Orange peel, which is a rich source of polyphenolic compounds, including hesperidin, is produced as waste in production. Therefore, optimization of the extraction of hesperidin was performed to obtain its highest content. The influence of process parameters such as the kind of extraction mixture, its temperature and the number of repetitions of the cycles on hesperidin content, the total content of phenolic compounds and antioxidant (DPPH scavenging assay) as well as anti-inflammation activities  was checked. Methanol and temperature were key parameters determining the efficiency of extraction in terms of the possibility of extracting compounds with the highest biological activity. The optimal parameters of the orange peel extraction process were 70% of methanol in the extraction mixture, a temperature of 70 °C and 4 cycles per 20 min. The second part of the work focuses on developing electrospinning technology to synthesize nanofibers of polyvinylpyrrolidone (PVP) and hydroxypropyl-β-cyclodextrin (HPβCD) loaded with hesperidin-rich orange peel extract. This is a response to the circumvention of restrictions in the use of hesperidin due to its poor bioavailability resulting from low solubility and permeability. Dissolution studies showed improved hesperidin solubility (over eight-fold), while the PAMPA-GIT assay confirmed significantly better transmucosal penetration (over nine-fold). A DPPH scavenging assay of antioxidant activity as well as inhibition of hyaluronidase to express anti-inflammation activity was established for hesperidin in prepared electrospun nanofibers, especially those based on HPβCD and PVP. Thus, hesperidin-rich orange peel nanofibers may have potential buccal applications to induce improved systemic effects with pro-health biological activity. Citrus aurantium (L.) (orange) is the world’s most widely grown and traded citrus species. The orange fruit is usually eaten whole or made into juice after peeling the outer peel. This peeling process leads to the generation of considerable peel waste [el waste . Due to el waste ,3. Orangel waste . Finallyel waste ,6.Hesperidin has been recently extensively evaluated for its health-promoting effects to reveal its antioxidant, anti-inflammatory, anticancer, antiviral, cardio-protective and neuroprotective properties . DespiteIn this nano era, numerous top-down and bottom-up nano methods have been introduced into the applications of manipulating drugs’ encapsulation, dissolution, permeation and controlled release behaviors . Some apThus, in this work, an attempt was made to produce rapidly soluble HPβCD/PVP-based nanofibers containing hesperidin-rich orange peel extract to improve its solubility and permeability. Full characterization of physicochemical properties as well as an evaluation of pharmaceutical properties has been carried out. The optimization of the orange peel extraction process preceded these studies.In recent years, interest in using herbal medicines in treating and preventing various diseases has increased worldwide, although in most cases, their quality may be questionable. Hence, there is a need to standardize plant product requirements and look for reliable tools to verify their quality. Therefore, there is an increasing emphasis on understanding the extraction process and determining the critical parameters of the process. In this work, a full factorial design model was created to assess the effectiveness of the extraction process. The influence of methanol content in the extraction mixture, its temperature and the number of repetitions of the cycle on hesperidin and total polyphenol content and the antioxidant and anti-inflammatory activity of extracts was assessed.The separation and the determination of 11 active compounds  contained in the orange peel extracts, as well as all concentration changes in hesperidin during dissolution and permeability studies of electrospun nanofibers, were carried out using high-performance liquid chromatography supported by a photodiode array detector. The retention times of all 11 compounds were compared with the retention times and UV spectra of the reference substances a,b. The Using the linearity equation of all 11 reference substances, it was possible to determine the content of all active compounds in all 9 extracts , whereas50), while anti-inflammatory activity decreased (observed as an increase in IC50).It is critical to ascertain the biological activity of the produced extracts. For this, the antioxidant  potential, as well as the anti-inflammatory activities , was evaluated. The complete results are shown in The technical aspects of the extraction procedure that produced the extract with the best characteristics and the highest activity could be identified based on the test results and statistical analyses. None of the analyzed cases showed that the number of extraction cycles was a critical parameter. Therefore, based on the utility contour profiles model, including all measured outputs, it was possible to predict the model and indicate optimized parameters of the orange peel extraction process, which are 70% of methanol in the extraction mixture, a temperature of 70 °C and 4 cycles per 20 min  . It was The E10 extract was created using optimized process parameters, and its activity was assessed to validate the model. The second phase of the experiment, which involved the creation of electrospun nanofibers, utilized the E10 extract.In the second part of the work, an attempt was made to optimize the electrospinning process. In a previous study, a significant effect of PVP and HPβCD on the physicochemical properties of nanofibers was demonstrated; therefore, the impact of these two excipients on the quality of nanofibers rich in orange peel extract was also examined in this study. Moreover, it was noticed that the addition of dichloromethane to methanol improves the quality of nanofibers ; hence, The produced electrospun nanofibers were examined using SEM to determine their morphology, XRPD to show structural characteristics and ATR-IR to identify any potential intermolecular chemical bond formation.A critical relationship was observed between the viscosity of electrospinning solutions and the diameter of the nanofibers. A too-low concentration of polymers (in the case of system no. 1) resulted in the formation of beads instead of nanofibers , which wIt is worth noting that the process ran smoothly in the case of N2–N5 nanofibers, which was also observed in the efficiency of the process . A speciThe ATR-IR spectra of nanofibers N1–N9 were analyzed and compared with the spectra of pure hesperidin, the lyophilized extract and excipients used for nanofiber fabrication, such as PVP and HPβCD .−1. The bands at 1518–1356 cm−1 are attributed to the aromatic C=C stretch and the aromatic C–O stretch at 1273, 1204, 1150, 1126 and 1067 cm−1. The ATR-IR spectra of native hesperidin showed characteristic bands because of the existence of different functional groups such as at 1645, 1518 and 1050 cm−1, which could be attributed to C=O stretching, C=C stretching and C–O stretching, respectively, as well as at 1018 cm−1 corresponding to C–C stretching in the rutinose ring and C–C stretching between glucose and rhamnose rings [−1 proving the existence of stretching of C–O. The C-H bending and CH2 wagging were observed at 1420 cm−1 and 1269 cm−1, respectively. The peaks at 1015 and 565 cm−1 were identified as the CH2 rock and N–C=O bending, respectively [−1 (H–OH bending vibrations), 1148 cm−1 (symmetric C–O–C stretching vibrations), 1080 cm−1 (C–O stretching vibrations), 1009 cm−1 (C–C stretching vibrations) and 947 and 847 cm−1 (presence of glucopyranose units). Furthermore, in the range of 1200–1500 cm−1, there are bands associated with bending vibrations of C–H and O–H bonds [The spectrum peak of carbonyl C=O stretch appeared at 1645 cmse rings . Most of–H bonds .−1 coming from PVP and bands at 1150 and 1080 cm−1 coming from HPβCD. Finally, some bands characteristic of hesperidin/extract at 1032, 934 and 841 cm−1 are still visible with a slight wave shift.When analyzing the spectra of N1–N9 nanofibers, the most intense are the bands at 1651, 1491, 1460 1423 and 1288 cmAnother important aspect in assessing electrospinning’s effect on nanofibers’ properties is the study of hesperidin content. The highest content was noted for the non-fiber system no. N1. A statistically significant relationship was shown that the increase in PVP concentration reduces the hesperidin content in the produced nanofibers .Pure hesperidin is classified in the IV class according to the Biopharmaceutical Classification System, i.e., as a poorly soluble and poorly penetrating substance. The nanofibers are designed as a rapidly soluble form to enhance permeation within the oral cavity. Therefore, in order to increase the penetration of hesperidin through the oral epithelium directly into systemic circulation, it is essential that the compound dissolves quickly in oral liquids. Therefore, hesperidin release from the prepared nanofibers was assessed . The disScientists put considerable effort into improving the permeability of active compounds through biological membranes. One approach is to amorphize the compound. However, amorphization alone is not always sufficient to achieve this goal . In the Biological activity studies (antioxidant and anti-inflammatory activities tests) have also been performed for obtained nanofibers. The results obtained for the electrospun nanofibers remained at the same level as the results for the starting material, which show no change in the biological activity of the extract as an active ingredient. Therefore, the electrospinning process had a negative impact on the activity of the entire formulation.Finally, an essential factor determining the application properties of the buccal preparation is the ability of the substance (PVP and HPβCD) to bind to the mucin found in the mucosa, which ensures that the product remains on the oral mucosa and does not cause the swallowing of the product into the digestive tract. For this purpose, rheological tests of nanofiber mixtures were used, the results of which are presented in To summarize the production step of electrospun nanofibers, a model of utility contours of profiles was made for all the tested effects . Based oAurantii amari pericarpium et mesocarpium (orange peel) was purchased from Natura Wita, Zdrowie z Zielnika  (lot no. 270821).® Reference Substance), hesperetin (≥95%), naringenin (98%), naringin , (+)-catechin , (−)-epicatechin , quercetin , rutin , kaempferol , caffeic acid  and luteolin  were obtained from Sigma-Aldrich . Excipients, such as (2-Hydroxypropyl)-β-cyclodextrin (HPβCD), average Mw ~1460, were supplied from Sigma-Aldrich , and polyvinylpyrrolidone (PVP) as Kollidon® 30 was supplied from BASF Pharma . Reagents for activity assays include the following: 2,2-Diphenyl-1-picrylhydrazyl (DPPH), potassium persulfate (K2S2O8), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt , neocuproine, ammonium acetate, copper(II) chloride (CuCl2·H2O), sodium acetate trihydrate (CH3COONa·3H2O), 2,4,6-tris(2-pyridyl)-1,3,5-triazine , iron(III) chloride hexahydrate (FeCl3·6H2O), sodium chloride, bovine serum, hexadecyltrimethylammonium bromide (CTAB) and hyaluronic acid (HA); for dissolution studies, they include the following: potassium chloride, sodium chloride, di-potassium hydrogen orthophosphate, magnesium chloride, calcium chloride and xylitol; and for mucoadhesive tests, they include the following: mucin from porcine stomach obtained from Sigma-Aldrich . Prisma™ HT buffer, Acceptor Sink Buffer and GIT lipid solution were obtained from Pion Inc. , whereas HPLC-grade acetonitrile and water were obtained from Merck . High-quality pure water and ultra-high-quality pure water were prepared using a Direct-Q 3 UV Merck Millipore purification system .Hesperidin . Independent factors were selected as the content of the extraction mixture, its temperature and the number of repetitions of the cycles (Using the Design of Experiments (DoE) approach, a factor experiment plan was developed for three independent variables, which were assigned three levels of values  as well as anti-inflammation activities .λ;max) of 280, 330 and 360 nm.The contents of 11 active polyphenolic compounds  were determined by using the modified HPLC-diode array detection method. As for equipment, the LC system (Dionex Thermoline Fisher Scientific) with Chromeleon software version 7.0 was used. Separations were performed on a LiChrospher RP-18 column, 5 μm particle size, 250 mm × 4 mm . The mobile phase was composed of formic acid 0.1% (A) and acetonitrile (B) with a gradient elution: 0–10 min, 12% B; 10–60 min, 12–60% B; and 60–65 min, 12% B. The flow rate of the mobile phase was 0.6 mL/min, and the column temperature was maintained at 30 °C. The detection was performed using a diode array detector at wavelength maxima , 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS) radical cation-based assays, Cupric ion reducing antioxidant capacity (CUPRAC) assay and Ferric ion reducing antioxidant parameter (FRAP) assay. All procedures were described previously .The procedure of hyaluronidase inhibition was determined by the turbidimetric method described previously .The electrospinning procedure was performed using NS + NanoSpinner Plus Electrospinning Equipment . The high voltage was set at 27 kV, the flow rate of the solution was 2 mL/h, and the distance between the syringe and rotary collector covered with aluminum foil was fixed at 12 cm. The experiments were carried out at room temperature (about 25 °C), and the humidity did not exceed 40%.2 full factorial design experimental plan and are presented in The amount of HPβCD and PVP and the solvent composition used to prepare nanofibers were selected based on Design of Experiment (DoE) data and the 3The dynamic viscosity of solutions was measured with a Brookfield DV2T viscometer at a predetermined temperature (25 °C).The surface morphology of the nanofiber was visualized using SEM. The nanofibers were sputter-coated with gold–palladium and then visualized by a scanning electron microscope .The diameter of nanofibers was measured on SEM images.The crystallographic structure of the samples was analyzed by X-ray diffraction  equipment with the copper anode (CuKα—1.54 Å) at a Brag–Brentano reflection mode configuration with 45 kV and 40 mA parameters. The measurement parameters were set up for 3–60° with a 45 s. per a step size of 0.05° in all cases.−1 and 4000 cm−1, with the resolution set to 1 cm−1, with a Shimadzu IRTracer-100 spectrometer equipped with a QATR-10 single bounce—diamond extended range and LabSolution IR software.The ATR-IR spectra were measured between 400 cmHesperidin content was determined by using the HPLC method described in Dissolution studies of electrospun nanofibers were performed using an Agilent 708-DS dissolution apparatus. A standard basket method was used at 37 ± 0.5 °C with a stirring speed of 50 rpm. Nanofibers were placed in 300 mL of artificial saliva solution at pH 6.8 (potassium chloride (1.20 g), sodium chloride (0.85 g), di-potassium hydrogen orthophosphate (0.35 g), magnesium chloride (0.05 g), calcium chloride (0.20 g), xylitol (20.0 g) and water up to 1 L; pH was adjusted to 6.8 by 1 M HCl). The liquid samples were collected at specified time intervals, and an equal volume of temperature-equilibrated media was replaced. The samples were filtered through a 0.45 μm nylon membrane filter. The HPLC method described above determined the concentrations of hesperidin in the filtered acceptor solutions. Sink conditions were preserved in the studies. The study was repeated six times.The permeability of active compounds enclosed in nanofibers through artificial biological membranes was investigated by using the gastrointestinal tract (GIT) PAMPA™  . Nanofibers were dissolved in donor solutions . The acceptor plates were loaded with an acceptor Prisma buffer at pH 7.4. The plates were put together and incubated at 37 °C for 15 min with continuous stirring at 50 rpm. Each experiment was repeated at least three times. The amount of permeated active compounds was determined using the HPLC method described above. The study was repeated six times.appP) were calculated from the following equation:DV—donor volume, AV—acceptor volume, equilibriumC—equilibrium concentration DC—donor concentration, AC—acceptor concentration, S—membrane area and t—incubation time (in seconds).The apparent permeability coefficients (The biological activity of electrospun nanofibers was determined by assays described in A viscometric method was used to quantify mucin–polymer bioadhesive bond strength. The evaluation was performed according to the method described previously . The stup < 0.05.Statistical analysis was carried out with Statistica 13.3 software. The normality of the results was checked using the Shapiro–Wilk test. The differences among the mean values were tested using the ANOVA test with post hoc Tukey’s range test for multiple comparisons. Differences between groups were considered to be significant at In the last few years, increased attention has been focused on industrial wastes, especially those containing residual phenols from used plant material. Therefore, the orange peel was selected for the study, which, in addition to being food waste, is also a good source of hesperidin. Based on the results of the DoE approach, it was possible to predict that the extraction efficiency increases with the increase in the percentage of methanol in the extraction mixture and temperature. Optimized parameters of the orange peel extraction process were selected as 70% of methanol in the extraction mixture, a temperature of 70 °C and 4 cycles per 20 min.Moreover, this study found that hesperidin contained in orange peel extract can be complexed with HPβCD/PVP and subjected to the electrospinning procedure to form stable nanofibers. The HPβCD/PVP-based formulation as well as the electrospinning process affected the solubility of hesperidin nanofibers, which significantly increased. In addition, an over nine-fold increase in the permeability of hesperidin contained in nanofibers was demonstrated. The best properties were obtained for nanofibers with the composition of HPβCD/PVP/25% dichloromethane/10 mL methanol, i.e., nanofiber no. N5. Therefore, hesperidin-loaded nanofibers can effectively improve hesperidin’s aqueous solubility and show enhanced penetration through the biological membrane, with the maintenance of its antioxidant and anti-inflammatory properties. Therefore, hesperidin-rich orange peel nanofibers may have potential buccal applications to induce improved systemic effects with pro-health biological activity.


Previous United Kingdom campaigns targeting antimicrobial resistance (AMR) recommended running multimedia campaigns over an increased timeframe. The 3-year-long Keep Antibiotics Working (KAW) campaign was a mass media campaign in England targeting the public and general practitioners (GPs).X2) and Mann–Whitney U tests, respectively.Every year, pre- and post-campaign questionnaire data were collected from the public, whereas post-campaign interview data were obtained from GPs. Data were weighted to allow pre- and post-campaign comparisons between independent samples. Significant changes in nominal and ordinal data were determined using Pearson’s chi-squared . Knowledge regarding declining antibiotic effectiveness when taken inappropriately improved following the campaign ; post-2019 = 77.6%; ; X2 = 5.753, p = 0.016). The proportion of individuals reporting concern for themselves or for children (≤ 16 years) about AMR increased by 11.2%  and 6.0%  respectively, pre- to post-campaign. Finally, in 2017, reported confidence to say no to patients requesting antibiotics differed significantly between GPs who were and were not aware of the campaign (net agree: 98.9% (182/184) vs 92.4% (97/105) respectively; X2 = 4.000, p = 0.045).Prompted campaign recognition was high, increasing by 6% from 2018 to 2019 ; 2019: 74% ; A high level of prompted campaign recognition was achieved. The KAW campaign improved aspects of AMR knowledge and certain attitudes towards appropriate antimicrobial use. It increased awareness of and concern about AMR, supporting GP confidence to appropriately prescribe antibiotics. Future determination of measurable behaviour changes resulting from AMR campaigns is important. What did you want to address in this study?We studied the impact of a national public health campaign in England on antimicrobial resistance and correct antibiotic usage. We assessed the public’s campaign recognition and if knowledge, awareness and understanding of antimicrobial resistance improved. We also checked if reports of correct antibiotic usage increased, if general practitioners felt more confident to decline antibiotics and if patients’ expectations for antibiotics reduced.What have we learnt from this study?We learnt that a campaign on antimicrobial resistance which uses marketing to encourage behaviour change that is beneficial to society  and which is promoted via multiple channels, including television, social media and patient resources (e.g. information leaflets and posters) helps to improve public knowledge on its key messages and supports general practitioners’ confidence to prescribe antibiotics appropriately.What are the implications of your findings for public health?Our findings suggest that campaigns using a multifactorial, social marketing approach may increase public knowledge and concern about antimicrobial resistance. However, whether campaigns actually lead to reduced antibiotic use and decreases in antimicrobial-resistant infections needs to be further investigated with outcomes that we can measure, such as events of inappropriate prescribing or occurrences of antimicrobial resistant infections.a massive global public awareness campaign on AMR”.Antimicrobial resistance (AMR) is a considerable threat to human health. Globally, an estimated 1.2 million people died in 2019 from antibiotic resistant bacterial infections [Previous United Kingdom campaigns, which aimed to optimise prescribing and reduce public demand for antibiotics, used simple single-channel approaches, such as distributing posters or leaflets to healthcare practices over short time periods ,4. EvaluMarket research with prescribers and the public to investigate pre-campaign knowledge, attitudes and behaviour towards AMR and antibiotic prescribing had identified that the public had limited understanding of AMR and misconceptions, for example that antibiotics are effective against viral infections, were common -13. FurtNational surveillance data from England in 2015 estimated that most antibiotics across England are prescribed in general practice (74%) . TherefoThe aim of the current study was to evaluate the national KAW campaign and in particular to assess whether it reached the target audiences in England, improved knowledge, awareness and understanding of AMR, increased reported action of appropriate antibiotic usage behaviours, strengthened GP confidence to decline antibiotics and reduced patient expectation for antibiotics.Development of the KAW campaign followed the Government Communication Service's Objectives, Audience/insight, Strategy/idea, Implementation and Scoring (OASIS) model and the Wellcome Trust's key principles for communicating AMR effectively ,19.Between February and April 2017, a pilot campaign was run through an Independent Television (ITV) called Granada Television, which broadcasts to all individuals living within Yorkshire and the North-West region of England. This pilot campaign was then evaluated to inform and support the development of the national KAW campaign.Supplementary Images 1 and 2. In the second and third years of the campaign, healthcare professionals could download from the Public Health England Campaign Resource Centre and customise for their surgeries. These resources were mostly aimed at primary care providers.The national KAW campaign then ran for three 3-month periods between November and January 2017, 2018, and 2019 using broadcast advertising (a television (TV) commercial, video on demand via YouTube, radio, newspaper advertising and posters) and advertising via social  and news media. People who were searching online for information on cold and influenza were served with an advert encouraging them to go to the pharmacy instead of the GP to promote self-care for mild colds and influenza virus infections. The direct-to-public communications were supplemented with posters, leaflets and ‘Treat Your Infection’ non-prescription pads providing advice on how respiratory tract and urinary tract infections can be managed at home if antibiotics are not required. In the first year of the campaign, these materials along with a letter outlining where to order additional resources, were distributed to all GP practices in England. The posters were also distributed to all community pharmacies in England. Illustrative examples of posters are provided in In addition to an overall evaluation of the campaign to determine its effectiveness and share learning with other countries, year-on-year optimisation was attempted to continuously improve outcomes towards the campaign aim. At each step, an informal evaluation was conducted followed by adaption of the channel mix and creative solutions.Supplementary Material 2. Key subgroups included mothers of children aged 0–16 years and adults aged over 50 years.Data collection from the public in England occurred in six steps; one pre and one post each wave of advertising in 2017, 2018 and 2019. Interviews with the public were conducted by Kantar Public, using a Computer Assisted Web Interviewing (CAWI) approach, with samples drawn from the Kantar Profiles (Lightspeed) Online Panel Network, an industry-leading double opt-in panel, built with highly validated and trusted sources and partners. Questionnaires were adjusted every year to account for changes to the campaign, updated audiences and messaging, and to incorporate learning or fill gaps from previous evaluations. A seven-point Likert scale, where 1 represented no concern at all and 7 represented a high level of concern, was used to determine the level of concern individuals felt regarding AMR. The CAWI questionnaire is available in The target sample size for questionnaire responses from the public was 1,000 respondents in each wave, providing a sufficiently large base size to allow robust analysis both of the overall sample, and of key subgroups. Each year, purposive sampling was conducted among mothers of children aged 0–16 years to increase the sample size of this sub-group and therefore allow for sub-group analyses.Supplementary Material 3. Samples were drawn from the GP practices’ database available on the National Health Service (NHS) Digital website. The GP sample was purchased from a specialist health professional database and quotas were set on GP practice size and region to ensure the sample was representative across regions and practice size.Three waves of quantitative research with GPs (following each year of the campaign) involved a target sample size of 300 participants to ensure a sufficient sample size. Interviews with GPs were conducted via telephone using Computer Assisted Telephone Interviewing (CATI) with a pre-defined questionnaire, which is presented in Supplementary Material 1) views was automatically generated every time the video was played on social media; this count was supplied by a media agency (Wavemaker). Media coverage was supplied by a Public Relations agency (Freuds) and included a count of mentions in a newspaper article or the news. Poster distribution data were supplied by the Resource Centre. Response levels and cost per click for the online campaign resources were monitored in real-time, to assess metrics of engagement with advertising and identify the best-performing adverts in the population.A count of video  collected at each campaign wave were weighted for the statistical analysis to ensure the samples were matched and nationally representative on the demographic variables of age (sample restricted to over 18-year-olds), sex (collected as a binary variable), region, and socioeconomic status (SES), allowing for pre- post-campaign comparisons to be made.For GPs, comparisons between individuals who were and were not aware of the campaign were made to determine any significant differences between the two groups. This is due to data only being collected from GPs following each year of the campaign. 2), or Mann–Whitney U tests, with the Z-score, were used to outline significant changes in nominal and ordinal data respectively for pre- and post-campaign measures of attitudes, knowledge, and concern regarding AMR. Tests were initially conducted to determine the effect of the campaign from pre-2017 to post-2019; tests were then conducted to outline the significance of year-by-year changes. Questionnaire response categories for questions assessing attitudes, knowledge, and reported behaviour (such as those further presented in tables 5 and 6) were pooled before statistical testing to give two response categories as opposed to four . A p value threshold of p < 0.05 was used to determine significance. Data were analysed using Microsoft Excel and IBM SPSS Statistics Version 27.0.Pearson’s chi-squared, Pearson’s cumulative test statistic   and 2019  compared with the public.There was a significant difference in unprompted recognition  of any AMR publicity by the public and the key subgroups from before the 2017 to following the 2019 campaign  . Unpromp2X = 8.742, p = 0.003; 2X = 9.283, p = 0.002). Prompted recognition was similarly as high in adults aged over 50 years as in mothers in 2018 but did not change significantly from 2018 to 2019. Prompted recognition was significantly higher post-2019 in mothers of children aged 0–16 years compared with the public . GP awareness of the campaign was lower than for the public following the campaign in 2018 and 2019. The TV campaign had the greatest recognition followed by information seen at the doctor’s surgery/clinic and newspaper articles, as shown in Supplementary Material 5.Prompted campaign recognition  was higher than unprompted recognition and increased significantly in the public and in mothers of children aged 0–16 years between 2018 and 2019 . Perceived knowledge of AMR was lower than that of antibiotics before the campaign (23.4% vs. 58%) but increased to a slightly greater extent following the campaign (9.2 vs. 8.1% increase). However, despite declining consistently over the duration of the campaign, the proportion of individuals who had never heard of the term ‘antimicrobial resistance’ following the campaign remained high  compared with 7.6% (74/969) for antibiotic resistance. There were significant differences in perceived knowledge of AMR depending on sex, with males reporting significantly greater perceived knowledge ; this difference remained following the campaign . Furthermore, a significant social gradient was identified for antibiotic resistance with higher SES groups reporting higher perceived knowledge ; the significance of this gradient reduced following the campaign .Perceived knowledge  of antibGPs’ perceptions of patient knowledge  improved during the campaign with a ca 27% reduction in the number of GPs who thought patients were unaware of issues relating to AMR following the campaign (post-campaign 2017: 45% (135/300) scoring ≥ 6 vs 56.7% (170/300) scoring ≤ 4; post-campaign 2019: 43.8% (152/347) scoring ≥ 6 vs 29.7% (103/347) scoring ≤ 4).2X = 9.772, p = 0.02) and 7.3%  respectively from pre-campaign levels.2X = 5.753, p = 0.016). The proportion of individuals identifying that ‘Taking antibiotics when you don’t need them puts you and your family at risk of antibiotic resistant infections’ is true increased by 5.6% from post-2017 to post-2019 .At the start of the campaign, more specific knowledge on antibiotics was poorer, including knowledge regarding appropriate antibiotic usage and the reduction of antibiotic effectiveness when they are taken inappropriately. There was a significant difference in responses to the statement ‘Antibiotics will stop working for you if taken for the wrong things’ following the campaign. The proportion of individuals answering true increased by 8.5%  about antibiotic resistance for themselves increased by 11.2% from 39.1 (376/962) to 50.3%   pre- to post-campaign respectively .The campaign had some impact on parents’ awareness and understanding of AMR as the proportion of parents reporting some level of concern  regarding AMR for children increased by 6.0% from 53.7 (495/922) to 59.7   pre- to post-campaign respectively. However, the proportion of parents who agreed they would always take their doctor’s advice on whether their child needed antibiotics remained stable over the duration of the campaign.2X = 6.067, p = 0.014) . However, this reduced to 57.6% (76/132) following the final year of the campaign.There was a significant 5.1% increase in the proportion of individuals reporting they were unlikely to ask for antibiotics following the first year of the campaign (= 0.014) . However2X = 4.672, p = 0.031). Furthermore, a greater number of participants reported using non-urgent NHS services first, such as a walk-in centre, instead of seeing their GP when they fell ill (post-2018: 3% (19/655); post-2019: 5.3% (38/713), 2X = 5.019, p = 0.025), as per the answer to Question 049 in Supplementary Material 2.There were significant differences post-campaign in hot state actions . A greater number of participants reported that they did not expect to receive antibiotics from their GP when ill (post-2018: 12.7% (83/655); post-2019: 16.8% (120/713); Most GPs agreed that the campaign supported them to say no to patients asking for antibiotics and that the campaign will make patients less likely to ask for antibiotics. Furthermore, almost all GPs who recognised the campaign felt it helped raise awareness of AMR .2X = 4.000, p = 0.045), however, this difference was no longer evident following the final year of the campaign. In 2018, a higher proportion of GPs who had seen the campaign explained the reasons why antibiotics are inappropriate when they were asked to prescribe antibiotics inappropriately (90.4% (161/178) vs 75.2% (82/109), 2X = 9.217, p = 0.002), although this difference was also not evident post 2019  vs 92.4% (97/105), ost 2019 .Our main findings are firstly, that campaign recognition increased following the 3-year campaign with slight differences seen in levels of recognition between the general public and within key subgroups. Adults over 50 years had significantly lower levels of unprompted recognition following the 2018 and 2019 campaign years, although there was no difference in their level of prompted recognition. Reasons for this are unclear but may be due to employed methods of communication with social media used more extensively to disseminate information in 2018 and 2019, or differing levels of interest in the campaign affecting participant recall. The level of prompted recognition for the present campaign was high with TV being the most common source of recognition, supporting the use of mass media campaigns to disseminate information to the public. This level of recognition was higher than for previous national antibiotic awareness campaigns “The English public antibiotic campaigns” , and simSecondly, aspects of participant knowledge, awareness and understanding of AMR increased following the campaign. Perceived knowledge of both antibiotic and antimicrobial resistance increased post-campaign. Perceived knowledge of AMR was lower before the campaign but increased to a greater extent. The differences in knowledge are likely due to an unfamiliarity with the term ‘antimicrobial resistance’ as it is typically more commonly used by subject experts, with public campaigns predominately focusing on the role of antibiotics and bacterial resistance to this class of drug  as this Previous research has shown that messaging related to a specific disease, such as respiratory tract infections (RTIs), may be more effective at reducing inappropriate antibiotic usage than more generic messaging . DespiteA positive effect on reported behavioural intentions was seen following the campaign with respondents reporting being less likely to expect an antibiotic prescription and the proportion of participants reporting that they would go to a pharmacy or use 111, a non-emergency number for urgent healthcare need that is not a life-threatening situation, because of seeing the campaign increased. Reported likelihood to request antibiotics was also affected with a 5.1 and 9.6% increase in the proportion of individuals reporting they were unlikely to request antibiotics from their GP for themselves or their child following the first year of the campaign, respectively. However, the significant differences which occurred following the 2017 wave of the campaign returned to baseline following the final year of the campaign. This suggests that a longer campaign duration may not result in greater improvements in reported knowledge, attitudes, and behaviours. It is difficult to determine the cause of the lack of further change in knowledge and attitudes in 2018 and 2019. However, the 50% reduction in spend and the year-on-year reduction in campaign video views from 10.3 million in 2017 to 5.6 million in 2019 may have contributed to this. Furthermore, some campaign fatigue was present by the end of the third year of advertising which may have also influenced attention paid to campaign messages . Future Finally, in the first 2 years of the campaign, KAW supported change among GPs with GPs who were aware of the campaign reporting greater confidence to say no to most patients asking for antibiotics. GPs who were aware of the campaign were also more likely to explain that prescription of antibiotics for viral infections was inappropriate when asked for antibiotics by a patient. Almost all GPs felt the campaign was important and helped to raise awareness of AMR. Supporting GPs and providing resources that can be used to facilitate healthcare professional and patient interactions in combination with messaging focused on the public has been shown to result in significant reductions in antibiotic usage . HoweverOverall, the campaign evaluation showed the key aims of the campaign were met with several significant changes in knowledge, attitudes, concern about AMR, and intentions to alter behaviour which would improve appropriate antibiotic usage and reduce pressure on GPs to prescribe unnecessary antibiotics.This study presents a comprehensive evaluation of a national AMR awareness campaign which are often not published. Nevertheless, the study does have some limitations. Firstly, the differing samples interviewed before and after each campaign year introduces individual variability; weighting of the samples attempted to reduce this variability. The use of self-report measures introduces the potential for social desirability bias in responses to attitude and behaviour-based questions. The lack of baseline measure for some variables also makes it difficult to determine the level of effect the KAW campaign had on all aspects of participant knowledge, attitudes, and behaviour.Furthermore, a sample size calculation was not completed. However, the sample sizes were deemed to provide a sufficiently large base size to allow robust analysis both of the overall sample and of key subgroups, while considering cost.Finally, the observational nature of the study and the lack of measurable behaviour change resulting from viewing the campaign messages means causation is difficult to determine. Several measures of changes in behaviour have been used in previous campaign evaluations, including GP attendance for colds, uptake of influenza vaccination, and antimicrobial usage . One sucChanges in healthcare seeking behaviour have occurred as a result of COVID-19, which, in combination with pandemic restrictions, have resulted in a reduction in antibiotic-resistant bloodstream infections and antibiotic prescribing . TherefoChanges in the public’s understanding of infection prevention and hand hygiene following the COVID-19 pandemic, as well as its positive impact on attitudes towards vaccination presents an opportunity for AMR messaging and interventions ,33. FutuThis paper highlights the strength of a multimedia, integrated social marketing and communications campaign in reaching its target audience, increasing awareness, and supporting GPs, and demonstrates that KAW was an important component in tackling AMR. The COVID-19 pandemic has changed the health landscape since the KAW campaign was launched, and further research should be undertaken to understand current attitudes towards and use of antibiotics, and to determine measurable behaviour outcomes to inform and allow the success of future campaigns to be determined with more certainty.


Antibiotic resistance is rising and poses a serious threat to human health on a worldwide scale. It can make it more difficult to cure common infections, raise medical expenditures, and increase mortality. In order to combat the development of biofilms and treat fatal bacterial infections, multifunctional polymeric nanofibers or nanotextured materials with specific structural features and special physiochemical capabilities have become a crucial tool. Due to the increased antibiotic resistance of many diseases, nanofibers with antibacterial activity are essential. Electrospinning is a flexible process able to produce fine fibers with specified properties by modifying variables such as the concentration of the solution, the feed flow, and the electric voltage. Substantial advancements have been made regarding the formation of nanofibers or nanotextured materials for a variety of applications, along with the development of electrospinning techniques in recent years. Using well-defined antimicrobial nanoparticles, encapsulating traditional therapeutic agents, plant-based bioactive agents, and pure compounds in polymer nanofibers has resulted in outstanding antimicrobial activity and has aided in curing deadly microbial infections. A plethora of studies have revealed that electrospinning is an effective technique for the production of antimicrobial fibers for the environmental, biomedical, pharmaceutical, and food sectors. Nevertheless, numerous studies have also demonstrated that the surface characteristics of substrates, such as holes, fibers, and ridges at the nanoscale, have an impact on cell proliferation, adhesion, and orientation. Nanoparticles and nanostructured materials represent a dynamic field of research, owing to their influence across a wide range of application domains. Significant advancements have been made due to recent breakthroughs in the preparation of nanostructured materials and in vital knowledge regarding their characteristics. Bacterial antibiotic resistance, and the rise of multidrug-resistant (MDR) microbes or bugs, have created a universal problem in recent decades. The use of inadequate antibiotics to treat MDR bacteria has resulted in the development of unusual resistances that have astonishingly propagated from corner to corner via the atmosphere, individuals, and faunae. Materials with a nanofibrous morphology have the potential to be employed to address a variety of environmental problems, including wastewater treatment, water disinfection, and air purification. In many biological systems, fascinating hierarchical structures frequently develop. Various hierarchical structures, including zero-dimensional particles, one-dimensional nanofibers, two-dimensional substrates, and even bulks, are now exploited in biomedicine. These include nanofibers that can create 1–3 dimensional substrates, and have a variety of sizes (nanometers or micrometers) and rich morphologies . For the purpose of creating nanofibers, a number of techniques, including electrospinning, chemical vapor deposition, melt spinning, solution spinning, and sintering, have been developed. A quick and affordable technique able to create micronanometer continuous nanofibers using a variety of polymers or compounds is electrospinning technology. The generated electrospun nanofibers have a high porosity, large specific surface area, and compact size, making them useful in a variety of biological applications, such tissue engineering, regenerative medicine, and wound dressings. An overview of the electrospun nanofibers used in antibacterial materials, tissue engineering and antimicrobial wound dressings is presented here. Additionally, new advances in the production of antimicrobial nanofibers utilized for aforementioned purposes are scrupulously described ,3,4. BioElectrospun nanofiber scaffolds have recently been shown to be excellent nanoscale therapeutic devices, owing to their physicochemical characteristics; these can be adjusted and made suitable for a variety of applications requiring antibacterial qualities. The design requirements and characteristics of electrospun nanofibers that can be used to enhance their therapeutic activities are briefly highlighted herein. Nanofibrous structures have several inherent qualities that characterize their use as functional materials, particularly for antibacterial or antimicrobial purposes. For instance, a fiber’s structure should be able to biomimic the extracellular matrix (ECM) of tissue at the nanometric scale, fashioning a favorable atmosphere for the regeneration of the target region and enabling healing processes. They can affect cell migration, linkage, distinction, and renewal, in addition to having topographical fibers that resemble the original ECM architecture. In addition, the porosity of nanofibers, which permits high-surface and wetting absorptivity and, consequently, affects cell proliferation, vascularization, and mechanical firmness, has a significant impact on their performance. Furthermore, the fractal structure of nanofibers, with its linked nanopores, superior surface energy, reactivity, and elevated thermal and electric conductivities, might deter the development of cells and inhibit microbial penetration. Electrospun nanofibers have the potential to be effective as antimicrobial materials for all the aforementioned reasons .Microorganisms are naturally able to modify their structural features and structures to lessen the effectiveness of antibiotics. By impairing bacterial walls and snooping with their DNA, RNA, or vital proteins, antibiotics kill bacteria. However, by altering their gene and protein expressions, bacterial cells may also acclimatize to environmental provocations. As more strains of bacteria develop multidrug resistance, antibiotic resistance in bacteria will rise. Due to this phenomena, it is urgent that non-antibiotic alternatives to antibiotics, to be employed as alternate antimicrobial therapies for these very resistant microorganisms, are found. Nanostructured materials are attracting interest and recognition in biomedical usage as a result of the development of nanotechnology. In particular, electrospun polymeric nanofibers have distinctive physical and chemical characteristics, such as specific dimensions, silhouettes, and apparent chemistries, that affect their healing action. As a result, they are flexible enough to be readily customized for antimicrobial treatment ,9,10,11.The aim and purpose of this Special Issue, entitled “Prospects of Bioinspired and Biomimetic Materials”, was to summarize the current state of ongoing research in the area of nanostructured and nanofibrous materials, with a distinctive focus on their antibacterial , bacteriHere is a summary of the contributions submitted to this Special Issue. 2 and SnO2 composite nanofibers. An analysis using scanning and transmission electron microscopy revealed that CeO2 and SnO2 nanofibers have an average diameter of 170 nm. The CeO2–SnO2 composite performed more efficiently when the methylene blue dye was subjected to photocatalytic degradation. Escherichia coli was also used as a representative bacterium to screen the aforementioned nanofibers for antibacterial activity, and the CeO2 and SnO2 composite nanofibers exhibited outstanding activity. The results of this research offer new opportunities for the purification of chemical and biological pollutants in water and the use of electrode materials in energy storage devices, which would significantly aid environmental remediation procedures [In one study, a successful electrospinning approach was employed to construct CeOocedures .E. coli and S. aureus, which cause acute waterborne illnesses. The results of this investigation revealed that, compared to virgin hydroxyapatite, the integration of manganese ferrite greatly enhanced both antibacterial and photocatalytic effects [Several deadly illnesses in humans and animals are induced by environmental pollution, particularly water contamination from dyes, heavy metal ions, and biological infections. The materials and techniques required for water purification are costly. As a result, there is an urgent need for low-cost materials for wastewater treatment. Considering this, butchered cow bone debris from the Najran area of Saudi Arabia were gathered and subjected to a thermal-based synthesis method for the preparation of hydroxyapatite. A composite composed of manganese ferrite and hydroxyapatite was then synthesized. The nanocomposite was classified using a variety of complex techniques, including X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray (EDX), transmission electron microscopy (TEM), UV–Vis, photoluminescence (PL), and Fourier transform infrared (FT-IR). This composite was found to have an incomparable photodegradation efficiency for industrial waste pollutants. Additionally, this composite demonstrated outstanding bacteriostatic activity towards the microorganisms  effects .E. coli, which is responsible for major waterborne illnesses, was another noteworthy feature. It was interesting that adding cerium oxide and chitosan to hydroxyapatite significantly enhanced its antibacterial and adsorption characteristics over that of pristine hydroxyapatite. Additionally, a unique heterostructure developed out of recycled superfluous camel bones that helps to reduce water pollution has mostly been engendered by the dye industry [Similarly, the main aim of this study was to create a unique heterostructure from camel waste bones using an affordable and environmentally friendly approach in order to purify various waste liquids. Camel bones were used to create hydroxyapatite by employing a hydrothermal process. The produced hydroxyapatite was used to create a heterostructure of cerium oxide–hydroxyapatite functionalized with chitosan, a biopolymer. Because chitosan is a plentiful natural polysaccharide, it has several impressive qualities that make it an excellent adsorbent for eliminating colorants and other unwanted molecules from water. These qualities include accessibility, affordability, hydrophilicity, biocompatibility, and biodegradability. The remarkable bacteriostatic capacity of this heterostructure regarding industry .In another study, the effectiveness of micro- and nano-clay as a cheap substance for removing crystal violet (CV) dye from an aqueous solution was investigated. Thermodynamic observations demonstrated the exothermic, spontaneous nature of CV dye adsorption on both micro- and nano-clays. Because of this, the natural micro- and nano-clays were used potent and promising adsorbents for the elimination of CV dye from an aqueous solution .Dodonaea viscosa and Juniperus procera were examined in one study. Two fractions were discovered to have potential anticancer and antibacterial properties. Further, modifications were made to active fractions in order to generate ZnO composites, and these were examined using SEM, XRD, TGA, and EDX. Furthermore, these nanocomposites were revealed to be safe when evaluated for cytotoxicity against typical fibroblasts. The active fractions were also subjected to GC-MS analysis to identify potential phytochemicals that may be accountable for the aforementioned activities [Apart from environmental and water pollution, cancer and microbial infections pose great risks to health and are a prevalent cause of mortality globally. A key component of the drug discovery process is the synthesis of medicinal molecules from natural and indigenous resources. As a result, the anticancer and antibacterial properties of extracts and fractions of tivities .Similarly, in another research study, the objective of the work was to evaluate the biocompatibility of polyurethane (PU) nanocomposites grafted with polyhedral oligomeric silsesquioxane (POSS) and their potential application as materials for muscle tissue regeneration. POSS nanoparticles exhibit effective nucleation and significantly enhance the mechanical, thermal, and biocompatibility of the resulting composites. This study examined the antibacterial capability, anchoring, propagation, communication, and differentiation of C2C12 on PU and POSS-grafted PU. As a result of the higher free energy of the POSS molecules and their anti-inflammatory potential, the abovementioned nanocomposites exhibited improved cell adherence. Because these nanofibers were found to be safe, biomimetic scaffolds offer great potential regarding cellular research and muscle regeneration .Interestingly, a green redox chemistry procedure was used to create gold (Au), silver (Ag), and palladium (Pd) nanoparticles by reducing the metal salt precursor with glucose, while also utilizing polyvinylpyrrolidone (PVP) as a stabilizing and capping agent. For the cytoskeleton, actin and mitochondria, respectively, the quantitative analysis of Mitotracker Deep Red and Actin Green exhibits a tendency for disturbance that is consistent with the results of the wound scratch experiment. With regard to the untreated control cells, the Pd NP-treated cells showed the greatest change, followed by Au NP-treated cells; meanwhile, the Ag NP-treated cells showed the least disruptive change, despite being statistically significantly different from the control in the case of mitochondria .Furthermore, in another investigation, a variety of Retinin-like proteins and techniques for their metallization, employing nickel, silver, and copper ions to enhance the adaptability of the bionic nanocoatings, were applied. The optimum methods for creating secure and anti-infective metalized bionic nanocoatings were identified via the comparative analysis of the ensuing bactericidal, antiviral, and cytotoxic capabilities. The more extensive application of these standards to various public surfaces may represent a secure and affordable strategy via which to minimize the risk of dangerous illnesses .Equally, for the same reason, two distinct nanotechnological methods were employed to create water-soluble preparations of pyrazole derivative 3-(4-chlorophenyl)-5-(4-nitrophenylamino)-1H-pyrazole-4-carbonitrile (CR232), which was revealed to exert an in vitro antiproliferative effect on several cancer cell types. Most significantly, CR232 was first trapped in a biodegradable fifth-generation dendrimer encompassing lysine (G5K), which is significant since this was performed without utilizing hazardous chemical solvents or additives that might be dangerous to humans .In summary, the studies included in this Special Issue demonstrate both the existing and flourishing thoughtfulness regarding the design, preparation, characterization, and possible applications of functional nanofibers or nano-textured materials. These significant contributions to fundamental research will open up diverse avenues for growth and innovation, enhancing the application of these nanotextured materials and fibers in technological fields.


Microplastics (MPs) and nanoplastics (NPs) are hotspots for the exchange of antimicrobial resistance genes (ARGs) between different bacterial taxa in the environment. Propagation of antimicrobial resistance (AMR) is a global public health issue that needs special attention concerning horizontal gene transfer (HGT) under micro-nano plastics (MNPs) pressure. Interactions between MNPs and microbes, or mere persistence of MNPs in the environment (either water or soil), influence microbial gene expressions, affecting autochthonous microbiomes, their resistomes, and the overall ecosystem. The adsorption of a range of co-contaminants on MNPs leads to the increased interaction of pollutants with microbes resulting in changes in AMR, virulence, toxin production, etc. However, accurately estimating the extent of MNP infestation in agroecosystems remains challenging. The main limitation in estimating the level of MNPs contamination in agroecosystems, surface and subsurface waters, or sediments is the lack of standardized protocols for extraction of MPs and analytical detection methods from complex high organic content matrices. Nonetheless, recent advances in MPs detection from complex matrices with high organic matter content are highly promising. This review aims to provide an overview of relevant information available to date and summarize the already existing knowledge about the mechanisms of MNP-microbe interactions including the different factors with influence on HGT and AMR. In-depth knowledge of the enhanced ARGs propagation in the environment under the influence of MNPs could raise the needed awareness, about future consequences and emergence of multidrug-resistant bacteria. The term “microplastics” (MPs) was first coined by Thompson in 2004 to describe the small (less than 5 mm in size) plastic fragments in the oceans . Since tSeveral recent studies describe the interactions between MPs and microbes in soil and aqueous environments ,22,23. MtrbBp and traF), global regulatory genes , outer membrane genes (ompA and ompC), etc. Recent transcriptomics studies showed up-regulation of genes controlling ARG conjugation (kilA and traI), cell permeability (ompR), and intracellular ROS production (recA) upon exposure to aged MPs relative to pristine MP exposures, contributing to enhanced HGT [Microorganisms stimulate multiple physiological responses in the presence of MNPs, such as enhanced production of intracellular reactive oxygen species (ROS), increased membrane permeability, and enhanced expression of certain genes. Physical and environmental factors such as size of MNPs, UV-aging, and leachates from MNP degradation are reported for stimulations of such reactions in microbes ,31,32. Mnced HGT ,30,33. Anced HGT .Pseudomonas aeruginosa, are reported to show increased expression levels of various efflux pumps leading to the release of resistance genes into the environment and enhanced AMR [MPs existing in several different forms, such as fibers, films, foams, or pellets, may serve as effective abiotic surfaces for microbial colonization due to their small size but bigger surface area, surface texture, hydrophobic composition, and long persistence. Recent studies have detected different forms of plastics and measured more biomass formation on MP-biofilm than on rock surfaces in river/lake environments ,37,38. Pnced AMR . Metagennced AMR . The rolnced AMR . Encapsunced AMR .E. coli are reported to be highly dependent on the size of MPs [E. coli [Recent studies have highlighted the role of MNPs in enhancing the mobility and transfer of ARGs and metal resistance genes (MRGs) in the ecosystem, causing the enhanced spread of AMR. Any agent or situation promoting AMR is considered a threat to global public health. A growing body of scientific evidence correlates the elevation of AMR indicators  in the presence of persistent MNPs in an ecosystem. However, elaborated information on underlying mechanisms and experimental evidence on the interaction of MNPs with microbes causing the propagation of AMR is still naive. e of MPs . UV-aged[E. coli . MP/NPs [E. coli . MPs als[E. coli . These pThe reports on the interaction of MNPs and microbial communities to date strongly show that MPs could highly influence the microbial resistome or changes in the resistome of environmental niches. Several other reports provide evidence that changes in the resistome profile of different groups of microbes due to the presence of MNPs could further enhance the propagation of AMR genes in the environmental compartments as a whole ,52.Nitrosomonas decreased significantly, while the abundance of Nitrospira and other nitrifying bacteria showed an increase. In another study, microbial community composition and abundance of ARGs and MRGs on the plastisphere borne biofilm and planktonic bacteria in wastewater treatment plant (by qPCR) revealed a higher abundance of potentially pathogenic bacteria in treated wastewater on the plastisphere than in the planktonic bacterial community [Several studies have reported changes in resistome of wastewater sludge systems  was inhibited by different concentrations and different types of MPs . DiffereAs listed in The persistence of MNPs in an ecosystem can disrupt the delicate balance that exists between its various components. In agroecosystems, MNPs can significantly affect the soil microbial biomass, microbial activity, and functional diversity . MNPs caThough MPs are classified as emerging pollutants, they may not be hazardous per se to living beings in their native chemical compositions in a pristine microenvironment. However, their omnipresence in the environment is a matter of concern owing to their role as a vector for several hazardous agents, including heavy metals , rare eaThe currently available methods for separating and detecting MNPs from a complex matrix such as soil have several limitations, restricting them mainly to qualitative enumeration (for MPs) or estimation of total mass (for NPs). Scientists worldwide are actively engaged in developing standardized protocols for separating MPs from the soil matrix and their identification . TechniqMPs are heterogeneous in nature; they can be formed by different polymers and present with various densities, shapes, and structures. Additionally, they often contain additives and are found in different aging states. These properties of MPs make their extraction, detection and quantification in complex matrices challenging. The smaller size of NPs further prevents their sampling and efficient detection from any complex matrix by techniques commonly employed for MPs detection. Furthermore, the optical detection of NPs is diffraction limited, impeding their analysis below the size of 300 nm. Moreover, when compared with MPs, the smaller size of the NPs poses further restrictions in the availability of reference materials . Samplin2O3/Pt/TiO2 microrobots for NP detection. They can trap NPs via magnetic attraction with their stacks and enable on-site electrochemical detection and screening. The use of a fluorescent molecular rotor for NP detection in radish sprouts and mussel tissues has been performed, paving the way for new perspectives to overcome challenges in this field [In recent years, a number of new techniques, alone or in combination with existing techniques, are increasingly used for MNP analysis. Some of these techniques are specialized ATR-FTIR with hyperspectral imaging, GC-ToF with thermo-gravitimetric analysis , Laser dis field . Researcis field .MPs provide a suitable surface for attachment of microbes and the formation of biofilms. The slimy nature of biofilm plays a major role in the subsequent attachment and adsorption of many other pollutants and pathogenic microbes, thereby becoming a hub or hotspot for gene transfer. Such close interactions among microbes, and between microbes and pollutants, greatly influence the resistome, thereby enhancing the spread of AMR. Several other properties of MPs, particularly their chemical composition, surface charge, or size, play an important role in gene transfer efficiency between microbes. Moreover, strongly aged MPs had higher ARG abundance compared to weakly aged ones. In agricultural soil, the persistence of MPs and their interaction with soil microbes also influence soil nutrient balance and crop productivity. This review highlighted several factors that have been studied and reported, specifically focusing on the mechanism of horizontal dissemination of ARGs, changes in resistome profile, and the resultant impact on the agroecosystem. However, this area of research is only in its initial phase, and we strongly believe that several other important factors may be yet to be identified. The existing body of research on the impact of MPs in the ecosystem shows that MPs may enter the food chain in several ways and affect human health. However, current research is still in the stage of exploration. Furthermore, accurate quantification of MPs in complex matrices from environmental samples is impacted by the lack of appropriate techniques and standard protocols for extraction and detection of MNPs. In this review, we summarized the promising recent advancements in the extraction and detection of MNPs. Overall, a deeper insight into MNP contamination in agroecosystems and its impact on different related constituents needs continued and extensive research. It is also crucial that knowledge from these should be utilized to benefit humankind and protect human health by implementing proper control measures for plastic usage in agriculture.


Mikania micrantha is one of the worst invasive species globally and can cause significant negative impacts on agricultural and forestry economics, particularly in Asia and the Pacific region. The rust Puccinia spegazzinii has been used successfully as a biological control agent in several countries to help manage M. micrantha. However, the response mechanisms of M. micrantha to P. spegazzinii infection have never been studied. To investigate the response of M. micrantha to infection by P. spegazzinii, an integrated analysis of metabolomics and transcriptomics was performed. The levels of 74 metabolites, including organic acids, amino acids, and secondary metabolites in M. micrantha infected with P. spegazzinii, were significantly different compared to those in plants that were not infected. After P. spegazzinii infection, the expression of the TCA cycle gene was significantly induced to participate in energy biosynthesis and produce more ATP. The content of most amino acids, such as L-isoleucine, L-tryptophan and L-citrulline, increased. In addition, phytoalexins, such as maackiain, nobiletin, vasicin, arachidonic acid, and JA-Ile, accumulated in M. micrantha. A total of 4978 differentially expressed genes were identified in M. micrantha infected by P. spegazzinii. Many key genes of M. micrantha in the PTI (pattern-triggered immunity) and ETI (effector-triggered immunity) pathways showed significantly higher expression under P. spegazzinii infection. Through these reactions, M. micrantha is able to resist the infection of P. spegazzinii and maintain its growth. These results are helpful for us to understand the changes in metabolites and gene expression in M. micrantha after being infected by P. spegazzinii. Our results can provide a theoretical basis for weakening the defense response of M. micrantha to P. spegazzinii, and for P. spegazzinii as a long-term biological control agent of M. micrantha. Mikania micrantha Kunth (Asteraceae), commonly called ‘mile-a-minute’, is a rapidly growing vine native to tropical America [Mikania micrantha has been reported in seven provinces in southern China, with the main infestations in Guangdong, Guangxi, and Yunnan provinces [Mikania micrantha is also known as a plant killer because its rapid growth enables it to completely smother crops or trees and block sunlight, preventing flowering and fruiting [ America ,2. It is America ,2 and ha America ,4. Mikanrovinces . It afferovinces . Mikaniafruiting ,3.Puccinia spegazzinii de Toni , has been introduced into several countries, including China, to help control M. micrantha [Puccinia spegazzinii is a microcyclic and autoecious rust with a life cycle of 19–21 days [P. spegazzinii have only been recorded in the field [Puccinia spegazzinii is known to attack only M. micrantha and to a much lesser extent, Mikania cordata (Burm. F.) B. L. Robinson. Its potential as a biological control agent is because it is particularly damaging to the leaves, stems and petioles of M. micrantha, reducing growth rates and flowering, and can tolerate a wide range of environmental conditions in which M. micrantha grows [The rust fungus, icrantha . Puccini–21 days . Teliosphe field . Pucciniha grows ,9,10,11.Puccinia coronata f. sp. avenae W.P. Fraser and Ledingham (Pucciniaceae) [Rust fungi  are one of the largest groups of plant pathogens and the most damaging to plants in the world ,13. Infeniaceae) .Medicago truncatula Gaertn (Fabaceae) following infection by Phakopsora pachyrhizi Syd. and P. Syd. (Phakopsoraceae) [Other responses relate to the phenylpropanoid, flavonoid and isoflavonoid metabolic pathway genes, which are involved in the production of phytoalexins as seen in oraceae) . In addioraceae) ,18,19. Doraceae) , and sevoraceae) ,22,23,24M. micrantha to infection by P. spegazzinii have never been studied. In this study, we have characterized M. micrantha as a model host pathosystem for P. spegazzinii to investigate the molecular mechanisms of the host response to infection. Using transcriptome and metabolome analyses, we show how levels of essential amino acids, ATP, and phytoalexins in M. micrantha change in response to infection by P. spegazzinii. In addition, we aim to show how genes related to biotic stress in M. micrantha are expressed following P. spegazzinii infection. This work will help understand the potential for the continuous use of P. spegazzinii in assisting in the management of M. micrantha in many countries.The metabolic responses in Puccinia spegazzinii IMI 393075 was imported into China from Australia in December 2019, and a culture was maintained on potted M. micrantha plants at the Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences. Mikania micrantha plants were grown in a greenhouse with lighting (day: 16 h and night: 8 h) and a daily temperature range of 20–35 °C. For inoculation, plant tissue  with mature P. spegazzinii pustules, indicated by a coppery-brown appearance, was suspended over healthy potted plants in a sealed plastic chamber maintained at 22 °C, 100% humidity, for 48 h of darkness . Then, g at 4 °C for 10 min. The supernatant was diluted using LC-MS grade water, so the final concentration was 53% methanol. The samples were subsequently transferred to fresh Eppendorf tubes and were centrifuged at 15,000× g at 4 °C for 20 min. The supernatant was injected into the liquid chromatography–tandem mass spectrometry (LC-MS/MS) system analysis.Following freezing by liquid nitrogen, the samples of infected leaves and uninfected leaves were ground into powder. A subsample of 100 mg powder from each of the three samples of infected and uninfected leaves was weighed, and the homogenate was resuspended with prechilled 500 μL 80% methanol and 0.1% formic acid by vortexing it well. The samples were incubated on ice for 5 min and were then centrifuged at 15,000× LC-MS/MS analyses were performed using an ExionLC AD system (SCIEX) coupled with a QTRAP 6500+ mass spectrometer (SCIEX) in Novogene Co., Ltd. . The extracts were injected into a column  with a 20 min linear gradient at a 0.4 mL/min flow rate for the positive/negative polarity mode.In the positive ion mode, samples were injected onto a BEH C8 Column  using a 30-min linear gradient at a flow rate of 0.35 mL/min for the positive polarity mode. The eluents were eluent A (0.1% formic acid–water) and eluent B (0.1% formic acid–acetonitrile). The solvent gradient was set as follows: 5% B, 1 min; 5–100% B, 24.0 min; 100% B, 28.0 min; 100–5% B, 28.1 min; 5% B, 30 min. The QTRAP 6500+ mass spectrometer was operated in positive polarity mode with a curtain gas of 35 psi, collision gas set to medium, ion spray voltage of 5500 V, temperature of 500 °C, ion source gas of 1:55, and an ion source gas of 2:55.In the negative ion mode, samples were injected onto an HSS T3 Column (100 mm × 2.1 mm) using a 25-min linear gradient at a flow rate of 0.35 mL/min for the negative polarity mode. The eluents were eluent A (6.5 mM ammonium bicarbonate–water) and eluent B (6.5 mM ammonium bicarbonate−95% methanol water). The solvent gradient was set as follows: 2% B, 1 min; 2–100% B, 18.0 min; 100% B, 22.0 min; 100–5% B, 22.1 min; 5% B, 25 min. A QTRAP 6500+ mass spectrometer was operated in positive polarity mode with a curtain gas of 35 psi, collision gas set to medium, ion spray voltage of −4500 V, temperature of 500 °C, ion source gas of 1:55, and an ion source gas of 2:55.Based on the Novogene Database , multiple reaction monitoring (MRM) was used to detect the metabolites in each sample. The product ion (Q3) was used for metabolite quantification. The parent ion (Q1), Q3, retention time (RT), declustering potential (DP), and collision energy (CE) were used for metabolite identification. The data files generated by HPLC-MS/MS were processed using the SCIEX OS Version 1.4 to integrate and correct the peaks. The main parameters were set as follows: minimum peak height, 500; signal/noise ratio, 5; and Gaussian smooth width, 1. The area of each peak represents the relative content of the corresponding substance.http://www.genome.jp/kegg/ (accessed on 10 October 2020)), Human Metabolome Database (HMDB) (http://www.hmdb.ca/ (accessed on 10 October 2020)) and Lipid Maps database (http://www.lipidmaps.org/ (accessed on 10 October 2020)). Principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed with metaX (a flexible and comprehensive software for processing metabolomics data) [2Y, Q2Y) were obtained through 7-fold cross-validation . R2Y and Q2Y parameters were used to evaluate the performance of the model, both of which vary between 0 and 1, where 1 represents a perfect fit [2 and Q2 values. Their regression lines can be obtained from the R2 and Q2 values after 200 times of mixing and modeling. If the R2 value is greater than the Q2 value and the intercept between the Q2 regression line and Y-axis is less than 0, it can indicate that the model is not overfitting, and the model is more stable and reliable [These metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (cs data) . Partialcs data)  to estabfect fit ,28. In ofect fit  is to rareliable .p-value < 0.05 (two-tailed Student’s t-test), a VIP > 1, and a fold-change (FC) ≥ 2 or ≤0.5 were considered to be differentially accumulated metabolites. VIP refers to the variable projection importance of the first principal component of the PLS-DA model and represents the contribution of metabolites to the groupings. For clustering heat maps, the data were normalized using z-scores of the intensity areas of differential metabolites and were plotted by the heatmap package in the R language. Z-score (standard score) is a value converted based on the relative quantitative values of metabolites, which is used to measure the relative quantitative values of metabolites on the same level. The Z-score was calculated based on the mean and standard deviation of the reference data set (control group), and the specific formula was expressed as Z = (x − μ) / σ, where x is a specific fraction, μ is the mean, and σ is the standard deviation.Metabolites with a P. spegazzinii were downloaded from NCBI (SRR17139378–SRR17139383) and used to explore the differentially expressed genes (DEGs).Transcriptome data of leaves infected for 17 days (A) and uninfected leaves by M. micrantha (GCA_009363875.1) [2 fold change| > 1 and false discovery rate (FDR) < 0.05 were considered DEGs. The KEGG enrichment analysis of functional significance terms based on the Kyoto Encyclopedia of Genes and Genomes ) database was conducted using a hyper-geometric test to find significant KEGG terms in DEGs for comparison with the genome background. GO enrichment analysis of DEGs was performed using the online OmicShare tool (http://www.omicshare.com/tools/index.php/ (accessed on 22 September 2022)). Hypergeometric and FDR multiple tests were utilized to identify significantly enriched pathways. Gene ontology (GO) terms and KEGG pathways with FDR-corrected Q-values ≤ 0.05 were considered to be significantly enriched.To obtain localization information for the reads in the reference genome, clean reads were compared with the reference genome of 63875.1)  using HI63875.1) , and the63875.1) . Genes wg and 4 ℃ for 10 min. The supernatant was placed into a new Eppendorf tube, and 500 µL trichloromethane was added before being mixed thoroughly and centrifuged at 10,000× g and 4 ℃ for 3 min. The upper suspension of each sample was kept and incubated on ice for the subsequent detection of ATP content in infected and uninfected leaves using an ATP content detection kit .Following freezing by liquid nitrogen, the samples of infected leaves and uninfected leaves were ground into powder. A subsample of 100 mg powder from each of the three biological samples of infected and uninfected leaves was weighed, and 1 mL buffer solution was added and homogenized in an ice bath using an electric tissue grinder. The mixed extracts were then vortexed and centrifuged at 8000× M. micrantha leaves were detected using quasi-targeted metabolomic sequencing, and the diverse set of detected molecules could be roughly grouped into 49 classes, predominantly organic acids and derivatives, amino acids and their derivatives, nucleotides and their derivatives, lipids, phenylpropanoids, and flavones  shows the separation between healthy and infected leaves  and VIP ≥ 1. Metabolites (61 downregulated and 13 up-regulated) significantly changed upon P. spegazzinii exposure  and non-infected (control) group, a total of 4978 DEGs  were identified, including 2054 significantly up-regulated genes and 2924 significantly down-regulated genes  and effect-triggered immunity (ETI), both of which are accompanied by a series of induced defenses [P. spegazzinii stress , MAP kinase cascades, and WRKY transcription factors are considered to be essential components of all PTI-triggered defense responses [2+ is considered to be an important early event in the response signal transduction cascade of plant infection pathogens, and the cyclic nucleotide-gated channel (CNGC) located in the plasma membrane (PM) contributes to the increase in cytoplasmic Ca2+during pathogen perception [M. micrantha, four CNGC genes are significantly up-regulated, which indicates that they may mediate Ca2+ influx  and genes (60%) had lower levels following infection, suggesting that infection of P. spegazzinii has some negative impact on M. micrantha.Despite these positive changes in levels of metabolites and gene expression, this study also showed that Puccinia spegazzinii is currently being used as an effective biological control agent and has been released against M. micrantha in many countries [M. micrantha using P. spegazzinii is seen as a benefit to many landholders, as plants can reshoot after slashing, and herbicides are harmful to humans and the environment [P. spegazzinii has been shown to reduce growth rates and flowering of M. micrantha [M. micrantha have decreased, offering hope that P. spegazzinii will continue to be able to suppress populations of M. micrantha in the field.ountries ,11,58,59ironment . In bothicrantha . The resM. micrantha as a model host pathosystem for P. spegazzinii to investigate the molecular mechanisms of host responses to a pathogen. We have provided the first description genome-wide of the different gene expressions and metabolite levels in the leaves of M. micrantha following P. spegazzinii infection. Interactions and coevolution between M. micrantha and P. spegazzinii may influence the overall pathogenicity of rust. Over time, M. micrantha may evolve a mechanism to resist P. spegazzinii, and the plant could once again become a problem for countries that already have released rust. Alternatively, the rust may develop ways to overcome the plant’s defense responses or resistance. Further studies may provide more insight. The results here certainly provide a better understanding of the interactions between M. micrantha and P. spegazzinii and the potential of P. spegazzinii as a long-term biological control agent for M. micrantha.In this study, we characterized M. micrantha to P. spegazzinii based on the analysis of plant metabolites and gene expression. The relative levels of essential amino acid, ATP, and phytoalexins were synthesized and accumulated in M. micrantha following P. spegazzinii infection. In addition, many key genes of the MAPK signalling pathway showed significantly high expression under the P. spegazzinii infection.In this study, we revealed the response and interaction mechanism of


The refinement of omics methods over recent years has enabled significant advances in microbiology. The functioning of the environment is extremely diverse, complex, and challenging to decipher, and consequently requires advanced molecular tools to address key scientific questions that remain poorly documented. Environmental omics aim at a better understanding of the metabolic processes of a wide range of organisms and/or complex microbial communities to improve phenotype-genotype linkages, thus providing novel insights into the key molecular players in response to environmental changes and invaluable information on microbial communities. In this context, this Research Topic showcases the power of environmental omics to characterize novel catalysts for biotechnological applications. The range of applications is large and includes the bioremediation of pollutants, design of innovative biosensors, screening for novel catalysts and therapeutic drugs, and bioproduction of novel chemicals and materials. We have encouraged scientists using omics to study different aspects of environmental processes to contribute to this Research Topic. The knowledge generated with these studies could be complemented by others with the ultimate goal of properly characterizing processes to develop knowledge-based biotechnological applications.Abdelmoneim et al. proposed in this Research Topic to replace the classical 16S RNA gene amplicon analysis with a taxon-specific qPCR for increasing the throughput of soil surveys which could also be used as a diagnostic tool for soil health. This study is an excellent illustration of how omics will become valuable diagnostic tools over the next decade, as recently envisioned . In this way, previously unknown functions could be discovered and annotated. However, improving methodologies and reducing their costs are of utmost importance for their widespread use. In this regard, Nitrospira has enabled the development of nitrogen removal strategies in wastewater treatment plants  into an efficient industrial process. Approaches such as design-build-test-learn cycles are well suited for this purpose (Dvorák et al., The works presented in this Research Topic illustrate different facets of the great utility of omics approaches for the rational development of a large variety of biotechnological applications. That the continuous progress in these methodologies are becoming more affordable will allow us to explore and understand new environments and processes to develop and optimize novel biotechnological applications.AO-A conceptualized and drafted the editorial. RM-E, JA, and SM-S provided critical review and insight to improve the writing. All authors approved the final paper.