Textile-supported nanocomposite as a scaffold has been extensively used in the medical field, mainly to give support to weak or harmed tissues. However, there are some challenges in fabricating the nanofiber/textile composite, i.e., suitable porous structure with defined pore size, less skin contact area, biocompatibility, and availability of degradable materials. Herein, polyamide-6 (PA) nanofibers were synthesized using needleless electrospinning with the toothed wheel as a spinneret. The electrospinning process was optimized using different process and solution parameters. In the next phase, optimized PA nanofiber membranes of optimum fiber diameter with uniform distribution and thickness were used in making nanofiber membrane–textile composite. Different textile fabrics  were developed. The optimized nanofiber membranes were combined with non-woven, woven, and knitted fabrics to make fabric-supported nanocomposite. The nanofiber/fabric composites were compared with available market woven and knitted meshes for mechanical properties, morphology, structure, and chemical interaction analysis. It was found that the tear strength of the nanofiber/woven composite was three times higher than market woven mesh, and the nanofiber/knitted composite was 2.5 times higher than market knitted mesh. The developed composite structures with woven and knitted fabric exhibited improved bursting strength (613.1 and 751.1 Kpa), tensile strength (195.76 and 227.85 N), and puncture resistance (68.76 and 57.47 N), respectively, than market available meshes. All these properties showed that PA nanofibers/textile structures could be utilized as a composite with multifunctional properties. Textile medical products are highly significant for the healthcare and hygiene sectors. Healthcare-associated infections (HAIs) are common adverse health events affecting hospital patients ,2. ThereSurgical meshes have been used to resolve hernia problems since 1891. The research in this field has expanded due to multiple difficulties after surgeries, such as infections, fibrosis, adhesions, mesh rejection, and hernia recurrence. Scientists, scholars, and researchers developed various methods for a wide range of mesh materials, their analysis, and implementation with different porosity and fiber size. Several implantation and surgical methods have been established , such asElectrospun-based nanofibers  offer a Some researchers worked on electrospun-based meshes; for example, Ebersole et al. developed an electrospun absorbable poly-caprolactone (PCL) scaffold in which two tale arrangements possessed elastic qualities and suture maintenance which were suitable for hernia fix applications . K. MolnThe authors fabricated the PVA nanofibers using multiple rings as the spinnerets and evaluated the effect of spinnerets on productivity and nanofiber morphology. It was stated the electric field mainly concentrated at the top side of all rings and directly affected productivity ,33. Fibe3) was purchased from Merck (made in Germany). Acetic acid was also purchased from Merck ; formic acid was purchased from Sigma Aldrich .Polyamide-6 , applied voltage  and spinning distance  because these factors have a prominent effect on results. A complete design of the experiment is given in w/w) and formic acid (50% w/w) solvents (w/w) solvents and continuously stirring at 1200 rpm for 24 h at 50 °C. The fabrication of PA nanofibers was carried out using a toothed wheel needleless electrospinning technique. The needleless electrospinning contains a toothed wheel, a grounded collector (rotating cylinder), a solution container that is made of Teflon, and a power supply (Gamma-high voltage DC power supply). The dimensions of the toothed wheel spinneret are as given thickness = 5 mm, exterior diameter = 60 mm, number of teeth = 14, and pitch = 13.45 mm. The other process parameters, such as toothed wheel rotation and collecting wheel rotation, were kept constant after the optimization of the process. The toothed wheel spinneret rotation was kept constant at 10 rpm, and the collecting cylinder speed was set at 50 rpm to collect uniform and even the nanofiber membrane. The applied voltage is connected to the toothed wheel spinneret, whereas the ground is connected to the rotating collector. The environmental conditions were also optimized and adjusted after pre-trials. The temperature and relative humidity were varied at 16 to 25 °C and 35 to 65% at the initial pre-trials during the electrospinning of PA6 nanofiber. After optimizing the environmental conditions, the temperature was set to 16 °C and 35% relative humidity and kept constant through the electrospinning of the PA6 nanofiber membrane. The membranes of different thicknesses were prepared initially, and the most suitable membranes with good tensile and bursting properties were selected for the development of composite structures.The development of electrospun nanofiber/textile composite was divided into 3 phases. The first phase objective was to obtain bead-free and smooth nanofibers from electrospinning. For this purpose, initially, pre-trials were carried out, varying the solution and process parameters such as solution concentration, applied voltage, and spinning distance to find out the optimized parameters. After pre-trials, these three factors were selected, i.e., polymer concentration  was performed to develop PP yarn. Then, weaving was performed to prepare PP fabric. Next, knitting of the same PP yarn was performed to develop knitted fabric. Non-woven fabric was developed using PP staple fibers through needle punched non-woven fabric system.After developing fabrics in phase III, the optimized nanofibers membranes with good tensile and bursting strength were attached to different textile structures through the calendaring process to fabric nanofiber nonwoven composite (NWC), woven composite (WC), and knitted composite (KC), respectively. The fabric substrate was sprayed with PA6 binder to obtain adhesion of the nanofiber membrane to the textile substrate. The calendaring speed was adjusted at 1.40 m/min, calendaring pressure at 12 bar, and the lower cylinder temperature was set at 120 °C. The combined layer of textile substrate and nanofiber membrane was passed between the calendars. The fabric substrate was toward a lower calendar (downward direction) during this process. The obtained composite of nanofiber membrane and fabric substrate has good adhesion properties and cannot peel off easily.For comparison purposes, two different hernia meshes were bought from the market. One was woven hernia mesh (MWM), and the second was knitted hernia mesh (MKM). Scanning electron microscopy (Quanta 250 FEI) was used to observe morphologies of electrospun PA Nanofibers. The diameter of electrospun PA nanofibers was calculated from SEM images with the help of an image analyzer, “Image J”, and a minimum of 50 fibers were tested from each image.Brunauer–Emmett–Teller (BET) analysis was performed to measure the surface area and porosity of the PA6 nanofiber membrane through Brunauer–Emmett–Teller (BET) analysis .The tensile strength of PA nanofibers was obtained with a (Tensiometric 2.5) single fiber strength tester with speed (10 mm/min) and gauge length (10 mm). Samples were tested at a temperature of 25 °C with 55% of relative humidity. Each sample was cut to dimensions 5 mm in width and 10 mm in length. Ten readings of each PA sample were tested.The universal tensile strength tester was used to evaluate the strength of textile materials. The testing standard for fabric tensile strength using the strip method is EN ISO 13934-1:1999. The fabric strip was cut according to the dimensions 10″ × 3″ in both directions of testing fabric minimum of two sample warp directions and another in the weft direction. The tear strength tester was used to measure the tear strength. The working principle of this machine is a ballistic pendulum (Elmendorf) and a cutter that places a cut of 2 mm on the specimen. The testing standard for tear strength is EN ISO 13937-1:2000.A bursting strength tester is an equipment that is used for measuring fabric bursting strength by applying pneumatic pressure on the sample until it bursts. Then, the reading is noted in Kpa. The testing standard for bursting strength is ISO 13938-2:1999. The testing standard for stiffness testing is ASTM-D4032.Fiber morphology is an important feature of the nanofiber, influencing the final nanofiber membranes’ performance. The solution concentration applied voltage and distance from nozzle tip to collector are primary factors for the structure and morphology of nanofibers fabricated from the multiple tooth wheel spinnerets. Initially, the electrospinning process was optimized with pre-trials under different process environmental conditioning. The temperature varied from 15 to 25 °C, and the relative humidity was 35 to 65%. At the high temperature (25 °C), the electrospinning process is not smooth due to the evaporation of the volatile solvent (acetic acid and formic acid) and fiber solidified before reaching the collector. Moreover, the solution concentration was also changed due to the evaporation of the solvent in the container during electrospinning. On the other side, the electrospinning of PA6 nanofiber was uniform and smooth at 16 °C temperature. Similarly, in the case of high relative humidity (65%), high moisture content between the spinneret and collector interacted with the polymer jets during its flight. This interaction caused the phase separation and solidified the PA6 nanofiber before reaching the collector, resulting in a coarser and uneven nanofiber membrane. Based on these trials, the chamber temperature was set at 16 °C, and relative humidity was kept at 35%. Solution concentration was also varied from 10 to 25%. It was observed that at low concentrations of 10 to 16%, beaded nanofibers were formed, while solution concentrations above 22% developed coarser ribbon-like nanofiber. Therefore, a solution concentration between 18 and 22% was selected for the further optimization of the PA6 nanofiber morphology. In the applied voltage, a minimum voltage of 30 kV is required to initiate the electrospinning process by using toothed wheel electrospinning. Uniform electrospinning of the PA6 nanofiber was observed for the voltage range from 35 to 45 kV. The spinning distance was also set between 16 and 24 cm after the pre-trials by analyzing the smooth electrospinning and evaporation of the solution.w/w% polymer concentration. When the polymer concentration was increased to 20 w/w%, then the diameter of nanofibers was also increased. The further increase in concentration to 22 w/w% also enhanced the diameter of the nanofibers. The formation of beads at low concentrations was due to the polymer solution’s low viscosity; hence, surface tension dominated and led to the formation of beads instead of nanofibers. While at a higher concentration level, the more viscous polymer solution formed the continuous uniform nanofibers, as shown in w/w% in comparison to the distribution of the diameter of the nanofibers. The average diameter of nanofibers found is 231 ± 65 nm, 343 ± 58 nm, and 418 ± 47 nm at a concentration level of 18, 20, and 22%, respectively. w/w%. The SEM images of the PA nanofibers showed that increasing the collecting distance from 16 cm to 24 cm has an inverse effect on the diameter of nanofibers keeping the concentration and voltage at a constant level. Fibers with uniform distribution were obtained on shifting from 16 cm to 24 cm. It can also be observed that the impact of collecting distance is not very significant during the electrospinning process while keeping the distance at the spinning range.w/w% concentration, and 24 cm collecting distance. Furthermore, these optimized nanomembranes were prepared at three different thickness levels, i.e., 80, 120, and 160 µm.The relation of nanofibers’ diameter with polymer concentration, voltage, and spinning distance is summarized in 3/g, and surface area of 36.043 m2/g. The pore size, pore volume, and surface area of the PA6 nanofiber membrane are very comparable, which can be very effective for biomedical applications.The tensile strength of nanomembranes of different thicknesses is given in As hernia mesh needs higher tensile and bursting strength, the nanofiber membranes with a thickness of 160 µm were chosen to fabricate nanomembrane/textile composites.The calendaring process combined PA nanomembranes with non-woven, woven, and knitted fabrics. The comparison between the developed nanofiber/textile composite and the market-available mesh (knitted and woven) is shown in Bursting is one of the most important mechanical properties that is used to measure how much a material can bear the force until it bursts. The graph shown in The stiffness of composites developed against reference meshes is compared and shown in In this research study, polyamide-6 nanofiber-based nanomembranes were synthesized using a needleless electrospinning process with the toothed wheel as a spinneret. Then different fabrics  were developed. Then the optimized nanofiber membrane was combined with each type of fabric to make a fabric-supported nanofiber composite. Based on characterizations, the morphology of the nanofiber membrane was affected by variations in applied voltage, polymer concentration, and collecting distance. Mechanical properties such as tensile, bursting, and tear properties of the PA nanofiber membrane were increased by increasing the thickness of nanomembranes. The tear strength and puncture resistance properties of the woven structure composite were much better as compared to other developed and reference meshes due to their stable structure. The knitted composite performed better in bursting strength, tensile, and stiffness properties than all other samples. It was concluded that hernia mesh should have high tensile strength, suture maintenance, excellent porosity to aid fibroblast colonization, minimum shrinkage tendency, stays flat without wrinkling, and have the correct design. When considering the obtained results, knitted composite has almost all these properties and was found to be potentially suitable for medical applications.


Antimicrobial resistance is a public health emergency and represents an impending pandemic. Implementing the lessons learned from responding to the COVID-19 pandemic is essential in accelerating the development of new antimicrobials and therapeutic strategies, rapid diagnostics and improved vaccines. A rededicated, coordinated and collaborative global effort of all stakeholders in academia, healthcare, government agencies, industry, finance and philanthropy is essential to halt the continued spread of antimicrobial resistance and to prevent further morbidity and mortality from drug-resistant pathogens. This staggering statistic included 1.27 million deaths directly attributed to bacterial AMR. These estimates suggest that bacterial AMR caused more deaths in that year than those resulting from HIV/AIDS and malaria combined. The major burden of AMR deaths is reported in low-resource areas—those with limited healthcare systems, laboratory infrastructure, sanitation, clean water and antibiotic stewardship.2 In the US, the CDC reports that there are more than 2.8 million antibiotic-resistant infections and greater than 35 000 deaths associated with AMR each year.3 A coordinated, global effort is needed to halt the continued spread of AMR and the resulting morbidity and mortality from drug-resistant pathogens. This Call to Action requires increased support for biomedical research, as well as significant expansion of and incentives for new and improved diagnostics, antimicrobials and vaccine candidates in the medical diagnostic and pharmaceutical pipelines, respectively.Antimicrobial resistance (AMR) represents an ongoing public health emergency and an impending pandemic of overwhelming proportions. The WHO has declared AMR among the 10 worldwide public health threats facing humanity.4 The COVID-19 pandemic has underscored the critical role of biomedical research and development in responding to public health emergencies with accurate, easy-to-use and rapid medical diagnostics, safe and effective vaccines and therapeutics, as well as public–private partnerships resulting in the essential infrastructure for community engagement in clinical trials and uptake of medical countermeasures.5 We need to build on these hard-learned lessons from responding to the COVID-19 pandemic and rapidly apply them to improving the response to the AMR global health crisis. Now is the time to halt the continued spread of AMR by advancing novel and innovative strategies for: improved surveillance of drug-resistant pathogens; discovery, preclinical and clinical evaluation of safe and effective antimicrobials and other therapeutics; development and validation of diagnostics capable of rapidly identifying drug-resistant pathogens and differentiating between bacterial and viral infections; identifying and testing preventive interventions including safe and effective vaccines; investigating and developing strategies utilizing bacteriophage therapy; combining genomic sequencing with artificial intelligence/machine learning tools, as well as educating healthcare providers and the public on the urgent need to increase antibiotic stewardship.During the past 2.5 years, the ongoing COVID-19 pandemic has served to further accelerate the AMR public health crisis with the increased use of antibiotics in the treatment of patients with SARS-CoV-2, the virus that causes COVID-19, as well as resulted in major disruptions in healthcare systems and infectious disease monitoring and treatment.6 As of late 2021, there were 77 antibiotic or combination agents and 217 antibacterial candidates in the clinical and preclinical development pipelines, respectively, targeting the 13 WHO priority pathogens, including Mycobacterium tuberculosis and Clostridioides difficile.6 The limited number of potential antimicrobials in these pipelines are inadequate to counter the rapid spread of AMR, as well as the continual occurrence of drug resistance and broad-spectrum AMR demonstrated by many pathogens.2The development of antimicrobials is an essential step in addressing the AMR crisis; however, from 2017 to 2022 only 12 new antibiotics have been approved by the US FDA and/or the EMA. The majority of these newly approved antibiotics demonstrate limited clinical benefit over existing treatments and approximately 80% of these drugs are from existing classes of antibiotics where mechanisms of resistance have previously been demonstrated.7 The NIH and the Biomedical Advanced Research and Development Authority (BARDA) AMR Diagnostic Challenge awarded 20 million USD to support innovative, accurate, rapid, point-of-need medical diagnostic tests to detect drug-resistant pathogens or differentiate between bacterial and viral infections.8 Additionally, public agencies  and private foundations and organizations worldwide are supporting key biomedical research that provides the basis for identifying new targets for antimicrobial agents, repurposing existing drugs, vaccines and other countermeasures to combat AMR. The UK and Sweden initially launched pilot subscription-based models to ensure pharmaceutical manufacturers of guaranteed contracts irrespective of the actual number of antibiotics sold. Based on the pilot program in the UK, the NHS has recently implemented the ‘Netflix’ subscription-style payment programme for two antibiotics, including cefiderocol and ceftazidime with avibactam.9 In the US, two congressional bills have been recently drafted, including the Developing an Innovative Strategy for Antimicrobial Resistant Microorganisms (DISARM) Act and the Pioneering Antimicrobial Subscriptions to End Upsurging Resistance (PASTEUR) Act, to stimulate the Federal Government’s and industry’s development and use of novel antimicrobial agents.10 At this time, only the PASTEUR Act is continuing to progress toward authorization.11During the past several years, numerous targeted initiatives have been launched to increase the number of antimicrobials and other strategies to counter AMR across different stages of the pipeline from basic biomedical research through to clinical trials and regulatory approval. These ongoing activities have included the Combating Antibiotic Resistant Bacteria Biopharmaceutical Accelerator , the Antimicrobial Resistance Multi-Partner Trust Fund , the Global Antibiotic Research & Development Partnership , the Replenishing and Enabling the Pipeline for Anti-Infective Resistance Impact Fund (estimated at 125 million USD) and the AMR Action Fund . Most recently, the AMR Action Fund has awarded funds to two companies with novel approaches utilizing bacteriophages or β-lactam/β-lactamase inhibitors.All of these programmes are highly commendable; however, more must be done now to fill the discovery and development pipelines and ultimately, pharmacists’ shelves, with the crucial antimicrobials, including oral and paediatric formulations, that are urgently needed. Our collective fight against AMR requires a reinvigorated, rededicated, coordinated and collaborative dedicated effort to accelerate the development of innovative and improved strategies to prevent, treat and diagnose AMR. Additional public and private funds are essential to support: continued biomedical research to identify new lead compounds and drug targets; strategies to de-risk pharmaceutical and biotech companies to fill the diagnostics, drug and vaccine pipelines; legislative and governmental incentives to encourage development of new and improved antimicrobials; and education of healthcare providers and the public on the need for antibiotic stewardship. This Call to Action requires that all of us—members of the pharmaceutical, biotechnology and medical diagnostic industries, along with healthcare providers, researchers in academia and government agencies, regulators, lawmakers, private investors and philanthropy communities join together to end the AMR crisis before it becomes an even greater public health emergency than the COVID-19 pandemic. Only through strategic and collective activities, at both the individual and global population level, can we hope to halt the further spread of AMR. These efforts targeting AMR must be a crucial component of our comprehensive plans and policies for pandemic preparedness and response. FasterCures and the Milken Institute stand ready to work with our public and private partners to stop AMR and prevent the impending AMR public health pandemic.


Materials and 24 in Applied Sciences) on emerging methods and technologies, the Special Issue of the Manufacturing Engineering Society 2021 (SIMES-2021) [Materials and Applied Sciences. After the complete success of the first , second ES-2021) ,7 was laOnce again, this Special Issue was promoted by the Manufacturing Engineering Society (MES) of Spain , with thIn this fourth edition, the joint Special Issue has gathered a total of 10 contributions (9 papers and 1 review) in the topics presented in Materials journal, six papers about cutting-edge advances in different fields of manufacturing engineering have been published after a rigorous review process. In particular, contributions have been published in “Advances and innovations in manufacturing processes”, “Robotics, mechatronics, and manufacturing automation”, and “Micro- and nanomanufacturing”. Regarding the specific contributions to the Special Issue in the Among them, the topic “Advances and innovations in manufacturing processes” received the highest number of contributions (representing 66.67%), followed by the topics “Robotics, mechatronics, and manufacturing automation” and “Micro- and nanomanufacturing” (each at 16.67%). Concretely, within the topic “Advances and innovations in manufacturing processes”, in the field of welding, García González et al.  presenteAnother interesting work in this topic, in the field of simulation and modeling of manufacturing processes, is presented by Fernández et al. . The objRelated to the topic “Robotics, mechatronics, and manufacturing automation”, the work by Calvo and Gil  introducMaterials in general, and among scientists and professionals from the industry in particular.Finally, the topic “Micro- and nanomanufacturing” received a contribution by Zamora et al.  within t


Background: Antimicrobial resistance (AMR) is a multidimensional phenomenon. The environment acts as a mixing pot of drug-resistant bacteria from many sources such as pharmaceutical, biomedical, veterinary, and agricultural sectors. In this study, we analysed the existing AMR-related policies/guidelines/legislations in India in the above domains and how the current practices are being guided by them. Methods: We used a convergent parallel mix method design. Quantitative data were collected through a review of policies/guidelines/legislations in the said domains and analysed using the SWOT tool parallelly supported by key informant interviews of domain-specific stakeholders. Results: Altogether, 19 existing AMR policies/guidelines/legislations were identified. The existence of few policies/guidelines in each domain indicated the evolving environment for policy interventions. However, the lack of capacity among farmers, inadequate provision for structured capacity building, high cost of alternatives to antimicrobials, and lack of provision of incentivisation in case of crop failure were identified as the major weaknesses prevalent across the domains. Opportunities for policy refinements/the introduction of new policies are ample. However, easy access to antimicrobials and injudicious use imposes threats to AMR containment in all sectors. Conclusions: Despite having a few policies for the containment of AMR, their implementation witnesses challenge due to the lack of collaborative approaches, the existence of policies disjointed from ground reality, infrastructural issues, and the lack of capacity and resources.  Since the introduction of antibiotics in 1930, they have gained immense importance in global health and food production . The indAMR is a multifaceted and complex global phenomenon, and the environment has been considered a mixing pot of drug-resistance bacteria from all sources . The phaIn recent times, AMR has been recognised as a “One Health” problem due to its interconnectedness between human and animal health and the environment. The burden of antimicrobial-resistant bacteria also varies with geographical location, further catalysed by different environmental variables  and seasAs a global initiative to contain AMR, the WHO, along with the FAO and the OIE, developed a comprehensive Global Action Plan (GAP) on AMR in 2015 . AdditioWe used a convergent parallel mix method design in the present study, where the quantitative data were collected through a review of policy-related documents/guidelines/legislations in various priority domains mentioned earlier, all of which are related to the environmental dimensions of AMR. The qualitative data were concurrently collected using key informant interviews of relevant stakeholders identified from similar priority domains and available during the period of study.The study was conducted during the period of April 2021–March 2022. We considered all the legislations, policies, and guidelines related to the use of antibiotics/antimicrobials; the allowable limits of antibiotics/antimicrobials in various items; the discharge limits of antibiotics from factories; waste management; and the monitoring of antimicrobial use in plant agriculture/animal husbandry/human health sector. Key informant interviews (KIIs) were conducted with stakeholders of the aforesaid priority domains at different levels, i.e., farmers, practitioners, executive or implementing officers/scientists, and policymakers. We initially searched the official websites of various line departments of the Government of India for the policies/guidelines/legislations directly or indirectly related to the containment of antimicrobial resistance in India. The electronic databases of the published literature such as PubMed/Medline, Google Scholar, Scopus, Embase, and Index Medicus were also searched for this purpose for the last ten years (2010–2020) . SimultaThe KIIs were conducted to understand how the current AMR policy landscape in India is guiding the practices and what could be the possible way forward. The key informants were selected based on their expertise and willingness to share information. A total of 17 KIIs were conducted in this study. The process of KIIs was initiated with the development of an interview guide for each domain encompassing key questions in the following segments: the existing policies/guidelines/legislation in India related to AMR containment in various domains; the status of implementation of those policies/guidelines/legislations; the challenges in implementing them; the gaps in the existing intervention strategies; and the way forward to improve the environment to contain AMR in India. The interviews were audio-recorded. Data were transcribed into text data, then arranged systematically, and coded based on the predetermined scopes  identified through the literature review. The defined codes were then gathered together by eliminating redundancy to develop the key themes. Until this point, two investigators ran this process independently. The SWOT  analysis is one of the tools that can be used for strategic management to appraise policies. We performed a SWOT analysis in this study to strategically identify the scopes of intervention in the mentioned domains for containing AMR in the environment in India, as this analysis considers both the internal and external factors and generates information for prioritising and facilitating decision making. In this study, we found 19 existing AMR policies/guidelines/legislations in India. Two were from the plant/agricultural sector, eight from animal husbandry, five from the human health sector, and four from the pharmaceutical sector. Two existing policies related to AMR were found in this domain. One was an act related to the use of insecticide along with antibiotics, which came into force in 1968 , regardiWe conducted a SWOT analysis of the policies available in this sector. It is evident from the presence of current policies that the system has started to evolve. However, implementation challenges persist. The ignorance among farmers regarding the optimum use of antimicrobials and the desire to make a profit are major weaknesses. During the KIIs, one of the key informants  mentioned, “the farmer believes that application of antibiotic in large amount keep the crops healthy and increase their yield, though the disease occurred in crops can be manageable also without any economic loss”. Another key informant  mentioned, “the farmers cannot identify the acute stage of infection when the antibiotic formulation is needed to apply on crops”. However, these weaknesses can also be used as opportunities for new policy introduction focusing on the capacity building of farmers. However, the absence of surveillance on sales of such preparation at the market may currently act as a threat to optimising the use of antimicrobials in the plant/agricultural sector .We reviewed 14 policies/ guidelines/ legislations related to AMR in the veterinary sector . Out of Upon analysis, it is understood that the concerned ministries are aware of the problem and already have started acting on it. However, the lack of provisions for quality checks of domestic products even seeds, the absence of structured capacity-building programmes for farmers, the absence of surveillance mechanisms for regulation of the administration of antibiotics and mechanisms for the regular checking of antibiotic residues in domestic food animals and monitoring of critically important antibiotics for humans which are used in animals as growth promoters are identified to be major weaknesses. Moreover, the lack of coordination among different authorised bodies, such as the Bureau of Indian Standards (BIS) and the Food Standards and Safety Authority of India (FSSAI) regarding the list of antibiotics to be used within a limit, may mislead the stakeholders on the administration of antibiotics on farm animals. One of the key informants  from this sector mentioned, “The farmers don’t get good quality seeds from agents and even farmers are completely unaware about the SPF (Specific Pathogen Free) testing reports of the seeds. Farmers do not have enough knowledge on farm management and treatment of animals, so they use antibiotics as preventive measures and apply those as they want”. However, the presence of strict surveillance on the use of antimicrobials for exportable food animals is the opportunity to introduce similar mechanisms for quality checks in domestic food animals. Presently, the absence of any provision for controlling the high cost of available growth promoters of animals acts as a threat, as it leaves no choice for the farmers but to use antibiotics as growth promoters . AnotherThe Ministry of Health and Family Welfare, Government of India, along with its line departments such as the Indian Council of Medical Research (ICMR) and the National Centre for Disease Control (NCDC), have adopted some approaches regarding the rational use of antimicrobials in humans to diminish the emergence of multidrug-resistant bacteria and their propensity of transmission in humans .The presence of a national action plan for AMR (2017–2021), few guidelines for infection control, and judicious antimicrobial use sets the field to develop policies to streamline the problem of AMR in the human health sector which will eventually reflect in other sectors too. However, the lack of policies mandating the rational use of antibiotics, weak regulatory control on the over-the-counter (OTC) sale of antibiotics, and the absence of structured utilisation of available platforms for generating public awareness on the consumption behaviour of antimicrobials are major weaknesses. The pressing demand for using information technology-based monitoring and surveillance on the administration of antibiotics in humans and AMR incidents in hospitals can be used as opportunities to create national-level data on it. Moreover, addressing the determinants of AMR in a national programme mode may create an environment in the country where all the states give due importance to the problem of AMR and bring out meaningful public health action. However, its absence in the country is currently acting as a major threat. We reviewed four policies/guidelines/legislations related to the pharmaceutical and biomedical waste management sector . HoweverUpon analysis, it is evident that newer policies can be introduced in both of these sectors, as few policies already exist, and there is a felt need for more. However, it is observed that the policies are mostly related to the disposal and management of hazardous substances and other solid and liquid wastes. This is a weak area where more clarity is required on the process of the disposal and management of antimicrobial drugs and related waste generated from hospitals, communities, and manufacturing units. In this regard, a hospital management personnel said, “All the discarded drugs are kept in the same place and sent to Central store and then outsourced further by a third party agency”. The Ministry of Environment, Forest, and Climate Change (MoEFCC) has very recently published the permissible level of antibiotic residues in effluent , and thiThe findings of the present study depicted the current status of AMR-related policies, guidelines, and regulations in India across different sectors and the major strengths, weaknesses, and threats present in the context of the available ones. However, the study also identified domain-specific opportunities for new policy frameworks or refinement of the existing ones. Trichoderma sp. as biopesticide and plant growth promoter agents [Pseudomonas fluorescens [A mechanism for the continuous capacity building of farmers remains a major requirement in all sectors. For example, it has been revealed by the Centre for Science and Environment (CSE) that the farmers in Delhi and Punjab rampantly use streptocycline as a fungicide for crop production . In a fer agents  and Pseuorescens  as biocoorescens  may presResearch and development are required in all sectors regarding the social implication of antibiotic use and to design interventions for behavioural change among stakeholders . HoweverPolicies regarding investment in the research and development of cost-effective newer methods of cultivation are required to provide some alternatives to the current practice of antimicrobials as therapeutics or growth promoters, and this need has been felt across sectors. In the veterinary sector, through the reinforcement of the existing policies, domestically consumed products must also be regulated for antibiotic residues, as it is carried out for exportable products, and the FSSAI may be identified as the agency to bring this change. However, regarding biomedical waste management, it is time to mandate the establishment of effluent treatment plants (ETPs) at hospitals and assess them to generate data on antimicrobial residues in effluents. Guidelines on maintaining a database on the usage of antibiotics in hospitals and other healthcare units, along with AMR cases, have become important for promoting the judicious use of antibiotics. The 2016 and 2018 Biomedical Waste Management (BWM) Rules did not incorporate the segregation of antibiotics from other solid waste. Therefore, antibiotic segregation from other solid wastes should also be given priority in the revised guidelines. In the pharmaceutical industry, policies are required for measuring the magnitude of AMR bacteria before the effluents undergo the recycling process. Continuous monitoring for priority pathogens in environmental samples can guide policymakers regarding policy refinements on antimicrobials. This study suffers the limitation of evaluating the gamut of policies/guidelines present in the important domains that contribute to environmental AMR in India which may be further investigated in each domain. However, this overall analysis generates evidence on how the existing policies guide the current practices and prepares the field for further discussion on policy requirements for the containment of environmental AMR in India. We conclude that though public health is the prime development indicator of our country, and the government allocates abundant funds for the accomplishment of national health policies, their implementation witnesses challenges due to the lack of collaborative approaches, the existence of policies disjointed from ground reality, infrastructural issues, and the lack of capacity and resources. This study highlighted the requirement of effective policies and implementable guidelines as well as coordination between state and central government agencies to address the present knowledge gaps on AMR to boost India’s potential towards leadership for limiting AMR across sectors.


Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Therefore, these PVDF membranes could find potential applications as filtration membranes in healthcare or the environment.Nanofibers have become a promising material in many industries in recent years, mainly due to their various properties. The only disadvantage of nanofibers as a potential filtration membrane is their short life due to clogging by bacteria in water treatment. The enrichment of nanofibers with active molecules could prevent these negative effects, represented by essential oils components such as Thymol, Eugenol, Linalool, Cinnamaldehyde and Carvacrol. Our study deals with the preparation of electrospun polyvinylidene difluoride (PVDF)-based nanofibers with incorporated essential oils, their characterization, testing their antibacterial properties and the evaluation of biofilm formation on the membrane surface. The study of the nanofibers’ morphology points to the nanofibers’ diverse fiber diameters ranging from 570 to 900 nm. Besides that, the nanofibers were detected as hydrophobic material with wettability over 130°. The satisfactory results of PVDF membranes were observed in nanofibers enriched with Thymol and Eugenol that showed their antifouling activity against the tested bacteria  Lately, there has been significant development of nanomaterials worldwide, including many interesting components with excellent physical and chemical properties. These materials are nanoparticles, quantum dots, nanowires, nanotubes, nanorods and nanofibers . The incHowever, the choice of polymer for the composite is significant; it plays a crucial role in nanofiber utilization . Among tNevertheless, the use of PVDF membranes is limited by two main problems: contamination during filtration and wetting. Clogging and wetting will reduce the membrane efficiency and its performance, which will increase operating costs and even cause failure. The solution to this issue may be an appropriate hydrophilic or hydrophobic treatment of PVDF membranes .New functional properties of the membranes can be achieved by the incorporation of other substances into the nanofiber structure, especially antibacterial properties that can limit membrane clogging. Scientists started to load active substances into the polymer matrix to enhance the final membrane with functional properties decades ago. To prevent the fast expiration, PVDF membranes commonly used in water treatment, for example, have the antibacterial substance implemented into the structure .Many studies have been dealing with incorporating different kinds of substances, such as fatty acids (FA), monoglycerides (MAG), proteins, types of nutrition and many others ,11,12,13Our study deals with the production of nanofiber membranes based on PVDF polymers, which are used primarily for filtration. The PVDF membranes with incorporated EOs should prevent the membrane from clogging through bacteria aggregation on the membrane surface.The physical properties of polymer solutions can influence the process of electrospinning. Their viscosity, electrical conductivity and surface tension are significant factors for nanofiber production .w/w concentration [The viscosity of PVDF solutions enriched with EOs was slightly lower than those without the EOs, which was probably caused by the addition of the EOs in liquid form. Per Fortunato et al., the rheological analysis of the PVDF/DMF solution demonstrated a Newtonian behavior up to 30% ntration .As was stated above, viscosity is a crucial factor for electrospinning and can influence the diameter range of nanofibers. The viscosity of the PVDF solution is (3724.6 ± 2.0) mPa·s and it might be predicted that the diameters of the nanofibers from this solution may take high values.However, the electrical conductivity of the samples was quite diverse. The PVDF solution with the conductivity of (9.90 ± 0.08) μS/cm has the highest value among all samples. The incorporation of the EOs decreased the resulting conductivity. The higher concentration of EOs in the solution, the lower the ability to induce an electric current .In addition, the surface activity of the PVDF solution was measured. According to the results given in E. coli and Gram-positive bacteria S. aureus. Inhibition zones were detected immediately, and after 3 and 6 months to reflect the EOs behavior loaded in nanofibers.PVDF solutions modified with EOs were used for the preparation of solvent cast films that were solid, flexible and homogenous with white and yellow coloring. Their antibacterial activity was determined by disk diffusion method against Gram-negative bacteria E. coli and S. aureus. Lin especially has the lowest antibacterial activity of all samples. Even though inhibition zones were observed at concentrations 3% and 5% w/w, the zones were not as significant for other tested EOs. The best results were provided by 5% w/w Thy against E. coli and S. aureus since the zones were over 40 mm in diameter. However, the efficiency of the EOs is different against Gram-negative and Gram-positive bacteria. Eugenol has higher antibacterial activity against E. coli because the zones were two times bigger than those against S. aureus.In all cases, the antibacterial effect of the EOs decreased over time, as the inhibition zones have lower values after 3 and 6 months after film preparation. Due to this fact, the theory of evaporation of the EOs from the films over time has been confirmed . MoreoveE. coli showed the highest sensitivity to the polymer film with the highest concentration (5% w/w) of EOs. Overall, PVDF films with Thy and Car showed the highest antibacterial efficiency of all tested polymer films.It can be concluded that w/w Thy, 3% w/w Cin and 5% w/w Eug. This could be caused by polymer solutions with active substances that achieved different viscous properties in comparison to the pure PVDF solution. As described previously, the process of electrospinning is affected by many parameters, especially the viscosity of the polymer solution, which can rapidly change the morphology of the nanofibers [After observing the antibacterial activity of EOs loaded into PVDF films, identical nanofibers were produced by electrospinning. nofibers .In addition to the smaller diameters of PVDF nanofibers with incorporated EO active substances, various deformations of these fibers were observed in the structure, which was affected by the volatile substances incorporated; this could occur when the solvent evaporated together with the active substances. In addition, it is observed that the diameters are highly variable due to various factors influencing the structure of the nanofibers during the spinning process .w/w EOs and pure PVDF nanofibers. A random arrangement of fibers in many layers can be observed. The fiber diameters of pure PVDF are visibly more extensive than others, except for nanofibers with 5% w/w Eug. The structure of pure PVDF nanofiber is formed by a thin network with a different distribution of diameters of individual fibers.Conversely, samples with incorporated EOs formed a denser network of fibers, with obvious structure deformations. A defect can be seen on nanofibers with Lin. In addition, Car nanofibers braided several threads together. In contrast, the Eug nanofibers are formed by a very dense network of fibers with large diameters of individual fibers, and the wavy orientation. Changes in fibrous structure after the incorporation of active molecules were observed also in the study of Peer et al., in which the PVDF-co-HFP based nanofibrous membranes were investigated .A hydrophobicity measurement was performed to better understand the material’s interaction with aqueous substances . FurtherFrom the result in Similar to PVDF films, the presence of an inhibition zone around the nanofiber membrane disk was evaluated. PVDF nanofibers with incorporated EOs alone proved the presence of these substances via sensory odor evaluation. However, no inhibition zone was confirmed in any sample on the tested bacteria. The absence of inhibition can be attributed to different structure and less surface contact of the nanofibers than in the case of the film. The nanofibers have smaller sizes, and they are distributed in many layers so that the fiber itself comes into contact with the agar surface only on a small surface. Following the results of the disk diffusion method, the nanofibers were subsequently tested to prevent biofilm formation.w/w Eug; the bacteriostatic effect was confirmed because of the suppressed growth of E. coli . Therefore, it was confirmed that the PVDF polymer itself does not exhibit antibacterial properties. Per the images of the PVDF nanofibers enhanced with EOs, it can be concluded that these nanofibers have a bacteriostatic to bactericidal effect. No bacterial cells were observed on the surface of 5% coli see . The othS. aureus. The only difference was in the higher number of S. aureus dead bacterial cells on the surface of individual nanofibers. According to these results, it can be concluded that the Gram-negative bacteria S. aureus is more sensitive to PVDF nanofibers than the Gram-negative bacteria E. coli.Similar results were also achieved with disks in the presence of w/w. S. aureus. Aggregation of bacterial cells is observed on the PVDF nanofibers without EOs, indicating the biofilm’s presence. The same conclusion was reached for the PVDF nanofibers with incorporated Cin, where densely grouped S. aureus cells can be seen. Nanofibers enriched with Car and Cin showed lower cell aggregation than pure PVDF nanofibers.Biofilm formation was evaluated using SEM images, a high concentration of bacterial cells confirming the presence of biofilm formation. SEM analysis revealed the occurrence of biofilm only on PVDF membranes with EOs concentration 5% Conversely, membranes with Thy and Eug have been evaluated as materials that can suppress biofilm formation. As can be seen in S. aureus and E. coli in the presence of PVDF nanofibers with 5% w/w EOs. As can be seen, pure PVDF nanofibers provide a comfortable surface for bacterial aggregation, the same as Cin nanofibers. In contrast, loading with 5% w/w Thy created unfavorable conditions for bacterial formation in the presence of both E. coli and S. aureus. Similar results were obtained by nanofibers loaded with Eug, but low biofilm formation was observed in the presence of E. coli.Further, Regarding the antifouling activity of PVDF nanofibers, it can be concluded that nanofibers with incorporated Thy and Eug could be potentially used for the production of a filtration membrane. Even though these nanofibers could not stop the bacteria growth completely, they prevent them from aggregation (biofilm formation).Nanofiber PVDF membranes are widely used, especially in wastewater treatment, but their lifetime and durability are threatened by bacteria that can impede the membrane by aggregating it on the surface. Since the PVDF polymer creates suitable conditions for bacteria and their adhesion, it is necessary to adjust the chemical composition of the membrane. Suitable substances are essential oils or their active compounds with antibacterial properties widely used in biomedical applications.w/w PVDF in DMF exhibited the viscosity of 3725 mPa·s, a comparable value with the result of previous study [Representatives of essential oils, Thymol, Linalool, Carvacrol, Cinnamaldehyde and Eugenol, were tested in this study. They were added to the polymer solution in various concentrations, solvent-cast and dried until a film formed. It is known that the properties of nanofibrous membranes are strongly affected by the character of the polymer solution. Thus, the surface tension, conductivity and viscosity of PVDF solutions were measured to complement the complex characteristics of investigated PVDF systems. Solution viscosity depends on the polymer character, its molecular weight and concentration. Control solution based on 23% us study . PVDF saus study ,28.Escherichia coli and Staphylococcus aureus model bacteria using a disk diffusion method. The individual results , the sensitivity of the model bacteria was demonstrated by fluorescence microscopy. The results showed lower presence of live bacterial cells on the surface of PVDF nanofibers enhanced with EOs than on the control. In addition, these samples were also tested to evaluate antifouling activity. Biofilm formation was suppressed by nanofibers with incorporated 5% . aureus . They foAlthough satisfactory results in suppressing biofilm formation have been achieved for PVDF membranes with incorporated Thy and Eug—and thus these membranes could find application as potential filtration membranes—further optimizations are needed for the production of nanofibers with antibacterial properties. The biggest obstacle is the incorporated substance itself, which is most likely to evaporate with the solvent during the spinning process and is not incorporated in a concentrated form.® (PVDF) was purchased from Arkema . As a solvent, N, N′-dimethylformamide p.a. (DMF) purchased from VWR Chemicals (France) was used. Active compounds of essential oils (EOs) with potential antibacterial properties were chosen: Thymol (Thy), Carvacrol (Car), Linalool (Lin), trans-Cinnamaldehyde (Cin) and Eugenol (Eug). These compounds were purchased from Sigma-Aldrich (USA) and used without further purification.Polyvinylidene fluoride KynarEscherichia coli ATCC 25922 and Gram-positive bacteria Staphylococcus aureus ATCC 25923. These bacterial strains were cultivated on Mueller Hinton agar  and Mueller Hinton broth  at 37 °C in a thermostat.Model bacterial strains chosen for testing antibacterial properties were Gram-negative bacteria w/w and dissolved in an organic solvent DMF using a magnetic stirrer Heildoph  . Subsequently, three different concentrations of EOs’ active compounds  were added into polymer solutions to their final concentrations of 1% w/w, 3% w/w and 5% w/w.A PVDF polymer solution was prepared at concentration 23% These compounds were quantitatively added to homogeneous, clear, viscous PVDF solutions and stirred on a magnetic stirrer Heidolph . Sixteen PVDF solutions were prepared.The polymer films were prepared as control of antibacterial activity of incorporated EOs in the polymer before the nanofiber production.After stirring, 5 g of each prepared polymer solution was cast into glass Petri dishes with a diameter of 60 mm, which were then dried at 40 °C for 24 h to evaporate solvent gradually. As a result, solid homogenous polymer films were formed, stored at 60% relative humidity and 22 °C ± 1 °C for up to 6 months. The storage of the films was observed to better understand the EOs evaporation during the time.Due to the incorporation of EOs compounds, the physical characterization of the PVDF solutions was determined to better understand EOs’ influence on electrospun nanofibers and their further behavior. The viscosity, electrical conductivity and surface tension were observed on these samples.The viscosity was measured by Brookfield DV—III Ultra Rheometer Ametek  at a shear rate of 20 rpm with spindle LV3. All measurements were determined at 26.2 °C ± 0.4 °C.® Cond 7100  at 25.6 °C ± 0.7 °C.The electrical conductivity was tested by WTW inoLabThe surface tension of the PVDF solutions was also analyzed. The samples were measured by KRUSS Easy Dyne Tensiometer  at 26.1 °C ± 0.5 °C.All the measurements were carried out in triplicate.Electrospinning with a needleless system was used to produce nanofibers from the PVDF solutions given above. The nanofiber layers were prepared on a laboratory device which consisted of a high voltage source Spellman SL70PN150 , a 10 mm diameter metal rod and a stationary flat grounded collector with aluminum foil . According to previous studies , PVDF soThe structure of the prepared samples of nanofibers shown in  was obseThe surface properties of PVDF nanofibers were studied using the sessile droplet method on Attension Theta tensiometer  in combination with OneAttension software at ambient conditions. Distilled water was used as the reference liquid with a droplet volume of 3 µL. All the measurements were tested in triplicate.Escherichia coli and Staphylococcus aureus using the disk diffusion method. First, disks of polymer films and nanofibers with a diameter of 9 mm were cut. Bacterial suspensions with 0.5 McFarland turbidity in saline solution were prepared from 24-h bacterial cultures incubated in MH broth . The prepared bacterial suspension was pipetted in a volume of 1 mL on the entire surface of MH agar. The excess suspension was pipetted off and the prepared disks of polymer films and nanofibers were placed in the Petri dishes. It was followed by a thermostat culturing the Petri dishes with samples at 37 °C for 24 h. After incubation, antibacterial efficiency was evaluated in the form of inhibition zones. This method was repeated after 3 and 6 months after the first experiment, each in triplicate.The antibacterial activity of the prepared polymer films and nanofibers was tested on ®9 and propidium iodide) for 10 s and then covering them with a square coverslip. The fluorescence microscope Olympus BX53  equipped with Microscope Digital Camera DP73  and the cell Sens Standard 1.18  software was used to determine this method. The live and dead bacteria cells were absorbed according to the dye’s interaction with the cell membrane.This method was performed only on PVDF nanofibers because of the inconvenient results from the disk diffusion method. Disks were removed from the Petri dish and placed on a glass slide. This was followed by dyeing them with fluorescent dye  and 60 μL of a 0.5 McFarland turbidity bacterial suspension with the sample of the PVDF nanofiber disk incubated at 37 °C for 72 h. After incubation, nanofiber samples were rinsed thoroughly from planktonic cells with distilled water. The antifouling activity was determined by the fluorescence microscope OLYMPUS DP73 and scanning electron microscope TESCAN VEGA 3.Biofilm formation was tested in glass tubes with 3 mL of BHI broth  + 5% p < 0.05. Data from the characterization of PVDF and antimicrobial activity tests (disk and well diffusion method) were expressed as mean ± standard deviation (SD). Statistical analysis was carried out by a one-way ANOVA followed by a Tukey test using Statistica software version 10, StatSoft, Inc.  at the significance level of S. aureus, biofilm formation was not confirmed in membranes with 5% w/w Eug and Thy. These results are positive for further research, focusing on a new approach to incorporating EOs into the nanofiber structure to achieve antibacterial properties. According to the results of antifouling activity, these membranes have great potential as filter membranes in healthcare or the environment in terms of applicability.During the experiment, PVDF-based nanofiber membranes enhanced with active compounds of EOs, namely Thy, Eug, Lin, Car and Cin at different concentrations, were successfully prepared by electrospinning. Additionally, the physical characterization of the PVDF solutions were determined to obtain better information about their impact on the nanofiber production. The fiber diameters of the individual nanofiber membranes were very diverse in how they were affected by the incorporation of EOs. The individual fibers showed a circular cross-section, a random arrangement and, in some cases, defects in the form of droplets and clumps of fibers. The prepared hydrophobic nanofibers were then subjected to antibacterial tests, such as the disk diffusion method and fluorescence microscopy. Even though no inhibition zone was observed in the presence of PVDF nanofibers, the results from the microscopy were valuable. The results show bacteriostatic and even bacteriocidic activity of EO-enhanced nanofibers. In addition to antibacterial activity, the biofilm formation on the surface of the PVDF membrane was assessed. In the case of


Phyllostomus discolor) and report advances in establishing this species as a tractable model for understanding vocal learning. We have taken an interdisciplinary approach, aiming to provide an integrated understanding across genomics (Part I), neurobiology (Part II), and transgenics (Part III). In Part I, we generated new, high‐quality genome annotations of coding genes and noncoding microRNAs to facilitate functional and evolutionary studies. In Part II, we traced connections between auditory‐related brain regions and reported neuroimaging to explore the structure of the brain and gene expression patterns to highlight brain regions. In Part III, we created the first successful transgenic bats by manipulating the expression of FoxP2, a speech‐related gene. These interdisciplinary approaches are facilitating a mechanistic and evolutionary understanding of mammalian vocal learning and can also contribute to other areas of investigation that utilize P. discolor or bats as study species.Vocal learning, the ability to produce modified vocalizations via learning from acoustic signals, is a key trait in the evolution of speech. While extensively studied in songbirds, mammalian models for vocal learning are rare. Bats present a promising study system given their gregarious natures, small size, and the ability of some species to be maintained in captive colonies. We utilize the pale spear‐nosed bat ( FoxP2, a speech‐related gene. Vocal learning is a key trait in the evolution of speech. Mammalian models for vocal learning are rare, and bats present a promising study system. We utilized the pale spear‐nosed bat to generate new, high‐quality genome annotations, examine structure and gene expression patterns in the brain, and created the first successful transgenic bats by manipulating expression of  FOXP2Vocal production learning  is the ability to produce modified or novel vocalizations, as a result of learning from the experience of the acoustic signals of others.Rousettus aegyptiacus) were shown to modify call frequencies toward playbacks, and their call development is abnormal in isolated situations in which juveniles were housed with mothers who were rarely producing social calls.Saccopteryx bilineata) learn territorial and courtship songs from adult males in their environment.Phyllostomus hastatus), adult calls that seem to denote group identity were modified following the experimental transfer of individuals between groups.Phyllostomus discolor), juvenile isolation calls used by pups to interact with their mothers were modified to be more similar to computer playbacks in hand‐reared individuals.P. discolor bats that were deaf from an early age had a modified vocal repertoire suggesting some reliance on hearing conspecific vocalizations to produce the appropriate repertoire.P. discolor bats were able to modify the properties of their social calls as adults.In recent years, bats have received increased attention as model systems for studying speech and language‐related traits such as vocal learning. We have previously outlined the benefits of employing these animals and the tools that would be needed to make them a powerful system for revealing biological and evolutionary mechanisms of vocal learning.P. discolor bat as a tractable species for studying the neurogenetic mechanisms underlying learned vocal communication. P. discolor has several features that recommend it for in‐depth study of the neurogenetic underpinnings of vocal learning. They are small, predominantly frugivorous , and thrive in captive breeding colonies.Herein, we aim to outline the progress that has been made in our own work to establish the P. discolor, including reporting their vocal repertoire in a social context,Our ultimate goal is to be able to understand the evolution and mechanistic underpinnings of learned vocal behavior, which necessitates an examination of the behavior as well as the underlying genetics and neurobiology. We have previously described our developments in behavioral aspects of vocal learning in ,Reference quality genomes are important for studying genotype–phenotype relationships, the molecular mechanisms underlying phenotypes, the evolution of traits, and for conservation efforts.P. discolor bat was originally published in 2020 by the Bat1K consortium as part of our release of the first reference quality bat genomes.P. discolor genome.The genome of the P. discolor annotations, we produced additional functional (transcriptomic) data from a range of tissues , and reprocessed ISOseq dataN = 21,516 to N = 25,058 , but miRNAs were also encoded within 10 Kb around the transcriptional start and end sites, and in exons, 5’UTRs and 3’UTRS  that represent a substantial improvement over previous bat genomes—including our prior P. discolor, it is crucial that we understand the basic brain morphology of the species and develop the tools necessary to build on this understanding to define specific circuitry underlying complex vocal learning behavior. To this end, we have been developing multiple complementary approaches in P. discolor, including electrophysiology, neuroimaging, connectivity tracing, and histology. Since electrophysiology is routinely employed in bats, we refer to the primary papers rather than presenting such data herein P. discolor brain. We report both T1‐ and T2‐weighted MRI images (see Table P. discolor bats.We have begun to utilize magnetic resonance imaging (MRI) and specifically diffusion tensor imaging (DTI)Auditory perception and processing are important components of vocal learning. Thanks to the extensive use of bats to study the neuroethology of echolocation, there is a good understanding of auditory pathways in the bat brain. The ascending auditory pathway in bats is broadly conserved with that of other mammals,P. discolor. Injecting a dextran tracer into the FAF . We expect any differences in expression related to vocal learning would be very subtle and that these changes would be found in specific populations of neurons rather than being large brain‐wide changes in expression. This likely would be due to the importance of these genes for general brain development and function. Indeed, this is what has been observed in songbird mapping studies where specific nuclei of the song circuitry show subtle changes in these genes. Work is now underway to perform a detailed comparison of the expression of these genes across bats, birds, rodents, and humans to uncover any convergent expression or patterns that are shared by vocal learning species.Overall, the patterns observed for FOXP2, FOXP1, PV, and GluR1 in the P. discolor so that future work can exploit them to address the neurobiology of vocal learning in bats. What is striking from all the approaches used is that the P. discolor brain shares strong structural homology with rodent brains. Albeit a much larger brain (about 1.5 times the size of a lab mouse brain), the gross structures of the P. discolor brain are easily identifiable when compared with rodents, which is highly beneficial for undertaking comparative work. There are, however, clear differences between species. For example, the AC in P. discolor  is greatly expanded compared to the mouse, and the FAF may be a specialized sensorimotor integration point for auditory processing and vocal‐motor production. Future work will apply the approaches discussed herein to explore vocal learning circuitry in the P. discolor brain and uncover the more subtle differences that may be present when comparing to rodents or other species.We have built upon the strong history of neuroethological research in bats to expand the tools that can be applied in P. discolor. Comparing our data with published histological atlas allowed us to validate the MR imaging approach and demonstrate its accuracy in revealing brain structures. Our findings will now make it possible to use neuroimaging to perform brain‐wide connectivity studies and unravel the P. discolor connectome. Such MR imaging and related connectivity analyses have limitations, such as relatively low resolution, and in the case of connectivity analyses, modeling the structure of the brain's connectivity indirectly by measuring the water density/diffusion rather than directly measuring it. By combining these data with complementary methods, such as PLI, which is a direct measure of connectivity with enhanced resolution, we can validate MR‐based data with histological data to circumvent these downsides.Neuroimaging approaches have been used in bats in a few prior studies,P. discolor brain will enable direct comparisons between vocal learning in bats and speech in humans—where there is a wealth of DTI data publicly availablein vivo to explore the whole brain activation pattern during vocal behaviors in P. discolor.In the future, targeted probabilistic tract‐tracing studies from brain areas involved in vocal communication in the P. discolor together with previous studies in other bat speciesP. discolor. Strikingly, we uncovered a novel projection of the pyramidal tracts pointing to possible involvement of the FAF in motor functions. This involvement has been speculated in the past as previous studies found strong projections from the FAF into the SC.Our tracing studies in P. discolor genome (Part I) mean that in most cases, the conservation of epitopes targeted by existing antibodies can be used to predict the specificity of an antibody for detecting bat proteins. Despite the ∼65 million years of evolution since the divergence of the bat lineage,The histological data presented herein demonstrate the feasibility of applying similar genetic mapping approaches in bats to those used in songbirdsObserving the natural state of a behaving animal can reveal potential mechanisms underlying that behavior. An effective way to demonstrate causal links between neurogenetic mechanisms and behavior is to perturb gene function. After genetic manipulations, effects on molecular pathways, brain development, and behavior can be measured.In widely used model organisms like flies or rodents, the creation of a germline transgenic animal to reveal causal mechanisms has become routine thanks to their ease and speed of generation.FoxP2 expression in the brain of living P. discolor bats to facilitate future exploration of its role in vocal learning behavior and associated neural circuitry. This was of particular interest given the identification of a role for FOXP2 in speech and language in humans and for vocal learning in zebra finch songbirdsHerein, we describe our efforts to manipulate FoxP2 is one of the most highly conserved genes between mice and humans.P. discolor Foxp2 protein is also highly conserved  was only intended as proof of principle and future work will explore the influence of manipulating FoxP2 expression on molecular pathways, cell morphology, neural circuitry, and vocal learning behavior after longer incubation times. This will give a chance for the changes that occur downstream of FoxP2, molecular or neural, to exert their influence on phenotypes using the toolkit we describe herein (Parts I and II). We will also extend these studies to the consequences of FoxP2 overexpression when introduced into other regions of the brain. For example, one benefit of the overexpression construct is that we can increase expression where FoxP2 is present but sparse , or in places where there is little FoxP2 expression .This is the first successful manipulation of gene expression in a living bat and the first generation of a transgenic vocal learning mammal. These data clearly show a successful manipulation of bat striatal neurons in vivo in the bat brain will allow a major step forward in understanding neurogenetic contributions to vocal learning. It will allow future exploration of not only FoxP2, but other coding genes, noncoding RNAs, and molecular pathways in this complex behavior. These include those implicated in songbird studies to determine if these are evolutionarily conserved mechanisms, as well as those from human studies—particularly genes implicated in speech/language disorders. This would reveal both fundamental mammalian mechanisms involved in complex communication, as well as mechanisms by which these genetic changes can cause disorders in children. As each new gene is addressed, it will be important to tailor the transgenic design and target the appropriate brain regions. For example, cross‐species comparisons could introduce the expression of a gene into a region where it is found in birds, but not bats. Studies based on clinical genetics could knock down gene expression to recapitulate the effects of the patient mutation. Building a toolkit of techniques and utilizing the newest technologies to generate these transgenics will provide maximal flexibility and greatly advance our ability to understand the neurogenetic mechanisms underlying mammalian vocal learning in bats and over evolution.The ability to manipulate gene expression P. discolor bat to explore the neural and genetic mechanisms underlying vocal learning. Many of these approaches are routinely applied in other animal systems like mice or songbirds but have not been utilized in this species before. If we are to harness the potential of bats to reveal the biological and evolutionary mechanisms of vocal learning, such tool development is crucial. Moreover, it is important that we do this in an integrative manner, linking the different levels of investigation from genes to brains to behavior. While we have addressed a few key areas from our current work, ongoing and future work aims to expand these tools through their exploration of techniques such as fMRI, monosynaptic and viral tracing, in vivo calcium imaging, and optogenetics. The tractability of P. discolor makes this an ideal bat species in which to develop and test these methods. However, we do not want to imply that studies should only focus on this species. We hope that any advances we make in tool development in P. discolor could be propagated and employed in other vocal learning bat species. As noted in the Introduction, several bat species have been identified with vocal learning abilities and importantly, the behaviors displayed by these bats vary greatly. Thus, to gain a true understanding of bat vocal learning, interrogation of bats across the family tree is crucial. Furthermore, studies in diverse bat species, and potential comparisons with other mammalian and bird vocal learners, are necessary to gain an evolutionary perspective on vocal learning.We have outlined approaches in the S.C.V. conceived and designed the experiments, contributed to data analysis and interpretation, and wrote the manuscript. P.D. and S.G.H. contributed to the conception and design of experiments and acquired and analyzed/interpreted data in Parts I/III and II/III, respectively. I.A.vT. contributed to the design of experiments and acquired and analyzed/interpreted data in Part II. N.H., J.M., and M.W. contributed to the design of experiments and acquired and analyzed/interpreted data in Part II. U.F. and P.H. contributed to the design of experiments and analyzed data in Part II. K.L. contributed to the design of experiments and analyzed data in Part I. M.H. and A.E.M. contributed to the data collection and analysis in Part I. G.M.H. contributed to the data collection and analysis in Part III. All authors contributed to the writing and editing of the manuscript and authorized the final version of the manuscript.The authors declare no competing interests.https://publons.com/publon/10.1111/nyas.14884.The peer review history for this article is available at: Figure S1. Mammalian species tree showing the diversity of the FOXP2 protein sequence across 48 mammals. Branches represent the number of differing amino acid sites relative to the Homo sapiens sequence. Each vertical line represents one amino acid change in the species. Species with some unknown residues are represented with “*”In vitro testing of FOXP2 transgenic constructs. Complete western blot image corresponding to Figure 7 of the main text.Figure S2. Table S1. Tissues used for transcriptomic studies.Table S2 List of species used for the annotation of known miRNAs.Table S3. Genomic locations of known and private miRNAs.Table S4. MR scanning conditions.Table S5. Antibodies used for immunostaining (IHC and IF).Table S6. Quantification of FoxP2 upregulation in bat striatum––individual replicate values.Materials and MethodsClick here for additional data file.P. discolor updated annotations.File S1. Click here for additional data file.File S2. FoxP2 Clustal Protein AlignmentClick here for additional data file.