Likewise, the presence of granuloma was associated with the type of anastomosis performed (VV or VE), with granuloma presence being more likely to undergo VV anastomosis. However, the presence of granuloma did not correlate with post‐VR maximum TMC or obstructive interval.Univariate analysis of the impact of granuloma presence and quantity is summarized in Table p = .005). Obstructive interval was significantly negatively associated with maximum TMC. For every year increase in obstructive interval there was a 4% decrease in TMC . Anastomosis type was also significantly associated with TMC. Patients that had VE had, on average, a 71% lower TMC when compared to patients that had VV anastomosis .A multivariate negative binomial regression, with TMC as the primary outcome was performed. After adjusting for age and smoking status, number of granulomas was not associated with TMC Table . BMI was4The impact of granuloma presence on post VR patency and pregnancy rates remains unclear. Prior series have been limited by patient number, as well as data on anastomosis type. In addition, all prior series report the presence of granuloma, but do not record laterality, or correlate this with the laterality of intraoperative vasal fluid quality, or anastomosis type. Our series represent the largest series of men undergoing VR for restoration of fertility, nearly 5000 men, of which nearly one‐fourth had granulomas.Prior data has shown that granuloma presence has been associated with better quality intraoperative vasal fluid .