Salmonella. The release of colicin Ib (ColIb), which is used by Salmonella to gain an edge against competing microbiota following infection, is coupled to bacterial lysis mediated by temperate phages. Here, we show that phage-dependent lysis and subsequent release of colicin and phage particles occurs only in part of the ColIb-expressing Salmonella population. This phenotypic heterogeneity in lysis, which represents an essential step in the temperate phage life cycle, has evolved as a bet-hedging strategy under fluctuating environments such as the gastrointestinal tract. Our findings suggest that prophages can thereby evolutionarily stabilize costly toxin release in bacterial populations.Bacteria are excellent model organisms to study mechanisms of social evolution. The production of public goods, e.g., toxin release by cell lysis in clonal bacterial populations, is a frequently studied example of cooperative behavior. Here, we analyze evolutionary stabilization of toxin release by the enteric pathogen  Enterobacteriaceae to kill competitors in the gut. While group A colicins employ a division of labor strategy to liberate the toxin into the environment via colicin-specific lysis, group B colicin systems lack cognate lysis genes. In Salmonella enterica serovar Typhimurium (S. Tm), the group B colicin Ib (ColIb) is released by temperate phage-mediated bacteriolysis. Phage-mediated ColIb release promotes S. Tm fitness against competing Escherichia coli. It remained unclear how prophage-mediated lysis is realized in a clonal population of ColIb producers and if prophages contribute to evolutionary stability of toxin release in S. Tm. Here, we show that prophage-mediated lysis occurs in an S. Tm subpopulation only, thereby introducing phenotypic heterogeneity to the system. We established a mathematical model to study the dynamic interplay of S. Tm, ColIb, and a temperate phage in the presence of a competing species. Using this model, we studied long-term evolution of phage lysis rates in a fluctuating infection scenario. This revealed that phage lysis evolves as bet-hedging strategy that maximizes phage spread, regardless of whether colicin is present or not. We conclude that the ColIb system, lacking its own lysis gene, is making use of the evolutionary stable phage strategy to be released. Prophage lysis genes are highly prevalent in nontyphoidal Salmonella genomes. This suggests that the release of ColIb by temperate phages is widespread. In conclusion, our findings shed new light on the evolution and ecology of group B colicin systems.Colicins are toxins produced and released by  Enterobacteriaceae family  that differ in a number of characteristics, including operon structure, plasmid type, and mode of entry into the target cell. Most notably, group A colicin systems encode a specific lysis protein which is generally lacking in group B colicins. In group A colicin systems, colicin production is inevitably coupled to colicin release by cell lysis. Therefore, colicin expression is tightly regulated  and takeSalmonella enterica serovar Typhimurium (S. Tm) strain (S. Tm SL1344 harbors a conjugative plasmid encoding ColIb (cib), which is expressed in response to iron limitation and the cell’s SOS response  in competition against closely related species, such as E. coli.Group B colicins lack a cognate lysis protein, and their mode of release was unclear for a long time . We prev) strain . S. Tm Sresponse , conditiamed gut . We demo strains . Moreovete phage . During response , which eresponse . TherebySalmonella at the single-cell level? What is the frequency of ColIb and prophage lysis genes in human-pathogenic Salmonella strains? On the basis of the results of our previous experimental studies  in S. Tm is unimodally expressed in the population upon induction (cib) and prophages at the single-cell level using a set of fluorescent gene reporters , prophages are induced by the SOS response upon addition of the DNA-damaging drug mitomycin C. A gfp reporter plasmid (pPcib gfp), reporting ColIb production at the single-cell level (S. Tm SL1344 (S. TmWT) and prophage-cured (S. TmΔPh) cells . Prophage-deficient S. TmΔPh pPcib gfp exhibited substantially higher green fluorescent protein (GFP) mean fluorescence intensities (MFI) compared to S. TmWT under all investigated conditions, particularly in the presence of mitomycin C (S. TmWT pPcib gfp and S. TmΔPh pPcib gfp was ruled out by plotting the cell size (forward scatter [FSC]) against the MFI  cells  and S2. omycin C . A size-10.1128/mBio.00912-20.1FIG S1S. TmWT pPcib gfp compared to S. TmΔPh pPcib gfp are independent of cell size. Examples of dot-plots (forward scatter [FSC]/GFP) obtained from the experiment described in S. TmΔPh pPcib gfp (black) exhibits higher, but size-independent (FSC) GFP signal intensity compared to S. TmWT pPcib gfp (gray). Download FIG S1, PDF file, 1.3 MB.Differences in GFP signal intensity levels of Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00912-20.4TABLE S1Table S1, PDF file, 0.1 MB.Bacteria and plasmids used in this study. Download Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00912-20.5TABLE S2Table S2, PDF file, 0.04 MB.Primers used in this study. Download Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the gfp expression dynamics of individual S. TmWT pPcib gfp and prophage-cured S. TmΔPh pPcib gfp cells under ColIb/prophage-inducing conditions using time-lapse microscopy in a CellASIC ONIX microfluidic platform. S. Tm microcolonies were grown in microfluidics chambers under ColIb-inducing conditions. Notably, lysis of individual S. TmWT cells was observed already within 2 h after mitomycin C exposure, while a part of the cib-expressing population did not lyse until the end of the measurement , microcolonies of S. TmWTpPcib gfp were initially grown in LB for 3 h. Subsequently, the medium was exchanged against LB supplemented with DTPA (100μM) to induce cib (gfp) expression only. Twenty minutes later, LB medium containing DTPA (100 μM) and MitC (0.5 μg/ml) was applied to induce full expression of cib (gfp) and in addition prophage-mediated cell lysis for 20 min. Afterwards, the medium was changed back to LB supplemented with DTPA (100 μM) only for the remaining time. (A) Merged bright-field and GFP channel. (B) GFP channel only. Download Movie S1, AVI file, 6.8 MB.CellASIC live-cell microscopy of Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00912-20.9MOVIE S2S. TmΔPhpPcib gfp. Using the CellASIC ONIX microfluidic system (Millipore), microcolonies of S. TmΔPhpPcib gfp were initially grown in LB for 3 h. Subsequently, the medium was exchanged against LB supplemented with DTPA (100 μM) to induce cib (gfp) expression only. Twenty minutes later, LB medium containing DTPA (100μM) and MitC (0.5μg/ml) was applied for 20 min to induce full expression of cib (gfp) and to show the failure of cell lysis in the absence of prophages. Afterwards, the medium was changed back to LB supplemented with DTPA (100 μM) only for the remaining time. (A) Merged bright-field and GFP channel. (B) GFP channel only. Download Movie S2, AVI file, 6.5 MB.CellASIC live-cell microscopy of Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the cib expression. S. Tm prophage ST64BΦ is from S. Tm ST64BΦ is a lambdoid phage often found in S. Tm virulent strains and of major importance in ColIb release (S. TmlysST::T7 pol pPT7 rfpPcib gfp) was constructed to simultaneously monitor ColIb production and induction of ST64BΦ lysis genes on the single-cell level  cells also expressed the lysis genes  fusion on a plasmid. Therefore, ST64BΦ lysis gene induction is now monitored via sfgfp expression. Using this reporter strain and S. TmWT pPcib gfp (S. Tm population (20.13%), while cib is expressed unimodally  and ST64B prophage lysis genes (rfp) simultaneously. Download FIG S2, PDF file, 1.9 MB.Examples of live-cell microscopy showing ST64B prophage lysis gene, and Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00912-20.10MOVIE S3S. TmlysST::T7 polpPT7rfpPcib gfp. Using the CellASIC ONIX microfluidic system (Millipore), microcolonies of S. TmlysST::T7 polpPT7rfpPcib gfp were initially grown in LB for 3 h. Subsequently, the medium was exchanged against LB supplemented with DTPA (100 μM) to induce cib (gfp) expression only. Twenty minutes later, LB medium containing DTPA (100 μM) and MitC (0.5 μg/ml) was applied for 20 min to induce full expression of cib (gfp) and ST64B prophage lysis gene induction (rfp). Afterwards, the medium was changed back to LB supplemented with DTPA (100 μM) only for the remaining time. (A) Merged GFP and RFP channel. (B) GFP (cib expression) channel only; (C) RFP (ST64B prophage lysis gene expression) channel only. Download Movie S3, AVI file, 10.1 MB.CellASIC live-cell microscopy of Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. Tm fitness by imposing a metabolic burden. Moving to study the effect of ColIb production on S. Tm growth, we extended our strain tool set. For this, we used S. Tm strain ATCC 14028 (S. TmWT 028) as the basis because this strain originally does not carry a ColIb plasmid. From there, we generated a derivative carrying ST64BΦ only  and a prophage-deficient variant (S. TmWT 028ΔPh). All strains were transformed with pColIb  as a proxy for growth hourly. During 4 h of growth, the most prominent differences between WT and phage-deficient S. Tm strains appeared under SOS-inducing conditions , the strain containing only the phage ST64B  and the prophage-free strain (S.TmWT 028 ΔPh). Strains carried either no plasmid, plasmid pColIb (p2) or were again cured of the plasmid (p2cured). Growth rates were determined from changes in OD600 during growth experiments in 96-well plates. (A) As an example, growth of all strains over 4 h in LB (0.5 μg/ml MitC) is shown. (B to E) From growth curves, the change in biomass per hour was calculated as the slope of the linear regression of OD600 values before (hours 0 to 2) and after (hours 2 to 4) lysis induction. (B) LB medium, (C) iron-limiting conditions (100 μM DTPA), (D) SOS-inducing stress alone (0.5 μg/ml MitC) or in (E) combination with iron limitation (MitC/DTPA) are shown. Download FIG S3, PDF file, 0.2 MB.Net growth rates of Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. Tm. Even though ColIb increases the competitiveness of an S. Tm population  (S. Tm (s), a plasmid encoding the colicin Ib (cib) and colicin Ib immunity (imm) genes, a temperate S. Tm phage, and a competing species (e), as, e.g., commensal E. coli (S. Tm (s0) to depict subsequent infections. The considered time scale of the interaction dynamics is ecological. Therefore, only S. Tm infection dynamics are addressed, while emerging S. Tm mutants are not considered. Even though S. Tm (s) may cause infections of different mammalian hosts in nature, pathogen host-to-host transition was not explicitly considered in the scale of this model.In order to study the dynamic interdependencies in this tripartite system of  E. coli . The mam10.1128/mBio.00912-20.6TABLE S3Table S3, PDF file, 0.1 MB.Parameter values used. Download Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00912-20.11TEXT S1Text S1, PDF file, 0.2 MB.Additional information and equations for the modeling. Download Copyright © 2020 Spriewald et al.2020Spriewald et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. Tm population in particular during inflammation-induced Enterobacteriaceae blooms, where S. Tm competes against close relatives  of all bacterial species. Furthermore, inflammation provides environmental cues , which trigger ColIb production and prophage-mediated cell lysis and induce expression of the ColIb receptor (cirA) on competing E. coli .ColIb can increase fitness of the elatives . During ironment . This ef E. coli , 23. ThuS. Tm host lysis, respectively (S. Tm harboring the colicin plasmid (s row) are immune to the colicin, by expression of the cognate immunity protein. However, they do not produce colicin, unless there is an environmental trigger, as induction by SOS stress (c). The temperate phage is integrated as prophage in the S. Tm genome (S. Tm (c), followed by lysis-mediated release of both colicin and free infectious phages (lysis rate δ). Upon infection of new hosts, the phage lyses new host cells or integrates into the bacterial genome with the probability ρ. Using parameter values determined from a combination of experimental data, literature research, and estimations  , s row aimations , the resin silico competition experiments between players using prototypical, fixed opposing strategies. Thus, evolutionary stability of colicin release via phage-mediated lysis can be studied by investigating the competition of a wild-type strain with a mutant strain that does not lyse at all  Upon a new infection by phages, the majority of the S. Tm population lyses, and a certain fraction becomes lysogenic. Therefore, the dynamics of phage-mediated lysis are determined by two parameters, the lysis rate δ of lysogenic S. Tm and the probability ρ to become lysogenic. To investigate evolution of phage-mediated lysis in our ecological model, mutant phages with altered lysis rates δm and ρm were introduced at very small numbers, appearing as free infectious phages or as prophages in S. Tm. Using the resulting altered set of model equations, the Lyapunov exponent (rate of separation) was determined numerically for every adaptation step. If the Lyapunov exponent was positive, the mutant is able to spread; if it was negative, the mutant dies out. The resulting dynamics of the trait (given by the parameters δ and ρ) are presented as pairwise invasibility plots (PIP)  Under stress conditions, prophage-mediated lysis of the lysogenic ts (PIP) .Enterobacteriaceae blooms  would influence the evolutionary fate of lysis. First, it was assumed that the SOS signal reliably indicates Enterobacteriaceae blooms, induced by inflammation  besides an inflammatory response could trigger the bacterial SOS response. Therefore, the SOS signal may not reliably indicate an S signal . Here, and ρ > 0 . The resS. Tm temperate phage-dependent lysis is not optimized to release the colicin but to maximize the spread of the phages. To investigate whether colicin release is required to convey evolutionary stability in the phage lysis system, we implemented an alternative scenario without colicin release and without competitor. Notably, a positive lysis rate (δstress > 0) was still found to reach an ESS  database, which we analyzed for the presence of colicin and lysis gene families . Thus, only 303 genomes lack lysis genes at all. In total, 1,213 S. enterica genomes (12.8%) show matches to colicin HMM profiles  seem to harbor colicin genes (group A and B) more often  than lysis-negative genomes , but this difference is not significant . Based on profile HMM matches, group B colicins are more abundant in S. enterica genomes  compared to group A colicins . We asked whether lysis-positive genomes  harbor group B colicins more often than lysis-negative genomes (n = 303). In the subset of lysis-positive genomes, 9.0% (n = 830) harbor a colicin from group B, while this is only the case for 6.6% (n = 20) of lysis-negative genomes. Group A colicins are present in 3.8% (n = 353) of lysis-positive genomes and 3.3% (n = 10) of lysis-negative genomes. We conclude that B colicins are not more prevalent in Salmonella enterica genomes that do contain lysis genes .Prophages are widely distributed in the genus lmonella . We alsoprofiles . InteresSalmonella enterica genomes by serovar to check for serovar-specific differences in colicin and lysis gene abundances , while colicin group B is found in 11.8% (n = 128) of genomes. In serovar Typhimurium, lysis-associated gene families seem to be omnipresent . This hindered us from analyzing whether group B colicins are also more frequently associated with prophages than group A. However, it suggests that due to high prevalence of prophages in serovar Typhimurium genomes, this serovar may generally be able to release and benefit from group B colicins.Finally, we focused our analysis on serovar Typhimurium, our experimental model system. Notably, only a small set of the 1,088 genomes harbor group A colicins  phage, another lambdoid phage, even higher lysis frequencies of up to 60% of the E. coli population were reported, which varied by growth state of the host bacterium .Using an evolutionary invasion analysis, er cells , sporulaer cells , or seeder cells . The lyter cells , 41. Froer cells . Under tbacteria . The sambacteria . Apart fbacteria , lysogenMaintaining evolutionary stability of cooperation by the release of public goods is a challenge for bacterial populations with phenotypic heterogeneity being an evolvable trait that can be shaped by natural selection. Theoretical and experimental analyses show that different mechanisms can maintain cooperative behaviors in microbial populations. These mechanisms include spatial proximity and local competition , pleiotrBesides group B colicins, a number of other substances, including DNA and host-directed toxins are known to be released by temperate phage-mediated lysis , 50–52. 50–S. Tm-ColIb-phage system forms an ESS in unstructured environments.Another well-known strategy that evolutionarily stabilizes colicin production is physical association of the cooperators and limited diffusion of the public good in a spatially structured environment , 53. ThiS. Tm fitness, at least within short periods of time , in particular in serovar Typhimurium   . This hiThe fact that the lysis is triggered by the phage and not the bacteria itself opens up many interesting questions. From the bacterium’s perspective, colicin release is clearly altruistic, while from the phagés perspective, lysis is an essential step of the replicative cycle and therefore a selfish act. Therefore, the question arises of whether bacterial altruism is not voluntary but enforced by the phage. There are examples of enforced altruism in insect societies . AlternaEnterobacteriaceae blooms. Interestingly, we find that group B colicins were far more prevalent than group A colicins in NTS. Since NTS are well adapted to growth in inflammation-induced blooms . For this purpose, plasmid pJLG2 was used as the template for the amplification of a T7 gene1 (T7 polymerase gene). To generate S. TmlysST::T7 pol (SJB34), oligonucleotides T7_ST64B_fwd/ST64B-sfgfp-rev (T7 gene1 (T7 polymerase gene), including a FRT site-flanked kanamycin cassette, from the template plasmid pJLG2. Correct exchange of the ST64B prophage lysis genes (SL1344_1955 to SL1344_1957) against the reporter gene T7 gene1 in SJB33 was verified by PCR and sequencing using the oligonucleotides Check_ST64_for/T7seq left 2/T7 seq right 2/Check_ST64_rev . The functionality of this reporter was controlled by the transformation of pJLG1 (carrying a T7 promoter sfgfp fusion) into S. TmlysST::T7 pol, following flow cytometry analysis.Bacterial strains and plasmids used in this study are listed in fgfp-rev  were useST64_rev . The allgfpmut3b. Generation of pPT7sfgfp (pJLG1) was achieved by amplification of the kanamycin resistance cassette-flanked sfgfp gene (encoded on pWRG7) using PCR and the oligonucleotides pJLG1 SFGFP SD BamHI Fw/pJLG1 SFGFP EcoRI Rev (T7 gene1), genomic DNA (gDNA) was extracted from E. coli BL21(DE3). Next, the T7 polymerase gene T7 gene1 was amplified by PCR using the oligonucleotides T7 pol FW NotI/T7 pol Rev XhoI , a PCR was conducted with oligonucleotides TagRFP-T_Fwd2/TagRFP-T_Rev . A culturing step in LB medium without selective antibiotics then ensured the loss of pM1451. Resulting strains were all confirmed by PCR to be cured of p2.To transform strains with the colicinogenic plasmid pColIb-P9 (p2), genomic DNA was isolated from eviously . Briefly600) of 0.5 and used to inoculate subculture I (ratio 1:20) in test tubes containing 3 ml LB (supplemented with respective antibiotics). Following an incubation time of 2 h at 37°C in a rotor wheel, subculture I was also normalized to an OD600 of 0.5. Normalized subculture I was used to inoculate subculture II. This was done in a 96-well plate containing LB medium only or LB supplemented with either mitomycin C (MitC)  or diethylenetriaminepentaacetic acid (DTPA)  or both in a ratio 1:1. Incubation of subcultures II was carried out for 4 h at 37°C and 180 rpm.In general, bacterial strains were at first cultured in test tubes , containing 3 ml LB medium (supplemented with respective antibiotics) for 12 h at 37°C in a rotor wheel. This overnight (o.n.) culture was normalized to an optical density at 600 nm  and incubated for 12 h at 37°C in a rotor wheel. This o.n. culture was used to inoculate 2 ml LB medium (ratio 1:20). The subculture was incubated for 2 h in a rotor wheel at 37°C. Next, a bacterial sample of this subculture was taken and diluted in 1× phosphate-buffered saline (PBS) to achieve the concentration of 106 CFU. Fluorescence-activated cell sorting (FACS) data were recorded by FACS CantoII running the FACSDiva software (Aria Becton Dickinson). The FlowJo software 8.8.4  was used for data analysis.Bacteria were grown in 96-well plates as described before and diluted in 1× PBS to a concentration of 102, 5 min). Next, the vacuum was removed, and wells 1 to 5 were filled with the test medium (300 μl/well): LB only, LB supplemented with 100 μM DTPA or LB supplemented with 0.5 μg/ml MitC and 100 μM DTPA. Finally, 100 μl of the cell suspension (106 cells/ml) was loaded in the cell inlet well 8. The manifold was attached to the plate, and the plate was vacuum sealed and placed onto a spinning disk confocal microscope  with Perkin Elmer UltraVIEW spinning disk system and Hamatsu Orca ER charge-coupled-device (CCD) or Orca Flash 4.0 complementary metal oxide semiconductor (CMOS) camera. The microscope was surrounded by a climate chamber with a temperature of 37°C. Bacterial cells were loaded (4 lb/in2 for 6 s) into the system followed by an additional priming step with LB medium only  to wash out untrapped bacteria. The CellASIC ONIX FG software was used to set up a protocol for the medium flow  was primed with LB medium. This was achieved by exchanging the buffer of the medium/buffer reservoir wells 1 to 5 against LB medium. The cell outlet well 6 and the waste well 7 were emptied. A drop of 50 μl of 1× PBS was placed in the bottom of well 7. The manifold was attached to the plate and by the CellASIC ONIX microfluidic perfusion system (model EV262); the plate was vacuum sealed and primed with LB medium of wells 1 to 5 simultaneously  test followed by the Kruskal-Wallis test with Dunn’s multiple-comparison test or the two-way ANOVA with Bonferroni’s posttest was used. −8). After a second burn-in phase with the mutant (2.75 periods), the growth of the mutant was measured for 2 periods. For some arbitrary parameter sets, it was checked that mutants with a positive Lyapunov coefficient took over the population as required by adaptive dynamics. The data were analyzed with R  using the search term “Salmonella enterica” and filter for “Prokaryotes” (not filtered for assembly level) and used for the quantification of colicin and lysis gene families. For this analysis, profile hidden Markov models (profile HMMs) (https://www.uniprot.org/uniref/). Amino acid sequences for colicin gene families were aligned using Clustal Omega   were genle HMMs)  (colicinle HMMs)  (lysis).al Omega  employinal Omega . We spot


In vitro cytotoxicity studies indicated that treatment with dopamine Lf-BNPs has relatively low cytotoxicity in SH-SY5Y and 16HBE cells. Qualitative and quantitative cellular uptake experiments indicated that Lf modification of NPs increased cellular uptake of SH-SY5Y cells and 16HBE cells, and borneol modification can promote the cellular uptake of 16HBE. In vivo pharmacokinetic studies indicated that AUC0–12 h in the rat brain for dopamine Lf-BNPs was significantly higher (p < .05) than that of dopamine nanoparticles. Intranasal administration of dopamine Lf-BNPs effectively alleviated the 6-hydroxydopamine-induced striatum lesion in rats as indicated by the contralateral rotation behavior test and results for striatal monoamine neurotransmitter content detection. Taken together, intranasal administration of dopamine Lf-BNPs may be an effective drug delivery system for Parkinson’s disease.Efficient delivery of brain-targeted drugs is highly important for successful therapy in Parkinson’s disease (PD). This study was designed to formulate borneol and lactoferrin co-modified nanoparticles (Lf-BNPs) encapsulated dopamine as a novel drug delivery system to achieve maximum therapeutic efficacy and reduce side effects for PD. Dopamine Lf-BNPs were prepared using the double emulsion solvent evaporation method and evaluated for physicochemical and pharmaceutical properties.  Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder characterized by deterioration of midbrain nigrostriatal dopaminergic neurons  encapsulated dopamine via nose-to-brain delivery to enhance therapeutic efficacy and reduce adverse effects. NPs can increase the stability and bioavailability of dopamine, allowing sustained release of dopamine, and avoid its peripheral metabolism. NPs, owing to their small size, are compatible with various administration routes including intranasal administration. An intranasal nose-to-brain delivery strategy may offer several advantages. Intranasal delivery systems are more efficient in noninvasively delivering drugs to the brain via the olfactory and trigeminal nerves from the nasal olfactory epithelium by circumventing the blood–brain barrier –poly(lactic-co-glycolic acid) (PEG-PLGA) copolymers have received FDA approval for their applications in drug delivery because of its biodegradability, biocompatibility, and versatility. PEG-PLGA nanoparticles were successfully prepared and it is found that PEG-PLGA nanoparticles accelerated blood clearance  encapsulated dopamine, with enhanced permeability and striatum-specificity, and evaluated the improvement in PD treatment efficacy following intranasal administration. Cytotoxicity and cellular uptake of the formulations were evaluated by SH-SY5Y and 16HBE cells in vitro, and pharmacokinetic and pharmacodynamic studies of the prepared formulations were also performed.Borneol was supplied from Yongye Biotechnology Co., Ltd. . Dopamine hydrochloride was a gift from Shanghai Keshun Biotechnology Co., Ltd. . 2-iminothiolane (Traut’s reagent), bovine Lf, 3--2,5-diphenyltetrazolium bromide (MTT), apomorphine, ascorbic acid, and 6-hydroxydopamine (6-OHDA) were purchased from Sigma-Aldrich . PEG-PLGA-maleimide  and methoxyPEG-PLGA  were purchased from Polyscitech . All other chemicals and reagents were of analytical reagent grade.Sprague-Dawley rats  were provided by the Experimental Animal Center of Luye Pharmaceutical Group and housed in a room kept at 22 ± 1 °C under standard 12 h light/dark cycles. All animal studies were performed according to the Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Yantai University.Dopamine-loaded BNPs were prepared using the double emulsion solvent evaporation method . The shape and surface morphology of Lf-BNPs were studied by transmission electron microscopy (TEM)  following staining with a 2% (w/v) sodium phosphotungstate solution.Entrapment efficiency was determined by high-performance liquid chromatography (HPLC) with a C18 reverse-phase column. The mobile phase was consisted of 2% methanol (v/v) and 98% PBS , and the effluent was monitored at 280 nm. Briefly, total drug content (free + entrapped) of formulations was studied by dissolving the formulation in acetic acid and free drug content was separated by centrifugation. The entrapment efficiency was calculated using the following equation:Dopamine release from the formulations was determined using the dialysis method  or the different NP formulations containing equivalent dopamine content for 24 h. Then, MTT (5 mg/mL) was added to per well and cells were incubated for 4 h. Dimethyl sulfoxide (200 μL) was then added to dissolve the formazan crystals, and the absorbance was measured at 570 nm on a Cytation 5 imaging reader .The cytotoxicity of free drugs and prepared formulations was evaluated by the MTT assay method. In brief, SH-SY5Y and 16HBE cells were seeded at a density of 10Qualitative analyses of cellular internalization of Nile red-loaded NPs, Lf-NPs and Lf-BNPs were performed by fluorescence microscopy , while quantitative analyses of coumarin-6-loaded NPs were performed by flow cytometry .4 cells/well and cultured for 24 h at 37 °C in a 5% CO2 environment. After incubation for 24 h, the cells were incubated with Nile red-loaded NPs (1 μg/mL) at different times. Subsequently, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 10 min. Finally, the cells were visualized by fluorescence microscopy.For fluorescence microscopy, SH-SY5Y and 16HBE cells were seeded in a 24-well plate at a density of 5 × 105 cells/well and treated as described earlier. After incubation with the same concentrations (6 ng/mL) of coumarin-6-loaded NPs, Lf-NPs or Lf-BNPs for 15 min, 1 h, and 2 h, the cells were washed 3 times with PBS. Then the cells were trypsinized and centrifuged at 1500 rpm for 5 min. After resuspension in PBS, the cells were analyzed by flow cytometry.For flow cytometry, SH-SY5Y and 16HBE cells were seeded in a 6-well plate at a density of 5 × 10For the pharmacokinetic study, Sprague-Dawley rats were divided into three groups and intranasally administered dopamine-loaded NPs (group I), Lf-NPs (group II), and Lf-BNPs (group III). Blood samples were collected for analysis by retro-orbital puncture at 0.25, 0.5, 1, 2, 4, 8, and 12 h post administration, and plasma was obtained after centrifugation. Then, the rats were sacrificed humanely at predetermined time points, and the brains were harvested and homogenized.m/z 154/137 for dopamine. The pharmacokinetic parameters were calculated by DAS v2.0 software .Dopamine concentrations in the plasma and brain were measured by HPLC – tandem mass spectrometry . The chromatographic separation was carried out on a C18 column under the following conditions: acetonitrile: 0.1% formic acid  at a flow rate of 0.8 mL/min; sample injection volume, 3 μL; column temperature, 35 °C. Mass spectrometer was operated in the positive ion mode by multiple reaction monitoring of the transition of Sprague-Dawley rats were examined one day before experiment to confirm that there was no rotation behavior. For the stereotaxic surgery, rats were deeply anesthetized by chloral hydrate (350 mg/kg) and then placed in a NEUROSTAR stereotaxic apparatus  to achieve a flat skull position. A small hole was drilled at the following coordinates: lateral (L) 1.65 mm and antero-posterior (AP) 0.4 mm, from the bregma point. 6-OHDA solution (4 mg/mL in 1% ascorbic acid) was injected unilaterally into the striatal at 0.5 μL/min. Three weeks after the surgery, contralateral rotations were recorded after injection of apomorphine hydrochloride (2 mg/kg). Rats exhibiting rotations of more than 75 rotations per 15 min are considered to have developed PD  and homovanillic acid (HVA)). Data are expressed as ng/g tissue weight.p < .05 and p < .01, respectively.All data are expressed as mean ± standard derivation (SD). Statistical analysis was performed by one-way ANOVA using SPSS 22.0 software . Statistically significant and highly significant differences are defined as Figure S1). The TEM images showed that the NPs were spherical and had a smooth surface. It may have led to a smaller diameter compared with DLS, because the NPs were dehydrated and shrank during TEM preparation. The mean diameters of formulations were not more than 200 nm, which indicated that the prepared formulations are suitable for intranasal administration . Free dopamine was rapidly released within 4 h (91.24 ± 4.03%). In contrast, the release profiles all exhibited the sustained release characteristics of dopamine from NPs. The cumulative release percentage of dopamine from these formulations can be up 70% in 72 h. NPs and Lf-NPs showed higher release rates than Lf-BNPs, and there were no significant differences when compared with the borneol modification of NPs.The release kinetics was studied for 5 days in PBS (pH 7.4) at 37 °C. The cumulative release percentage of dopamine from dopamine solution, dopamine-loaded NPs, Lf-NPs, and Lf-BNPs at many time points was shown in Supporting Information . The Lf receptor is overexpressed on the apical surface of respiratory epithelial cells and in the capillaries and neurons related to neurodegenerative diseases, thus Lf modification of NPs increasing cellular uptake of SH-SY5Y cells and 16HBE cells. There was no obviously difference between Lf-BNPs and Lf-NPs for SH-SY5Y cells, while the mean fluorescence of Lf-BNPs was significantly higher than that of Lf-NPs (p < .05) for 16HBE cells. These results demonstrated that borneol modification can promote the cellular uptake of 16HBE.Cellular uptake of NPs in SH-SY5Y and 16HBE cells was qualitatively evaluated using fluorescence microscopy  and quanPlasma and brain dopamine levels post intranasal administration of dopamine-loaded NPs, Lf-NPs, and Lf-BNPs at various pre-determined intervals up to 12 h in rats are shown in The plasma concentration-time curves of NP, Lf-NP, and Lf-BNP groups were similar , indicattmax of 1, 1, and 0.5 h, respectively, and the peak plasma concentrations of dopamine in the brain post intranasal administration of dopamine-loaded NPs, Lf-NPs, and Lf-BNPs were 149.3 ± 21.7, 200.4 ± 13.7, and 219.4 ± 13.3 ng/g with tmax of 1, 1, and 0.25 h, respectively. The lower tmax values for the brain (0.25 h) than that for blood (0.5 h) may also be attributed to the preferential nose-to-brain delivery post-nose-to-brain delivery of dopamine Lf-BNPs. The AUC0–12 h (1696.4 ± 119.3 ng.h/g) of Lf-BNPs was higher than that of NPs and Lf-NPs. The intranasal delivery of dopamine Lf-NPs promoted dopamine concentration in the brain compared with the equivalent dose of NPs, and intranasal delivery of Lf-BNPs enhanced the delivery of dopamine to the brain compared with NPs and Lf-NPs.The peak plasma concentrations of dopamine in blood post intranasal administration of dopamine-loaded NPs, Lf-NPs, and Lf-BNPs were 160.2 ± 16.5, 168.3 ± 17.5, and 175.3 ± 16.8 ng/mL with The results demonstrated that Lf and borneol co-modified NPs significantly increased dopamine delivery to the brain via the intranasal route. In the previous report, drug uptake from nose to the brain mainly occurs via three different pathways: a systemic pathway of absorption and subsequently into the brain by crossing the blood-brain barrier, and a direct pathway from nasal mucosa epithelium into the brain mainly along the trigeminal or olfactory nerves that bypass the blood-brain barrier  compared with those in the PBS group on day 10. Moreover, the apomorphine-induced rotations in the Lf-BNP group were statistically significant difference from the Lf-NP group on day 20 (p < .05).The results of the contralateral rotation behavior test are shown in In order to evaluate the change in the content of dopamine in the striatum, an HPLC-MS/MS method was used to detect dopamine and its metabolites in the striatum. After day 20, the levels of dopamine, DOPAC and HVA in the PBS group were significantly lower than that in the normal group, while post-treatment with dopamine Lf-BNPs, the contents of dopamine, DOPAC, and HVA were significantly higher in comparison with the PBS group . These rOur finding revealed that Lf-NPs and Lf-BNPs can deliver effective doses of dopamine into the lesioned striatum after intranasal administration. Moreover, our results indicated that the therapeutic efficacy of dopamine Lf-NPs was better than that of dopamine NPs, and the therapeutic efficacy of dopamine Lf-BNPs was greater than that of dopamine Lf-NPs, indicating that modification with borneol and Lf can deliver more drugs into the striatum to achieve ideal therapeutic efficacy.In the present study, we proposed borneol and Lf co-modified nanoparticles encapsulated drug to be an effective nose-to-brain drug delivery system for enhancing dopamine delivery into the brain for the treatment of PD. Cell viability evaluation results showed the relatively low toxicity of NP formulations. Qualitative and quantitative cellular uptake experiments demonstrated that Lf modification of NPs increased cellular uptake of SH-SY5Y cells and 16HBE cells, and borneol modification can promote the cellular uptake of 16HBE. Pharmacokinetic studies showed significantly increased absorption of dopamine Lf-BNPs into the brain after nose-to-brain delivery than that of dopamine NPs, which demonstrated that borneol and Lf co-modification could together enhance drug transport to the brain via intranasal administration. Pharmacodynamic study indicated that 6-OHDA-induced PD rats treated with dopamine Lf-BNPs showed decreased apomorphine-induced contralateral rotations and increased dopamine content in the lesioned striatum. In conclusion, the results demonstrated that the drug delivery system in this study could provide an effective noninvasive approach to encourage the access of dopamine to the brain for treatment of PD.


C. elegans vulva is formed from divisions of three vulval precursor cells (VPCs) – P5.p, P6.p, and P7.p – arranged along the anteroposterior axis in the ventral epithelium . Previous analyses show the orientation of P5.p and P7.p descendants is determined by the interaction of multiple Wnt signals. egl-20/Wnt is expressed in the tail  and forms a posterior-to-anterior concentration gradient . It has previously been shown that EGL-20 acts instructively during vulva development by imparting directional information, as opposed to being permissive, where it would only be required for polarization . By moving the source of egl-20 expression from the posterior of the worm to the anchor cell, the axis of symmetry of the developing vulva, we can reorient the daughter cells of P5.p and P7.p toward the center in a wild-type configuration.The egl-20 from the center of the axis of symmetry partially suppresses the lin-17(n671) phenotype . To test whether LRP-2 acts downstream of EGL-20, we ectopically expressed egl-20 from the anchor cell in a lin-17(n671); lrp-2(gk272) double mutant background and compared it to a lin-17(n671); lrp-2(+) strain. If LRP-2 acts downstream of EGL-20, then anteriorly-expressed EGL-20 will not be able to suppress the lin-17 phenotype, with is the result observed (Table 1). Thus, like CAM-1 and VANG-1, LRP-2 likely acts downstream of EGL-20.Expression of Strains:VC543: lrp-2(gk272). Strain obtained from the CGC and provided by the C. elegans Reverse Genetics Core Facility at the University of British Columbia, which is part of the international C. elegans Gene Knockout Consortium.MT1306: lin-17(n671) PS5800: lin-17(n671); syEx1031[Pfos-1a::EGL-20::GFP] MT1488: lin-17(n671); unc-13(e1091)lin-17(n671); lrp-2(gk272) double mutant constructed by crossing lrp-2(gk272) males with strain MT1488 hermaphrodites.The


Polyvinylidene fluoride (PVDF) is among the most attractive piezo-polymers due to its excellent piezoelectricity, lightweight, flexibility, high thermal stability, and chemical resistance. PVDF can exist under different forms of films, membranes, and (nano)fibers, and its piezoelectric property related to its β phase content makes it interesting for energy harvesters and wearable applications. Research investigation shows that PVDF in the form of nanofibers prepared by electrospinning has more flexibility and better air permeability, which make them more suitable for these types of applications. Electrospinning is an efficient technique that produces PVDF nanofibers with a high β phase fraction and crystallinity by aligning molecular dipoles (–CH2 and –CF2) along an applied voltage direction. Different nanofibers production techniques and more precisely the electrospinning method for producing PVDF nanofibers with optimal electrospinning parameters are the key focuses of this paper. This review article highlights recent studies to summarize the influence of electrospinning parameters such as process , solution , and ambient (humidity and temperature) parameters to enhance the piezoelectric properties of PVDF nanofibers. In addition, recent development regarding the effect of adding nanoparticles in the structure of nanofibers on the improvement of the β phase is reviewed. Finally, different methods of measuring piezoelectric properties of PVDF nanofibrous membrane are discussed. The PVDF crystalline structure exhibits five different polymorphs, i.e., α, β, γ, δ, and ε phases, depending on crystallization conditions [PF6] added to pure PVDF nanofibers and found that although pure PVDF nanofibers are still dominated by the β phase, there is a small amount of the γ phase. For PVDF nanofibers with [BMIM][PF6], there is almost the 100% β phase without the γ phase. This is because in the electrospinning process, the PVDF just ejected was still in the state of solution. Under the high energy provided by an electric field, the unstable TTTT configuration of the β phase is converted to the TTTG configuration of the γ phase. Due to the strong interaction between [BMIM][PF6] and PVDF, the unstable β phase configuration of PVDF is stabilized, thereby preventing the β phase’s conversion to the γ phase.Organic nanomaterials are used as additives to improve the β phase of PVDF nanofibers in many researches, including graphene , carbon Mahdavi et al. added 0.25, 1, and 4 wt % of 1-octadecyl-3-methylimidazolium Bromide ([OMD]Br) to 22 wt % of a PVDF/DMF/acetone solution . The resCNTs have a strong interaction with PVDF molecular chains and are widely used as nanoparticles in PVDF electrospinning . AlthougIn addition to the interaction between additives and PVDF, hydrogen bonds are also generated to act on the PVDF molecular chain to stabilize and improve the β phase of PVDF. Nasir et al. added 6 wt % of 2,3-dihydrodecafluoropentane to a PVDF/DMAc solution for electrospinning. In this solution, the positive charges of PVDF (hydrogen atoms) interact with the additive (fluorine atoms) negative charges . The hydThe commonly used test method is piezoelectric force microscopy (PFM) to investigate piezoelectric properties on nanofibrous membranes. The tip of the PFM is fixed on an electrospinning fiber, and a voltage is applied to the fiber. The voltage causes the deformation of the internal structure of the fiber and is detected by the tip. The images of the amplitude and phase obtained by the piezoelectric reaction reflect the piezoresponse magnitude and polarization direction within the fibers. The hysteresis loop obtained under voltages provides information about the ferroelectric properties of the fibers. The maximum amplitude of the fiber can be obtained by PFM amplitude and DC voltage hysteresis loops.Baji et al. used PMF to determine the nanofibers’ piezoelectric properties  152]. T. T152]. Gebrekrstos et al. have determined the maximum amplitude of three nanofibers PVDF/GO, PVDF/GOCOOH, and PVDF/GOF by PFM . The d33In addition to PFM, three other types of methods are also used to measure the piezoelectric properties of nanofibrous membranes, including frequency, laser interferometry, and quasi-static methods . Soraya Another method of measuring piezoelectric properties is the laser interferometry method. Fabriani et al. have used this method to measure PVDF nanofibrous membranes’ piezoelectric properties . The PVDCompared with the frequency method and the laser interferometry method, the quasi-static method does not require such high experimental accuracy, and the equipment is simple and easy to set up at low cost. Therefore, among the current methods for measuring PVDF nanofiber membranes’ piezoelectric properties, the quasi-static method is very common. The principle is to deform a static nanofiber membrane through a specific method  and then measure the output voltage change through an oscilloscope as a function of the piezoelectric response.3 fibrous bundle) on two stages with an interval of 1 mm and connected electrodes at both ends to measure the voltage generated by piezoelectricity [Lee et al. have placed their sample (PVDF/BaTiOctricity . One stactricity . The sam−1) [Some quasi-static method equipment deforms the membranes by changing the pressure, thereby testing the membrane’s piezoelectric properties ,158, and−1) .Due to its high piezoelectric constants, flexibility, the variability of the manufacturing processing, and the moderate cost compared to high specialty copolymers, PVDF has garnered research interest for their potential use and advantages in applications as energy harvesters, sensors, etc. To maximize the PVDF’s piezoelectric performances, the total polar phase contents (β and γ phases) of this polymorph polymer need to be enhanced. A current solution to reach this objective is to use the electrospinning process and to develop nanofibers. By applying a strong electrical potential between a polymer and a collector, a jet of the polymer under nanofibers is obtained with a simple process called solution or melt electrospinning. Three significant forces, i.e., the viscoelastic force, the electric force, and the surface tension, govern the electrospinning process, and three major parameters  tailor the electrospun nanofibers. Solution electrospinning is often preferred to melt spinning, because it requires working with higher electric fields and with polymers with specific viscosities. In the solution electrospinning, the preparation of the solution is one of the keys to obtaining nanofibers with desired properties. A suitable solvent with adequate properties  as well as the selection of PVDF (the molecular weight and molecular weight distribution) and the concentrations of these two components needs to be considered carefully. As all these parameters interact, it is complex to find a consensus to ensure the best stretchability of a PVDF jet leads to the highest β phase content. The process parameters also need to be adjusted very carefully, as they affect the total polar phase content of the PVDF and ultimately its piezoelectric performance. Indeed, depending on the chosen voltage or even the flow rate, the stretching of the polymer jet is modified and consequently the PVDF crystalline structure .Furthermore, some studies pointed out that RH or ambient temperature could also affect the formation of the β phase of PVDF. In addition, in order to optimize the piezoelectric response of PVDF, some research teams have been interested in new ways by modifying some aspects of the electrospinning process  or investigating the influence of additives (organic or inorganic nanoparticles) on the PVDF crystalline structure. Besides the difficulty of choosing the right set of elements to obtain electrospun PVDF nanofibers with the highest beta phase content, the piezoelectric properties’ measurements are still complex and primarily performed by using PFM.


The World Health Organization launched a recent global campaign to combat ageism, citing its ubiquity and insidious threat to health. The historical context that promoted this pernicious threat is understudied, and such studies lay the critical foundation for designing societal-level campaigns to combat it. We analyzed the trend and content of aging narratives over 210 years across multiple genres—newspaper, magazines, fiction, nonfiction books—and modeled the predictors of the observed trend.A 600-million-word dataset was created from the Corpus of Historical American English and the Corpus of Contemporary American English to form the largest structured historical corpus with over 150,000 texts from multiple genres. Computational linguistics and statistical techniques were applied to study the trend, content, and predictors of aging narratives.p = .003), over 210 years. There are distinct shifts: From uplifting narratives of heroism and kinship in the 1800s to darker tones of illness, death, and burden in the 1900s across newspapers, magazines, and nonfiction books. Fiction defied this trend by portraying older adults positively through romantic courtship and war heroism. Significant predictors of ageism over 210 years are the medicalization of aging, loss of status, warmth, competence, and social ostracism.Aging narratives have become more negative, in a linear fashion (Though it is unrealistic to reverse the course of ageism, its declining trajectory can be ameliorated. Our unprecedented study lay the groundwork for a societal-level campaign to tackle ageism. The need to act is more pressing given the Covid-19 pandemic where older adults are constantly portrayed as vulnerable. Ageism—the discrimination of older adults based on their age —has undeMore recently, campaigns to combat ageism have continued to persist in the same vein. At the individual level, there has been a significant progress in designing intergenerational programs to reduce ageism . These pThe effectiveness of such laudable campaigns depends on the ability to target the deep-seated drivers of ageism that have percolated over the last 200 years—a time period that traditional methods cannot measure. Against this historical backdrop, we analyze how older adults have been portrayed in various media genres across these 200 years. Recent grants by the National Endowment for the Humanities (NEH) and the National Science Foundation (NSF) provided unprecedented linguistic datasets, and innovative scholars have created “Psychomics”—a methodology to analyze societal stereotypes and narratives using high throughput sematic data .We leverage these developments to study aging narratives to fulfill three research objectives, and test three hypotheses: First, we analyze the trend of aging narratives over 21 decades (1810–2019) by integrating the Corpus of Historical American English , and the Corpus of Contemporary English  to form the largest historical corpus of American English—600 million words—with over 150,000 texts from newspapers, magazines, fiction, and nonfiction books. We hypothesize that aging narratives have become more negative over 21 decades (Hypothesis 1). Second, we analyze the narratives underlying the trend, specifically, the evolution from the 19th to 20th centuries, across multiple genres—newspapers, magazines, fiction, and nonfiction books. We hypothesize that the content of aging narratives in the 19th century will contain a majority of positive themes while those in the 20th century will contain a majority of negative themes (Hypothesis 2). Third, we test multiple hypotheses of the declining trend in aging narratives over 200 years: loss of status, warmth, competence, social ostracism, and the medicalization of aging (Hypothesis 3). Hypothesis 3 expands the literature on sociological drivers of aging narratives beyond previous studies e.g.,  that focOur study is significant in three ways. Conceptually, this is one of the first known studies to analyze the content of aging narratives across 210 years in a massive and comprehensive historical corpus spanning multiple genres. Previous studies, though comparable in historical horizon , analyzeWe synthesize the extant literature on the historical content and measurement of aging narratives to highlight the need for more rigorous datasets and methods that our study provides. Scholars painted a declining image of the old person: from the pedestal of prestige in the early 19th century to the plains of negativity in recent decades. Archival evidence from selected 19th century sermons suggested that growing old was a natural process to be celebrated . The advA vast library of scholarly work have been devoted to investigating perceptions through situational judgment of tests e.g., , and surAgeism also manifests in many qualitative studies: Birthday cards hinting that an older adult’s best days are behind them, a booming beauty industry designed to hide signs of aging, health care practitioners harboring preferential bias against older adults . AdvertiRecent advancements in corpus linguistics and quantitative social sciences enabled scholars to go beyond single sources to leverage multiple genres. For example, Our 600-million-word corpus spanning 21 decades from 1810 to 2019 was created by integrating the respective decades from the COHA (1810–2009) and the COCA (2010–2019). This is the first known integration of both prominent corpora to form the largest structured historical English corpus.COHA is 100 times larger than other equivalent historical corpora and used by major studies to test historical shifts in narratives . It consAll synonyms of “older adult” from 1810 to 2019 were compiled from the Historical Thesaurus of the Oxford English Dictionary , and theN = 14,678) was rated on a scale from 1 (very positive) to 5 (very negative), a method found to be valid and reliable to measure age-stereotype associated words , neutral collocates were rated 3 , and very positive collocates were rated 5 . The inter-rater reliability using Cronbach’s alpha was .984  for the scoring method. For every synonym per decade, we calculated a mean score that was subsequently weighted (by the number of times the synonym appeared in that decade) to yield a Cumulative Aging Narrative Score (CANS) for the respective decade, and we conducted a trend analysis of the respective aging narrative score across 21 decades.To test Hypothesis 1, each collocate that met the study criteria , 2 , and 3 (high status). Examples include “lowly” rated as “1,” “military-looking” rated as “3.” The Stereotype Content Model proposed that social perceptions form along the dimensions of cold/warm and incompetent/competent  to 5 (very warm). For example, “sincere” and “trustworthy” will be rated “very warm” while “aloof” and “unfriendly” will be rated “very cold.” Loss of Competence is measured by “does this collocate describe the ability of a person?” on the following 5-point scale from 1 (very incompetent) to 5 (very competent). For example, “smart” will be rated “very competent” while “simple-minded” will be rated “very incompetent.” Medicalization of Aging is measured by “does this collocate describe or relate to the physical health of a person?” on a binary scale of 0 (No) and 1 (Yes). Sample words that are rated “1” are “sick.” Social Ostracism is measured by “does this collocate describe a person’s group membership?” on a binary scale of 0 (No) and 1 (Yes). The inter-rater reliability using Cronbach’s alpha for all the aforementioned variables ranged from .982 to .991 .To test Hypothesis 2, we ran a Latent Dirichlet Allocation (LDA)—a well-established machine learning technique for topic modeling—on the collocates of the 19th and 20th century, respectively. Hypothesis 3 was tested by a linear mixed model with the cumulative aging narrative score as the outcome, decade as the random factor and fixed factors—loss of status, warmth, competence, social ostracism, and the medicalization of aging, controlling for covariates. ompetent . Againstp = .0029). Neither the quadratic (β = −0.00026 p = .49) or the cubic (β = −0.00009 p = .19) models reached significance. The unstandardized intercept of the linear trend was 3.08  indicating that aging narratives were positive in the early 1800s. However, with each passing decade between 1810 and 2019, there was a 0.142-unit  decrease. The proportion of decades with CANS above the neutral threshold fell from 66.7% in the 1800s to 30% in 1900s. Taken together, this suggests that aging narratives have become more negative over 210 years, providing support for Hypothesis 1  is marital affairs, and likely about the legalities behind marriage ; Topic 2 related to marital status . Another pertinent topic is courtroom proceedings: Topic 3 contained collocates about the judicial branch ; Topic 4 about official documents  and official affairs . Another salient topic is familial affairs: kinship terms are employed  in Topic 5; and peppered throughout the dataset .The topic of societal issues appears in Topic 1, with the use of words that convey a reliance  on assistance ; and in Topic 2 on crime ; and Topic 3 on dissatisfaction with societal problems like the cost of living  and external stressors . The remaining topics are centered on public health issues and public welfare, where Topic 4 contains words about receiving medical attention ; Topic 5 about institutional  spending on welfare ; Topic 6 on monetary aid  for older adults in need ; Topic 7 on societal support  for the less fortunate .These prevalent topics suggest that older-adult-related news in the 1800s had a focus on family and the law. Conversely, 1990s news topics are centered on public health and societal issues.Topics encompass war and family honor: Topic 1 covers aspirations  in the war; Topic 2 about postmortem honor  and words of veneration ; Topic 5 of national history  and family . The topic of illness in the family is in Topic 3, with words about death (die) and sickness  in the family , and Topic 4, where sickness (sick) is present in the family (relative).The topics of war and family honor are present in Topic 2 , and on family ; Topic 5 in honoring a generation of nation-builders . Topic 1 contains the topic of family, with kinship terms  and motherhood-related words ; Topic 6 about growing up . Another prevailing topic is of death and illness: Topic 3 contains words about medical welfare ; Topic 4 on imminent death ; Topic 7 about severe illness .While the key older-adult-related topics in magazines are similar , the topic of death and illness in the 1900s contains more severe collocates  and collocates about public health  compared to the 1800s; thus suggesting a subtle shift toward negativity.The topic of family and marriage is present in Topic 1, with father–son dyadic relationship terms  and the cycle of life and death ; Topic 3 on marriage ; Topic 5 about the monarchy (king) and familial terms . The topic of death and illness is present in Topic 2, on sickness  in the family ; and in Topic 4, with words connoting to mortality  in the family  and postmortem arrangements .The topic of death and illness is common: Topic 1 relates to illnesses  among the population ; Topic 2 on medical care ; Topic 3 on the medical circumstances of the less fortunate ; Topic 4 on sickness ; and Topic 5 on public health .In sum, older-adult-related topics in the 1800s had a family-heavy focus on genealogy and kinship terms; and took a darker tone in the 1900s with its most prominent topics being entirely focused on death, illness, and health.Fiction in the 1800s revolves around courtship and romance: Topic 1 contains words about women ; Topic 3 on romance ; Topic 4 about courtship and physical appearance . Other topics include heroism and wartime stories: Topic 2 contains words connoting to veneration for the departed ; Topic 5 about honoring veterans  and appreciating peacetime . Another topic is daily interaction: Topic 6 contains everyday exchanges  and Topic 7 contains words on the indoor setting and activities .The topic of daily interaction is found in Topic 1, related to the indoor setting and household furniture ; Topic 3 about feeling happiness ; Topic 6 contains quotidian tasks . The topic of death is present in Topic 2, with words about death  in the family . The topic of showbiz is present in Topic 4, with words about filming equipment  and stage directions . The topic of romance is present in T5, about couples  interacting ; Topic 7 about beloved individuals .Both the 1800s and 1900s contain themes on entertainment  and daily life . Thus, fiction was the only genre of texts that did not present any notable negative changes in the way the older adults have been portrayed.p = .003; physical health descriptors have increased, indicating the medicalization of aging, β = −1.26, p = .013; social ostracism descriptors have increased, β = −0.79, p = .033; warm  and competent  descriptors have decreased significantly.We used linear mixed models to analyze predictors of the declining trend of aging narratives and found support for hypothesis 3. High status descriptors of older adults have decreased, indicating the diminishing status of older adults, β = 0.68, This is one of the first known studies to analyze the historical content of societal ageism over an unprecedented time horizon of 210 years from 1810 to 2019. This is achieved through the innovative merger of well-used corpora to create the largest structured corpus of historical English containing 600 million words across multiple genres—and the creative applications of computational linguistics and machine learning to the study of societal narratives . The keyOur study made three important contributions. First, our findings lend credence to existing scholarship—albeit mostly on single sources —that older adults, since the late 1800s, were largely viewed as the dominant occupants of almshouses who were often described as deserted by their children and too infirm to work . While tSecond, our study contributed to understanding the historical underpinnings of societal ageism. In addition, our study expanded the sociological drivers of the negative aging narratives beyond the single sociological variable—medicalization of aging—that ok boomer,” contributing to intensifying intergenerational tensions  and the AARP should front, vigorously. The need to act is more pressing given the Covid-19 pandemic where older adults are constantly portrayed as the vulnerable and a high-risk group . If unchWe are grateful for the financial support from the Social Science Research Council’s SSHR Fellowship (MOE2018-SSHR-004), and National University of Singapore Lloyd’s Register Foundation IPUR Grant (IPUR-FY2019-RES-03-NG). The funders had no role in study design, data collection, analysis, or writing.None declared.


Tanzania launched its first National Action Plan (NAP) on antimicrobial resistance (AMR) in 2017 to reduce the burden of AMR in the country and contribute to the global response. We aimed to analyze the implementation of the NAP on AMR in Tanzania using the governance framework. In-depth interviews were conducted with human and animal health practitioners and national-level policy actors. We adapted Chua’s AMR governance framework to analyze the development and implementation of the NAP in Tanzania. Implementation of the NAP has realized several achievements, including: (i) the establishment of a functioning Multi-Sectoral Coordinating Committee for coordinating the implementation of AMR activities; (ii) existence of governance structure; (iii) establishment of human and animal surveillance sites; (iv) creation of AMR awareness in the community and (v) availability of guidelines at the health facility level to ensure AMR stewardship. However, some dimensions of the governance areas, including reporting and feedback mechanisms, accountability, transparency and sustainability of AMR plans, are not effectively implemented. Addressing these challenges should involve strengthening the collaboration of the different sectors involved at different NAP implementation levels by careful planning and coordination, and provision of adequate resources to ensure sustainability. Antimicrobial resistance (AMR) is a global public health threat. Several studies have reported the causes of rising AMR, including self-medication ,2, antibIn several LMICs, including Tanzania, implementation of NAPs for AMR remains fragmented and lacks comprehensive multi-sectoral and multi-pronged approaches . MoreoveDespite widespread agreement that tackling AMR domestically can only be achieved through a strong governance framework, it is not clear how various efforts to implement the NAP in Tanzania are following the framework. We adapted Chua’s AMR governance framework to analyze the development and implementation of the NAP for combating AMR in Tanzania . The fraThe analysis of the implementation of the NAP adapted to Chua’s governance framework consists of five main governance areas: policy design, implementation tools, sustainability, monitoring and evaluation and one health engagement. In the next few sections, we detail our findings according to each governance area.The policy design analysis focused on strategic vision, coordination, participation, accountability and transparency and equity.Strategic vision implies that the NAP contains clearly defined goals and objectives to direct interventions. The Tanzania NAP identifies ten priority actions and specific interventions for each activity with quantifiable targets . The fivIn Tanzania, the Ministry of Health is the central coordinating ministry, while the Multi-Sectoral Coordinating Committee (MCC) is responsible for coordinating, facilitating and overseeing the implementation of all AMR activities. According to the Tanzania national AMR surveillance framework , the impThe reviewed literature and interviewees indicate that with the exception of community members, who were not involved, all other key stakeholders were involved in the development of the NAP. These included ministries responsible for public health and animal health, universities and research institutions, regulatory authorities , government agencies ; international organizations ; professional councils , representatives of health facilities  and the private sector  .Interviewees had varying views on the involvement of different sectors in the preparation of the AMR NAP. Some reported that key sectors like public health, livestock and fisheries were involved. They stated that because every key sector was involved, it was easy to outline their roles in the implementation of the plan.“I think all sectors were influential because each sector is directly involved and each sector has advantages in the action plan because we have seen that the issue of bacteria resistance doesn’t concern one sector, it affects all sectors”.(Key Informant (KI): scientist from National Laboratory). Another respondent claimed that all sectors had equal influence during the preparation of the NAP:“Of course, when you talk about these issues involving more than one sector, the human health sector has often been taking a big part (okay)…, even in the plan it took a big part but at the end of the day it has to touch other sectors because they provide a response to human health. Human health is leading because it is a priority”.Several interviewees raised concerns that not all sectors were involved in the preparation of the NAP. The environmental sector was not represented despite the fact that waste management is a substantial concern within the NAP. The health sector was reported as the dominant sector throughout the development process and was given priority compared to other sectors.“There was no direct community involvement but the government prepared action plan. After the plan is ready the community will be informed and the community will be educated on what the plan is all about”.Most of the key informants said categorically that community members, who are key in the implementation of the AMR NAP as consumers of antimicrobials, were not included in preparing the plan.“I’m not sure about the community involvement. In the sessions which I attended I did not see a participant specifically from the community”.Another respondent said:To ensure accountability and transparency within the NAP, a clear governance structure under the supervision of MCC has been established . The NAP“The guidelines have helped to us observe drug resistance among patients, … we get information through weekly reports, daily reports and monthly reports”.Facility-level respondents reported that during their weekly clinical meetings, they get feedback about drug resistance.“What I know is that every clinician must have guidelines that he/she uses to provide treatment to patients, guidelines for all the diseases. So, when the clinician finds that a patient has used a certain medicine for a long time without getting better, then he moves a patient to another medicine which is stronger”.According to the NAP, the Medicines and Therapeutic Committee (MTC) in each health facility is mandated to ensure the rational use of medicines, including antimicrobials. The MTCs should monitor prescribing practices to ensure they adhere to the standard treatment, and ensure the availability of up-to-date important reference materials for medicine information including the Standard Treatment Guidelines and Essential Medicines List . Health “So, we came up with a strategy that a pharmacist should make sure we have all the required medicines. When there is shortage, we should take immediate actions to get alternative medicines for our patients”.Regarding equal access to medicines, including antimicrobials, interviewed health workers reported that the facility management attempts to ensure that all medicines required are available to all patients who need them.Tanzania also has the Standard Treatment Guidelines and National Essential Medicines List, which reflect the policy of the government of ensuring the availability of safe and efficacious essential medicines to all its citizens. This is a tool used to promote access to essential medicines to achieve maximum therapeutic benefit and optimize patient outcomes .The NAP outlines six dimensions of interventions, which are understood as tools for implementation. These are surveillance, antimicrobial stewardship programs, IPC measures, education, AMR research, public awareness activities and medicine regulation. The following sections examine each intervention.The National AMR Surveillance Framework aims at generating evidence on the burden of AMR among priority pathogens isolated in hospitals from patients. The strategic objectives of this framework are aligned with the National AMR Action Plan based on national needs and priorities . The fraEight and three surveillance sites for humans and animals, respectively, have been established in Tanzania. These surveillance sites consist of five zonal referral hospitals, one national hospital and three regional hospitals. The surveillance sites are coordinated by the National Public Health Laboratory. The Sokoine University of Agriculture and two Zonal Tanzania Veterinary Laboratory Agency’s centers in Mwanza and Arusha are the only surveillance sites for animals. The 2020 GLASS reports show that Tanzania did not submit data on any priority pathogens in 2019 because the country had recently begun surveillance, with the participation of a few laboratories [“We have now developed antimicrobial stewardship guidelines which will be released at any time”.Policy makers stated that in ensuring that there is responsible use of antimicrobials by healthcare professionals, and more specifically, selection of the most appropriate antimicrobials, duration, dose and route of administration, the MoH has distributed the guidelines to all health facilities, including dispensaries. The MoH provides education to health workers through on-the-job training programs. The government has also introduced the guidelines for Health Training Institutions to impart knowledge on AMR to both the instructors and students. The idea behind it is to ensure that when students graduate, they know and can implement these standards and guidelines. One of the key informants stated:“We already have a guideline and have begun to distribute it to various regions in preventing and controlling the infection. We are also conduct sensitization campaigns to create awareness on personal hygiene”.Infection prevention and control (IPC) across human and animal sectors is one of the key priorities in the NAP. A number of activities have been identified, including supportive supervision to all health facilities by quality assurance teams. These also include facilitating the availability of IPC-related equipment, supplies and guidelines, establishing a functioning vaccination program for human and animal health and engaging communities in the implementation of personal hygiene and environmental sanitation . NationaThe NAP has prioritized AMR research and development activities, which include engaging relevant stakeholders to identify current gaps in knowledge and potential research areas, developing national research guidelines on AMR and undertaking research related to AMR. The literature showed that more research on AMR has been conducted after introduction of the NAP, indicative of the growing importance of AMR in both academic and policy arenas. The Tanzania national budget allocates 1% of the country’s GDP to finance research activities . This le“Community health education has increased and it is not only from the Ministry but also from individual and other stakeholders. For example, there are Pharmacy student’s association collaborating with others people to educate the public about AMR”.Interviewees reported that there are ongoing education programs to train Health Training Institutes’ tutors on guidelines and standards regarding antimicrobial use (AMU) and AMR so that they can train their students. In turn, students also participate in educating the community about AMU and AMR:“There is a technical working group whose main task is to provide education on AMU and AMR to the general public and students organizes concerts and symposia and involves students who go around sensitizing the community on AMR”.Different models have been used to create AMR awareness in the general public. These include local television and radio programs, and newspaper articles produced by different media houses. There are also regular trainings to ensure proper understanding and awareness of AMU and AMR among the media.Nane Nane (Farmers’ Day) exhibitions, the government and other stakeholders participate in creating awareness of drug resistance in both animals and humans.“Government officials go direct to farmers and livestock keepers to provide education about antibiotics resistance”.(KI: Ministry of Livestock and Fisheries)Participants reported that during Antibiotic Awareness Week every November, many awareness activities are conducted by different stakeholders, including the MoH. When there are public events such as the “We expect that no person will be prescribed antibiotics before performing culture and sensitivity in order to be sure the medication you are prescribing is going to work. However, we failed because some hospitals are not equipped. I also think district hospitals do not have the capacity”.A list of medicines is used at all levels of the healthcare system to ensure appropriate use of antimicrobials. According to the NAP, Tanzania has legislation for the regulation of the quality, safety and efficacy of medicines including antimicrobial agents. The Tanzania Medicine and Medical Devices Authority is responsible for managing all drugs, including antibiotics, in the country. The interviewed policy makers reported that, although the medicine regulations may be in place, they may only be partially implemented. One of the main reasons for low levels of implementation is a lack of capacity within primary healthcare facilities . These facilities often do not have adequate financial and human resources, which results in a lack of diagnostic tools and laboratory equipment to perform culture and antimicrobial susceptibility testing, as recommended by the guidelines.Monitoring and evaluation is an important governance area in the implementation of the NAP as it generates evidence to inform policy makers, planners and implementers whether the implementation of the NAP is effective or not. It also involves dissemination of information and reports to different stakeholders about the implementation of the NAP.There is an established collaboration between the government of Tanzania and the WHO, and AMR data are transmitted to the international surveillance system through WHONET software. While the human health sector reports findings to GLASS on a monthly basis, interviewees pointed out some difficulties in accessing this information. According to the NAP, national-level data ought to be used to inform decision-making processes, however, the existing limited data on AMR are still a challenge in Tanzania.“The national level AMR focal persons do conduct supervision of the lower levels. This is because, currently, only the zonal levels are functioning. It will take time before the programme is rolled out nation-wide and to all levels”.(KI: MCC member)Interviewees reported that efforts have been made to put in place AMR focal persons for human and animal sectors at the national, zonal, regional and lower levels who are responsible for conducting supervision of AMU and AMR and provide feedback to the national-level organizations. The national AMR focal persons have the additional responsibility of reporting on the prevalence and trends in AMR to GLASS. One of the key informants stated:The effectiveness dimension requires that nations should put in place mechanisms to monitor and evaluate the effectiveness of various AMR interventions and specific policies. For instance, nations should strive to measure the effect of AMR on human and animal health . The AMR“Another challenge is insufficient funds compared to existing activities. And if I look clearly, I see that this AMR is more of donor funded project than the government so when donors leave, the situation will be difficult”.(KI: FAO representative to MCC)Sustainability of the NAP depends much on the allocation of resources and a dedicated budget. However, interviewed policy makers reported that the MCC receives funds from development partners to implement activities identified in the NAP. Most of the interviewed policy makers raised a concern that the sustainability of AMR interventions is questionable because the majority of them receive funds from development partners.“The preparation of this plan was coordinated by the Ministry of Health, as I explained it was the issue of one health so we were together with the Ministry of Livestock and Fisheries”.In a tripartite approach, the FAO, OIE and WHO emphasize that addressing health risks at the human–animal–plant–ecosystem interfaces requires strong one health engagement . AMR is Our study has revealed that the implementation of the AMR NAP has realized several achievements, including: (i) the establishment of a functioning Multi-Sectoral Coordinating Committee for coordinating, facilitating and overseeing the implementation of all AMR activities; (ii) existence of governance structure; (iii) establishment of surveillance sites for humans and animals; (iv) creation of AMR awareness in the community; and (v) availability of guidelines at the health facility level to ensure AMR stewardship. The analysis reveals that some of the dimensions of the governance areas, including reporting and feedback mechanisms to monitor the NAP progress, accountability, transparency, resource mobilization for research activities and sustainability of AMR plans, were not effectively implemented, which poses challenges towards effective implementation of the NAP. Resolving these challenges requires a systematic governance approach to successfully achieve the desired goals and objectives.Tanzania’s AMR NAP 2017–2022 has a strategic vision to provide long-term direction. This implies that the NAP has clear goals and objectives to provide direction to interventions . The strParticipation in the development and implementation of the AMR NAP is an important dimension, which increases ownership and acceptance of the plan, thus facilitating its implementation. The study findings reveal mixed results. On one hand, the formulation of the NAP engaged one health key stakeholders such as health, livestock and fisheries, although the health sector dominated the process. Unfortunately, the environmental sector, which is a critical actor, was not involved. It was noted that some of the sectors still work in silos which implies that there is a weak inter-sectoral collaboration and accountability system. Other sectors prefer to be accountable to their seniors who are in the same sector and they are hierarchically accountable to them for other responsibilities, making one health engagement a challenge, which limits effective implementation of the NAP. Notably, cross-sectoral coordination is problematic in LMICs because the governance structures and mechanisms to bridge sectors, not only within government but also with nongovernmental partners and programs, are fragmented or inflexible ,28,29. TIn Tanzania, community members, who are key in the implementation of the AMR NAP as consumers of antimicrobials, were not included in preparing the NAP but were involved during the implementation of the plan, especially during awareness creation campaigns, which aim at informing the community on the effects of AMR. In Tanzania, different stakeholders participated in a series of community awareness-raising activities calling for responsible use of antibiotics in humans and animals in order to reduce the effects of antibiotic resistance. A similar dilemma was revealed in a systematic review of several European countries that indicated that citizens were expected to be recipients of awareness activities or education interventions stipulated in the AMR NAP documents, rather than having an active role in taking proactive measures, including those targeting the reduction of the need for antibiotics .We report the existence of frameworks for providing regular reports on AMR surveillance data. However, the findings revealed that in Tanzania, data for funding of AMU/AMR surveillance are not always publicly available , which lOur study reveals that despite the existence of standard treatment guidelines and Medicines and Therapeutic Committees that could have ensured equity in accessing antimicrobials, there is no evidence to suggest that the implementation of the NAP is equitable. Access to qualified service providers, including doctors, laboratory technicians and pharmacists, may partly limit equitable access to existing essential antimicrobials. In LMICs, unskilled health workers and limited laboratory facilities have been reported as among the factors contributing to the unnecessary prescription of antimicrobials, thus increasing the incidence of AMR ,34,35.Our findings highlight a lack of surveillance infrastructure and capability as most of the health facilities, especially in the rural areas, do not have well-equipped laboratory facilities with skilled personnel. Thus, laboratory infrastructures need to be strengthened to extend surveillance sites to more public and private healthcare facilities. Similar issues arise for AMR surveillance in the animal health sector. These challenges are not unique to Tanzania, but studies indicate that several African countries face similar obstacles ,37. FurtIn Tanzania, IPC is one of the key priorities in the NAP and the government has put in place IPC-related equipment, supplies and guidelines for the implementation of personal hygiene and environmental sanitation. However, there are no documented studies showing the effectiveness of these efforts in Tanzania. Similarly, Ghana has a national policy on IPC , howeverThe reviewed literature demonstrates contrary findings about AMR education and public awareness in most African countries. Notably, there is little continuing education on AMU for key stakeholders such as prescribers and livestock keepers, including poultry farmers . In manyDespite the existence of legislations and a list of medicines and pharmacopoeia books to ensure the quality, safety and efficacy of medicines and to guide the management of drugs, we noted weak enforcement of these guidelines and legislations, which has resulted in mismanagement of antibiotics in Tanzania. This includes the dispensing of antimicrobials by unauthorized people and their sale in the auction markets like any other commodity . Most LMOur findings reveal that the reporting of AMR data in Tanzania is limited as there are no effective mechanisms that feed information to the various centers responsible for decision and policy making, hence limiting the monitoring and evaluation of the extent of AMR. Both GLASS and WHONET were identified as reporting systems used in Tanzania. A systematic review on AMR in the Central African Region highlighted that there is a paucity of data because of the poor reporting system, which is partly a result of the limited and poor surveillance system ,46. TanzIn Tanzania, the NAP includes a section on monitoring and evaluation to measure the effectiveness of interventions. The Interagency Coordination Group on AMR reported that it is difficult for poor countries to put in place a robust and accountable system for monitoring and evaluating NAPs because many of the countries lack systems and capacity and where systems are available and functional, changes in political power or in governance systems at different levels become a threat to their sustainability . Like M&This is the first study in Tanzania assessing the implementation of AMR NAPs using a structured governance framework modified from Chua’s framework (2021) to better adapt to our study findings. The framework allowed a structured format of analysis of various dimensions of governance areas, which helped to provide Tanzania’s context-specific analysis of best practices regarding implementation of the NAP. However, our study has one limitation. Some important information, such as how many resources are allocated for carrying out the NAP’s activities, could not be obtained. This further speaks to issues of transparency and accountability and the lack thereof within the NAP, but also to wider debates on public policy making.This exploratory qualitative study analyses the implementation of the NAP on AMR in Tanzania using the governance framework. It involved in-depth interviews with animal and human health practitioners at a sub-national level and senior officials from human, animal and environmental sectors at the national level. A review of relevant documents, including guidelines, policies and national and international AMR reports, was conducted to complement information generated from primary data sources. The exploratory study design was considered appropriate for this study because it helped the researchers to conduct an in-depth exploration of views from different key informants at the facility, district and national levels.Three districts, namely, Ilala, Kibaha and Kilosa were selected for the sub-national-level actors. Ilala is an urban area, densely populated, with informal housing, transport infrastructure, dump sites, agriculture, industrial commercial activities, fishing and sand mining. It is highly polluted by effluents from Msimbazi River tributaries originating from different sources, leakage of effluent from waste dumps, abattoirs and domestic wastewater from septic tanks and pit latrines that are used by about 85% of the city population. The selection of the district was based on the following factors: many different livelihood activities and environmental contamination by effluents and wastes from different sources . Kibaha We used a purposive sampling strategy to draw the key informants from each group of actors, namely, laboratory technicians, livestock officers, in-charges of health centrers and dispensaries, pharmaceutical assistants and dispensers in Ilala, Kibaha and Kilosa districts. We identified key informants (practitioners) through a mapping exercise and under the guidance of officials from the district and ward/village levels. The policy makers at the national level were purposely selected from the ministries responsible for human health, livestock and fisheries, food and agriculture. The other national key informants included members of the AMR MCC and some from the National Regulatory Authority and National Health Laboratory Quality Assurance and Training Centre. The national-level key informants were selected because of their roles in the preparation and supervision of the implementation of the national action plan for AMR.We conducted 111 in-depth interviews with key informants from national, district and health facility levels. Interviews lasted 45 to 90 min and were run by 12 research assistants. All research assistants were trained according to Guidelines on Ethics for Health Research in Tanzania  and we eAll study participants provided their informed consent for inclusion before they participated in the study and they were informed about anonymity and confidentiality issues, and that they could withdraw from the study at any time they wished. The Medical Research Coordinating Committee of the Tanzania National Institute for Medical Research approved the protocol (Ref. No. NIMR/HQ/R.8a/Vol IX/3147). The study was conducted following the principles of the Declaration of Helsinki.This study uses a governance framework to analyze the development and implementation of Tanzania’s AMR NAP according to three governance areas: policy design, implementation tools and monitoring and evaluation. The NAP is an important first step in fighting the increase in the prevalence of AMR in the country. While the NAP outlines strong objectives, operationalizing and implementing them require careful planning with attention to detail to ensure Tanzania can achieve its goals. This analysis indicates that despite the availability of the NAP, there are challenges in its implementation. These include weak AMR surveillance, inadequate resources for plan implementation sustainability and a lack of funding for research and monitoring the progress. Strengthening the one health approach involving different NAP implementation levels, combined with careful planning with adequate allocation of resources, should be given top priority in policy and decision making for the effective implementation of the NAP.