Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional composite nanofilm that facilitates visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈ 3.2 µm  and are observed with a mean spatial frequency of one hotspot per ≈ 7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots co-localized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals. To form the vast and complex network necessary for an organism to sense and react to the world, neurons must connect at highly specialized junctions. Individual cells communicate at these ‘synapses’ by releasing chemical signals (or neurotransmitters) such as dopamine, a molecule involved in learning and motivation.Despite the central role that synapses play in the brain, it remains challenging to measure exactly where neurotransmitters are released and how far they travel from their release site. Currently, most tools available to scientists only allow bulk measurements of neurotransmitter release.To tackle this limitation, Bulumulla et al. developed a new way to measure neurotransmitter release from neurons, harnessing a technique which uses fluorescent nanosensors that glow brighter when exposed to dopamine. These sensors form a very thin film upon which neurons can grow; when the cells release dopamine, the sensors ‘light up’ as they encounter the molecule. Dubbed DopaFilm, the technology reveals exactly where the neurotransmitter comes from and how it spreads between cells in real time. In particular, the approach showed that dopamine emerges from 'hot spots' at specific sites in cells; it also helped Bulumulla et al. study how dopamine is released from subcellular compartments that have previously not been well characterized.Improving the sensors so that the film could detect other neurotransmitters besides dopamine would broaden the use of this approach. In the future, combining this technology with other types of imaging should enable studies of individual synapses with intricate detail. Chemical neurotransmission generally falls under one of two broad categories: fast synaptic transmission or neuromodulation. Synapses that mediate rapid communication between most excitatory and inhibitory synapses in the brain primarily employ glutamate or γ-aminobutyric acid (GABA). Such chemical communication occurs at highly specialized synaptic structures that have nanoscale spatial organization and operate with millisecond temporal precision . In contGenerally, the nature of the chemical synapse is less well understood for neuromodulators, which differ from their classical counterparts not only in their mechanisms of action on receptors but also in their secretory apparatus and spatiotemporal dynamics. For dopamine, one of the most important neuromodulators in the brain, the challenge is compounded by certain unique features pertinent to dopamine neurobiology. Dopaminergic neurons are known for their large size and exhibit highly ramified axonal arborizations and dense varicosities . Studies2+ dependent, suggesting gating of dopamine release by a Ca2+-mediated release machinery, reminiscent of classical active zones. Dopamine efflux was observed at major dendrites, fine dendritic processes, and at junctions of the soma and dendrites but rarely directly from the cell body itself. Soma of dopamine neurons were observed to receive strong dopaminergic input due to release and diffusion from proximal dendrites. Retrospective super-resolution imaging at identified release sites shows that Bassoon, long established as a presynaptic scaffolding protein of the cytomatrix in presynaptic active zones, is also enriched at the vicinity of dopamine release sites in dendrites and dendritic arbors. The expression of vesicular SNARE protein synaptobrevin-2 correlated with dopamine release activity in dendritic segments, establishing its functional utility in dopamine neurons. Therefore, active zones in dendrites appear to utilize a constellation of presynaptic and SNARE-complex proteins that are responsible for coordinating release, similar to those observed in classical synapses. This study offers a technology that facilitates visualization of the spatial and temporal efflux of dopamine and deploys the technology to shed light on somatodendritic dopamine release, a facet of dopaminergic neurobiology that has been insufficiently characterized.In this work, we developed an assay that facilitates visualization of the efflux of dopamine from active zones with synaptic resolution and quantal sensitivity. Here, we define efflux to mean the two-dimensional (2D) broadening of released dopamine quanta. In axonal arbors, we observed that dopamine release arises from a sparse set of varicosities, and we were able to assign the observed spatially defined effluxes to individually identified boutons. In dendrites and dendritic arbors of dopamine neurons, we similarly visualized spatially resolved effluxes of dopamine from putative dendritic active zones, which have been less well understood than release zones in axons. Our results show that dopamine neurons can sustain robust levels of release from their dendritic processes. Indeed, we demonstrate that dendritic release is fast and CaTo a first-order approximation, once released from a synaptic active zone, the temporal evolution of the released chemical should approximate that of diffusion from a point source, characterized by an isotropic expansion from the point of release but constrained by transporter activity and local three-dimensional ultrastructure. Such a signal can only be fully measured if the sensing platform sufficiently samples the underlying signal in both the spatial and temporal domains. However, the inability of current technologies to measure chemical efflux sufficiently in the spatial domain limits our ability to study chemical synapses. We addressed this challenge by using near-infrared (NIR) fluorescent dopamine nanosensors to image single dopamine release sites from rat primary midbrain neuronal cultures. The nanosensors are assembled from oligonucleotide-functionalized, single-wall carbon nanotubes in solution phase  and havedK) of 268 nM (50 )values of ~ 1 µM for D1-like and ~ 10 nM for D2-like dopamine receptors, suggesting that DopaFilm is sensitive to chemical secretions that have physiological relevance  . Dopaminf 268 nM . This coelevance . In ordeWe asked if dopamine neurons grown on DopaFilm can be evoked to release dopamine, and whether DopaFilm fluorescence transients can recapitulate the predicted spatiotemporal evolution of dopamine efflux. To drive this effort, we virally co-expressed TH-GFP (GFP expressed under the control of the rat tyrosine hydroxylase [TH] promoter) and Syn-ChrimsonR-tdTomato . This co-expression paradigm facilitated identification and optical stimulation of putative dopamine neurons. About 79% of TH-GFP+ neurons were confirmed to be dopaminergic in retrospective immunofluorescence against TH, the rate-limiting enzyme in dopamine biosynthesis. On the other hand, all of the TH-immunoreactive neurons expressed the virally delivered TH-GFP transgene. During seeding, we optimized density of cells on DopaFilm such that mature neurons formed a monolayer of cells on the substrate. The seeding density allowed us to record activities arising from isolated dopamine neurons where no other neurons in the vicinity of the neuron of interest were TH+, ensuring that detected activity can be assigned to single identifiable processes with minimal crosstalk. We used 561 nm LED (5 pulses and 25 Hz) to depolarize dopaminergic neurons. A subset of dopamine neurons exhibited spontaneous spiking activity, and we applied no external stimuli in those cases. Furthermore, most dopamine neurons exhibited stochastic, temporally uncorrelated release events that appeared to be action potential independent, and those events were also included in our data. All imaging experiments were carried out 3–4 weeks postviral infection, and neurons were 4–5 weeks in vitro at the time of activity imaging.–6 cm2 s–1 (mean ± SD), in reasonable agreement with estimated values of diffusion coefficient for dopamine (peak) and 3.83 ± 0.8 s (mean ± SD) for first-order decay time constant (τoff) (DA (≈ 100 ms) and dLight (reported as τ1/2 of ≈10 ms followed by a plateau of ≈100 ms) (1/2 ≈ 100 ms) and comparable to or faster than those reported for GRABDA (≈ 3–17 s for variants). For comparison, G-protein-gated inwardly rectifying K+ (GIRK) current-based dopamine dynamics measurements exhibited τpeak ≈ 250 ms, whereas carbon fiber recordings peaked in τpeak ≈ 300 ms  . The tur≈100 ms) . On the ≈ 300 ms . This su≈ 300 ms . Both eve’ media .We next explored the consistency of DopaFilm ∆F/F hotspot dynamics over multiple stimulation epochs and asked if repeat stimulations in the same FOV generated DopaFilm hotspots that localize to the same set of boutons. We carried out imaging in an FOV where we applied the same optical stimuli to drive hotspot activity over separate imaging sessions. In axon terminals, we observed that repeat stimulations can be carried out with rest periods of ~ 2–3 min between stimuli, giving rise to a consistent set of DopaFilm hotspot activities, and suggesting that release-competent dopamine neuron boutons likely have a high probability of release . To evaliK ≈ 100 nM). Application of NOM altered the clearance kinetics of DopaFilm transients, defined by a first-order decay time constant, τoff -type glutamate receptor antagonist 2,3-dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX) and N-methyl-D-aspartate (NMDA)-type glutamate receptor antagonist D-2-amino-5-phosphonovalerate (D-AP5) were bath-applied to the co-culture system while imaging release from dopamine neurons. We first examined neurons from which DopaFilm activity can be detected from spontaneous spiking events in which we applied no external stimulus to generate activity. We imaged from these neurons under ACSF  and then bath-applied NBQX (10 µM). DopaFilm activities that were detected before application of NBQX were absent in the postdrug imaging sessions . Applica–4 in unpaired t-test) (off) for TTX transients were smaller than those of evoked releases (mean ± SD (s): 3.83 ± 0.8 vs 1.52 ± 0.53, off values, we created a cumulative frequency distribution of τoff values with and without TTX (for evoked release) and defined a parameter, τ10/90, as the range of τoff for values that fall in the 0.1–0.9 quantile window in the cumulative distribution curve  0.29 ± 0.24 s–1 per release site, which contrasted with a frequency of (mean ± SD) 0.16 ± 0.13 s–1 per release site for spontaneous activities before TTX addition  . The decon curve . In axonaddition . NotablySD of F0 . IncidenSD of F0 . In densSD of F0 .1=5.7%, µ2=9.6%, and µ3=15.1% ∆F/F offered the best fit  and clearance (τoff) were similar when compared to transients in axonal release sites  and active zone protein complexes that dock and prime synaptic vesicles  as well as SNARE-complex proteins  that carry out vesicle fusion in the final exocytotic step . We wantWe similarly explored whether DopaFilm activity co-localizes with proteins involved in the exocytotic release of neurotransmitters. A myriad of cellular functions that require membrane fusion, including subcellular compartmentalization, cell growth, and chemical secretion, involve the use of SNARE-complex proteins . SNARE pAmong design criteria for biological sensors is the requirement that activity be read out in temporal and spatial domains at sufficiently high sampling rates to recapitulate the underlying dynamics. From a temporal perspective, designing probes with suitable kinetics is carried out routinely during biosensor development. However, targeting the designed probes to read out the biochemical processes at appropriate spatial scales is not always trivial. As it pertains to chemical synapses, the nature of chemical effluxes demands uniform delivery of probes to the synapse and peri- and extra-synaptic spaces because such signals are inherently incompatible with cell-centered experimental paradigms. In this study, we introduced DopaFilm as a technology that facilitates visualization of chemical effluxes from dopamine neurons. DopaFilm inherently covers a large 2D plane and is therefore capable of detecting chemical efflux at the source (synapse) and tracks the signal to the end of its inherent spatial extent, subject to its limit of detection. We therefore circumvent the non-trivial demand of targeting the probe to the synapse and its surroundings. This design approach, coupled with the film’s favorable temporal properties and quantal sensitivity, has facilitated visualization of synaptic dopamine chemical effluxes with unprecedented resolution. We used these capabilities to shed light on the nature of dopaminergic chemical synapses. We demonstrate that dopamine varicosities serve as beacons that broadcast dopamine release into neighboring peri- and extra-synaptic spaces. DopaFilm registered these release sites as hotspots of fluorescence activity and further afforded tracking of the evolution of the hotspots in the spatial domain, in real time.To demonstrate the utility of DopaFilm, we applied it to study release in somatodendritic compartments of dopamine neurons. Dopamine release in midbrain regions, particularly in the substantia nigra pars compact (SNc) and ventral tegmental area (VTA), is a critical component of dopamine signaling for two important reasons. First, dopamine released in SNc and VTA is predicted to activate D2 autoreceptors, which in turn tune the excitability of dopamine neurons through GIRK channels . This se2+-dependent, and is broadcast from hotspots that are enriched with the presynaptic active zone protein Bassoon. Our study ascribes an important role for the SNARE-complex protein synaptobrevin-2 in dendritic dopamine release, consistent with predications from previous studies , as well as fine dendritic processes and dendritic arbors participate in dopamine release. Remarkably, dendrites with no apparent varicose morphologies participated in dopamine release as did dendritic arbors with axon terminal-like bouton structures. Dendritic activity shared much in common with dynamics observed in axons, but dendritic hotspots appeared localized in their spatial extent to the immediate vicinity of the release site in contrast to release in axonal processes, which propagated to a larger spatial extent. These differences may be driven by the dense axonal arbors of dopamine neurons, which give rise to higher number of release sites per unit area or could be driven by intrinsic differences between axonal and dendritic release sites. Dendritic release was observed to be robust, Ca studies . Establi2+ recordings from postsynaptic processes can help us study presynaptic chemical synapses but these methods are lacking in throughput, represent summed synaptic input from multiple release sites, and are not suitable for neuromodulators that signal through G-protein coupled receptors  is spectrally compatible with existing optical technologies, as we have demonstrated in this study. However, the unique spectral properties of DopaFilm means that microscopes that typically rely on silicon technology for photon detection (cameras and photomultiplier tubes) are not compatible with its use. DopaFilm requires use of detectors with InGaAs sensors for photon detection and additional optimization of optical components to facilitate transmission of NIR and SWIR photons. This requires an investment by the end user, albeit a modest one. Furthermore, single-wall carbon nanotube-based nanosensors, from which DopaFim is fabricated, have been developed for electroactive neurochemicals , and HiPco single wall carbon nanotubes were purchased from Nano Integris (batch #HR35-141). Solution-phase nanosensor synthesis was carried out by first mixing 1 mg of (GT)6 and 1 mg of HiPco SWNT in 1 mL of 1 × PBS. Then the solution was bath-sonicated (Branson 1800) at room temperature for 20 min followed by probe-tip sonication (Sonics Vibra Cell) for 15 min in an ice-bath. Resulting suspension was centrifuged at 20,000 rcf (Eppendorf 5430 R) at 4°C for 60 min, and supernatant was carefully transferred into a new Eppendorf tube and stored at 4°C until further use. For characterization, each sensor batch was diluted to a working concentration of 10 ppm in 1 × PBS. Fluorescence and absorbance measurements were carried out on NS Super NanoSpectralyzer (Applied Nanotechnologies). Solution-phase fluorescence measurements and dopamine response tests were carried out on a custom built near infrared plate reader using flat clear bottom black 96-well plates . Solution-phase ∆F/F values were calculated from integrals of fluorescence counts between 875 and 1300 nm for pre- and post-dopamine fluorescence spectra.Single-stranded 5′-GTG TGT GTG TGT-3′  plating media to NbActiv4 [BrainBits-NB4]), and cultures were maintained in a 5% CO2 humid incubator at 37°C to facilitate cell adherence. After ~ 2 hr, attachment media was aspirated and growing media consisting of plating media: NbActiv4 at 1:20 ratio (v/v) was used. After the first week, cultures were fed twice a week by replacing old media 1:1 (v/v) with fresh NbActiv4 media. To express opsins and identify dopaminergic neurons, cultures were infected at 5 days in vitro (DIV) with titer-matched viruses using pAAV-Syn-ChrimsonR-tdT (Addgene #59171) and pAAV2.5-TH-GFP (Addgene #80336), respectively . Plasmid DNA preparations and virus packaging were performed by Molecular Biology and Viral Tools facilities at Janelia Research Campus.Primary rat neuronal culture work was conducted according to the Institutional Animal Care and Use Committee (IACUC) guidelines of Janelia Research Campus of the Howard Hughes Medical Institute. Sprague Dawley (Charles River Laboratories) neonatal rat pups were euthanized, cortical and hippocampal hemisphere tissue and SNc tissue dissected, and dissociated in papain enzyme  in neural dissection solution  at 37°C water bath. After 30 min, enzyme solution was aspirated, and tissue pieces were subjected to trituration in 10% fetal bovine serum containing MEM media. Following trituration, cell suspension was filtered and resulting single-cell suspension was centrifuged to yield a cell pellet. Cell pellet was resuspended in Plating media  imaging, we developed a custom microscope based on Thorlabs Bergamo microscope body. We modified the Bergamo to facilitate optimal imaging in 400–1400 nm with widefield epifluorescence and confocal laser scanning modalities, using commercially available or custom-ordered optical components that maximize photon collection, transmission, and detection in the 400–1400 nm range. We used the widefield epifluorescence modality of the microscope for all our imaging experiments in this study. The microscope is equipped with two fiber-coupled NIR lasers to excite far red, NIR and SWIR fluorophores: a 671 nm laser  and a 785 nm laser (Thorlabs S4FC785). Additionally, the microscope is equipped with the following 4-wavelength LED light source (Thorlabs LED4D067) to excite and/or actuate visible range fluorophores and opsins: 405, 470, 561, and 625 nm. The microscope is equipped with an InGaAs camera with optimized sensitivity in the SWIR range , and an sCMOS camera  for visible range imaging. SWIR images were acquired with µManager, an open source microscopy software .2PO4, 24 NaHCO3, 12.5 glucose, 5 HEPES, 2 CaCl2, 2 MgSO4, and spiked with 5 ppm of nanosensor solution for 1 hr. We then replaced the media with fresh ACSF and mounted the culture on the microscope for imaging. We used a 10× objective  to identify a dopaminergic neuron from the co-culture system. We then switched to 40× objective  to carry out activity imaging in the identified dopamine neuron. Before each imaging session, we record a GFP image of the TH signal in the green channel. We then switch to the NIR/SWIR channel for activity imaging. Imaging is carried out at frame rates of 10–20 frames per second depending on the brightness of the DopaFilm substrate (50–100 ms exposure time). If the neuron exhibits autonomous spiking activity, we do not apply any external stimuli. If the neuron does not exhibit spiking activity, we apply optical stimulus to evoke activity. Optical stimulation is driven through a custom-built MATLAB code that simultaneously controls and synchronizes the camera and optogenetic stimulation light source through external TTL pulses and time-locks stimulation times with specific camera frames. Optical stimuli applied to evoke activity are: 5 pulses, 25 Hz, 5 ms pulse width, and at power of 1 mW/mm2. We use ChrimsonR to evoke activity and use 561 nm LED (Thorlabs LED4D067 with DC4100 driver) of the four-wavelength LED light source for stimulation. For repeat stimulation experiments, we used a rest period of ~ 2 min in between sessions. All imaging sessions were carried out at room temperature in ACSF. When imaging in Ca2+-free media, we switched our imaging buffer to Ca2+-free ACSF. When imaging in NOM or TTX, we spike the imaging ACSF with a known volume and concentration of a NOM or TTX solution to attain the target concentration of NOM or TTX. We incubate the culture for 10 min at room temperature before carrying out postdrug activity imaging.Primary midbrain neuronal cultures were imaged 35–40 DIV. At the beginning of a typical imaging experiment, we replaced the NbActiv4 media with ACSF composed of, in mM: 124 NaCl, 2.5 KCl, 1.25 NaH2, 1 MgCl2, and pH 7.4 adjusted with NaOH; osmolality 300 mOsm. Pipette internal solution contained (mM): 130 K-gluconate, 10 HEPES, 10 KCl, 1 EGTA, 4 Mg-ATP, 10 phosphocreatine, 0.3 Na-GTP, and pH 7.3 adjusted with KOH; osmolality 280 mOsm. Whole-cell patch pipettes had tip resistances of 3–7 MΩ and data were collected from cells with access resistance <25 MΩ. Membrane potentials were offline corrected for a –12 mV liquid junction potential. The electrophysiological parameters of neurons including capacitance, input resistance, and access resistance were determined in voltage clamp configuration by a 5 ms pulse from –80 mV to –70 mV. In current clamp, the resting membrane potential was recorded prior to current injection with series resistance compensated up to 80%. Rheobase firing threshold was measured by injecting cells with sequentially increasing +10 pA current injections of 100 ms duration. The minimum current required to evoke an action potential was defined as the rheobase. The firing rate, voltage threshold, and action potential properties were determined from a 1 s injection of current at ~ 2× rheobase. The minimum time between current injections under each protocol was 5 s. Data are presented as mean ± SD, and statistical comparisons were performed by unpaired Mann-Whitney U tests.For electrophysiological recordings from hippocampal neurons, cultures were prepared as described above but with tissue extracted from the hippocampus only. Recordings were performed 14–16 days after plating at room temperature in the whole-cell voltage clamp and current clamp configurations using an Axopatch 200B amplifier (Molecular Devices). Data were acquired using pCLAMP 10 software and a Digidata 1440 A (Molecular Devices), filtered at 5 kHz, and digitized at 20 kHz. Cells were bathed in external solution containing (mM): 140 NaCl, 10 HEPES, 5 glucose, 4 KCl, 2 CaCl0 stack and generates correlated pixel components that we identify as hotspots of activity. To compute ΔF/F0, we first convolve the fluorescence time series movie  with a 2D gaussian (σ = 0.5 pixels). To obtain F0, a leaky cumulative minimum is calculated for each pixel in F, followed by repeated lowpass filtering to converge on a smooth F0 that obeys the minima. ΔF is then calculated as F–F0. Next, we partition the image into correlated components by applying non-negative matrix factorization (NNMF) to ΔF with sparsity and contiguity constraints. This partitions the image into largescale correlated regions (i.e. into pixel clusters with correlated activity). See 0 is then calculated using the component’s clustered pixels. Heatmaps and timeseries of ΔF/F0 traces are then generated for each movie file using native MATLAB capabilities. For simplicity, we refer to ∆F/F0 as ∆F/F in most of our figures and text. To calculate temporal FWHM and determine amplitude of ∆F/F traces, we used MATLAB’s peak finder program. To calculate τoff, we fit first-order decay curves to identified local maxima. To determine τpeak, we used temporal difference between the location of the peak and the maxima of the second derivative of the trace. Statistical model fits and model discrimination tests were carried out using code written in MATLAB. All data analyses were done in MATLAB 2020 using the code located at the following repository: https://github.com/davidackerman/nnmf; swh:1:rev:cff3d16a70b4fd4354e486347e38e7a9a1d167be). To compute centroids of hotspots and to generate overlay images, we used Fiji  were computed and reported from Student’s unpaired two-tailed t-test unless noted otherwise. We used MATLAB’s built-in boxplot algorithm to display distributions for some of our data. The definitions for relevant features of the box plot are as follows: red line = median, edges of box: 25th and 75th percentile, top and bottom hash lines: minimum and maximum values of non-outlier data, and red points: outlier data.1) antibody. Anti-mouse and anti-guinea pig secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor plus 647-(Invitrogen) were applied at 1:2000 dilutions, and. anti-chicken secondary antibody conjugated to Brilliant Violet 421 (Jackson Immunoresearch laboratories) was added at 1:1000 dilution. Super-resolution microscopy was performed at the Janelia Imaging Facility on a Zeiss LSM 880 inverted confocal microscope equipped with a Plan-Apochromat 63×/1.4 NA oil objective in Airyscan mode using a hexagonally packed 32-channel gallium arsenide phosphide photomultiplier tube . Dopaminergic neurons of interest from the activity images were located by using MatTek dish grid location in the reflection mode.After activity imaging, cultures were fixed using 4% paraformaldehyde in 1 × PBS for 15 min at room temperature and washed twice with 1 × PBS. Next, cells were permeabilized/blocked in with 2% BSA in 1% Triton X-100 in 1 × PBS (PBST) for 1 hr at room temperature. All primary antibodies were incubated overnight at 4°C and secondary antibodies for 1 hr at room temperature in PBST containing 1% BSA. Primary antibodies used in this study include chicken anti-TH , mouse anti-TH , rabbit anti-TH , mouse anti-Bassoon , chicken anti-MAP2 , and guinea pig anti-synaptobrevin-2 . TH-GFP signal (after activity imaging) was amplified using a rabbit anti-GFP  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Lu Chen as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Bianxiao Cui (Reviewer #3).Thank you for submitting your article "Visualizing Synaptic Dopamine Efflux with a 2D Composite Nanofilm" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) All reviewers agree that the key claims of the manuscript are well-supported by the data, therefore, no new experiments are required for the revision.2) There are a few inconsistencies in the data presentation, and some aspects of data analysis could be improved. Please see the detailed comments/suggestions made by the reviewers below.Reviewer #2 (Recommendations for the authors):The following are suggestions for additional analyses that may make the presentation of the data more clear and more broadly useful.Release probability. The stimulated DA signals here provide an excellent opportunity to examine release probability for a single release site. If there were many stimuli delivered during each experiment, the authors should consider reporting the probability of seeing release events in the axon and dendrites.Quantal release – axons. Data presented in Figure 4 suggests detection of the quantal release of DA . Please quantify the frequency of release events in +TTX from a single release site and/or across release sites. Do 'quantal release' events have roughly the same kinetic profiles across imaging sites? Seems like this should be true if a single vesicle is being released.Figure 4F shows a frequency histogram for dF/F amplitudes in TTX. Are the data values from the same release site or from multiple release sites? Also, is there a statistical method that may be used to demonstrate that the fits provided represent clearly distinct fits rather than a skewed distribution?Did the authors test for quantal release from the dendrites in the presence of TTX? If so, please comment.Reviewer #3 (Recommendations for the authors):In this article, the authors monitored the spatiotemporal dynamics of dopamine release in dendritic processes of dopaminergic neurons using synthesized DopaFilm nanosensors. The strength of this work is included in the "Public Review" section and won't be repeated here. Overall, this is a high-quality study and the experiments were systematically carried out. I support the publication of this work with revisions. Below are some questions and comments that shall be addressed.1. During the co-culture of dopaminergic neurons with hippocampal and cortical neurons, are the density well controlled such that these neurons mostly form single-layer on the DopaFilm such that all dopamine release measured is from the actual dopaminergic neurons in direct contact with the film?2. In Figure 3, the authors showed the localized dopamine release after stimulation. Would the time interval between stimulation events affect the localized signal?3. Following up on the previous point, the authors also showed some spots with no dopamine release. What is the percentage of these 'silent' sites compared to all observed dopamine release events?4. The authors mentioned the dendritic release of dopamine is less diffusive than axon release. However, if the diffusive coefficient of dopamine is constant within the DopaFilm, then what is the biophysics of this observation? Would this have anything to do with, for example, the effective surface area/numbers/spatial distribution of the release sits? More comments are needed here.5. The authors mentioned DopaFilm transients clearly localized to MAP2-positive and TH-GFP- processes, but later showed dopamine release is more directly related to Bassoon puncta. More clarification between dopamine release and these processes is needed. Reviewer #2 (Recommendations for the authors):The following are suggestions for additional analyses that may make the presentation of the data more clear and more broadly useful.Release probability. The stimulated DA signals here provide an excellent opportunity to examine release probability for a single release site. If there were many stimuli delivered during each experiment, the authors should consider reporting the probability of seeing release events in the axon and dendrites.We find this possibility intriguing and did consider it. However, we hesitated to calculate release probability because we are not certain about the number of action potentials that are evoked by our optical stimulation protocol, which likely evokes multiple action potentials. This is reflected in release probability estimates based on current data, which are very close to one (100%). Depolarization of dopamine neurons under electrophysiological control and simultaneous imaging from DopaFilm could permit evaluation of release probability with the resolution of a single bouton, which will permit examination of variance of synaptic release and estimation of intrinsic release probability under single spike conditions. Future studies from the lab will pursue this line of inquiry. For the current study, we will note that release from boutons appears to have a high probability of release based on our data that tracks time course of response across repeated simulations .Changes to manuscript in response to this comment:1. We added a phrase at the end of an existing sentence in the Results section:“In axon terminals, we observed that repeat stimulations can be carried out with rest periods of ~2-3 minutes between stimuli, giving rise to a consistent set of DopaFilm hotspot activities, and suggesting that release-competent dopamine neuron boutons likely have a high probability of release .”Quantal release – axons. Data presented in Figure 4 suggests detection of the quantal release of DA . Please quantify the frequency of release events in +TTX from a single release site and/or across release sites. Do 'quantal release' events have roughly the same kinetic profiles across imaging sites? Seems like this should be true if a single vesicle is being released.off) for TTX quantal events and compared those with τoff from axons in the same field of view before TTX application (for evoked release activities). We created cumulative frequency distributions of τoff values in TTX and without TTX (evoked activities) . We defined a parameter, τ10/90 as the range of τoff for values that fall in the 0.1 to 0.9 quantile window in the cumulative distribution curve. In axon terminals without TTX, we determined τ10/90 <inline-graphic mimetype="image" mime-subtype="png" xlink:href="media/image1.png" /> 1.95 s, whereas τ10/90 for TTX was 1.15 s, suggesting that quantal events have kinetic properties that are statistically less spread, consistent with the reviewer’s expectations.We now quantify the frequency of TTX quantal events pooled across many release sites. To provide better context, we also quantified the frequency of spontaneous release events and provided a comparison of their frequency of occurrence with those of TTX quantal events. Here, by spontaneous release events, we mean those that are not generated by stimulus-evoked nor cell-autonomous spiking events. These events tend to be stochastic and temporally uncorrelated when they occur, so they are easy to identify. Their peak ∆F/F also tends to be smaller. We showed that these spontaneous events have amplitudes that are comparable to those of TTX quantal events but exhibited temporal characteristics that are different . Intriguingly, we find that TTX events occurred with nearly twice the frequency of these spontaneous events . To show that TTX kinetics have “roughly the same kinetic profiles” compared to evoked release activities, we compute decay time constants . The spatial extent of DopaFilm fluorescence hotspots were diminished after application of TTX , and the peak amplitude of transients were smaller compared to evoked release . TTXp < 10-4 in unpaired ttest) . The decay time constants (τoff) for TTX transients were smaller than those of evoked releases (Mean ± SD (s): 3.83 ± 0.8 vs. 1.52 ± 0.53, Figure 4—figure supplement 1E). To evaluate the statistical spread of the observed τoff values, we created a cumulative frequency distribution of τoff values with and without TTX (for evoked release) and defined a parameter, τ10/90, as the range of τoff for values that fall in the 0.1 to 0.9 quantile window in the cumulative distribution curve . In axon terminals without TTX (for evoked release), we determined that τ10/90 ≈ 1.95 s, whereas τ10/90 for TTX data was ≈ 1.15 s, suggesting that TTX events have kinetic properties that are more similar to each other. Interestingly, we observed that TTX transients occurred with a frequency of (Mean ± SD) 0.29 ± 0.24 s-1 per release site, which contrasted with a frequency of (Mean ± SD) 0.16 ± 0.13 s-1 per release site for spontaneous activities before TTX addition .”∆F/F peaks were comparable to spontaneous activity peaks detected before TTX addition (Peak ∆F/F (%) (Mean ± SD): 5.3 ± 1.6 for spontaneous activity vs. 7.9 ± 4.6 for TTX) but exhibited different kinetic characteristics as measured by FHWM (s)  as a statistical model to fit the experimental data. We now provide a justification for choosing three Gaussians from among a possible set of GMM selections. Akaike Information criterion (AIC) scores are often used to discriminate among statistical model fits for experimental data, with lower AIC values corresponding to statistical models that explain the most variation with the least number of model parameters. We calculated AIC scores for GMMs containing one (single Gaussian) to four Gaussians . AIC scores decreased as the number of Gaussians increased, hitting a minimum for three component GMM. A three component GMM had much lower AIC score than a two component GMM . On the other hand, a four component GMM had a higher AIC score than a three component one, suggesting overfitting. Therefore, we conclude that three component GMMs are better than the alternatives explored.To explore the possibility that our experimental data can simply be explained by a skewed distribution instead of a GMM, we compared our three-component GMM with a lognormal distribution. Lognormal distributions are positively skewed and are frequently encountered in biological processes. A single lognormal distribution is a two-parameter statistical model (µ and σ) and would therefore be treated favorably in AIC scoring compared to an eight parameter three-component GMM . This is because AIC scoring rewards a model’s predictive power, while penalizing the number of parameters used to dissuade overfitting. When we compare the best fitting lognormal with our chosen three component GMM, the GMM still returned a lower AIC score than a lognormal fit, suggesting that it explained experimental data variance better than a skewed distribution. In addition to AIC, we used a secondary model discrimination method called a Q-Q plot (quantile-quantile plot) to support our conclusion that GMM is a better statistical model for the experimental data. In conclusion, while we do not know if our chosen model is the best statistical model from all models that can be considered for this task, it appears to be clearly better than the few alternatives we have considered here.Changes to manuscript in response to this comment:1. We produced a new supplementary Figure 4—figure supplement 3 to go along with the main Figure 4.2. We added a new paragraph to the Results section.The new text now reads:1 = 5.7%, µ2 = 9.6%, µ3 = 15.1% ∆F/F offered the best fit . The spacing of the mean parameters, µ, suggested that DopaFilm fluorescence transients in TTX are likely driven by a quantized biological process where each upward deflection in DopaFilm fluorescence represented some integer multiple of a unitary fusion event. A four component GMM returned a higher AIC score than the three component AIC, suggesting overfitting . We considered the possibility that our experimental data may simply be better described by a positively skewed distribution function instead of a GMM. To evaluate this possibility, we compared our chosen three-component GMM with a lognormal distribution. Lognormal distributions are positively skewed and are frequently encountered in biological processes.35 A single lognormal distribution is a two-parameter statistical model (µ and σ) and would therefore be treated favorably in AIC scoring compared to an eight parameter three-component GMM . AIC rewards a model’s predictive power, while penalizing the number of parameters used. In this way, multiple models fitting the same data can be compared. Here, we saw that the GMM’s AIC score was better than that of the lognormal model , suggesting that GMM explained the observed experimental variance better than a simple skewed distribution. In addition to AIC, we employed a graphical model discrimination method called Q-Q plot (quantile-quantile plot), which also supported our conclusion that GMM is a better statistical model for the experimental data . In sum, our experimental results and statistical analysis demonstrate that DopaFilm transients that are measured in TTX represent quantal release events of dopamine and, to our knowledge, offers the only experimental observation of putative single-vesicle fusion events in which the released neurochemical can be visualized in both spatial and temporal domains.”“We pooled peak ∆F/F values obtained from TTX hotspot traces across the field of view of imaging and generated a histogram distribution of the peak ∆F/F values . Inspection of single traces and the pooled histogram data suggested that DopaFilm transients in TTX may represent quantal events of different sizes or integer multiples of the same quantal size. To rationalize our experimental observation, we explored the suitability of statistical model fits for our experimental data. We first considered the use of Gaussian mixture models (GMM), which are often employed in quantal analysis of synaptic transmission events. Using Akaike information criteria (AIC) as a discriminating score, we evaluated the relative qualities of GMMs composed of one to four components. We found that a three-component GMM whose mean parameters (µ) are µDid the authors test for quantal release from the dendrites in the presence of TTX? If so, please comment.TTX data that we have so far collected in somatodendritic regions eliminates activity. However, we have not fully explored if TTX quantal events can be seen in dendrites and we’re unable to make a conclusion based on the data that we have. We do observe stochastic spontaneous events in dendrites, which we already state in the manuscript.Changes to manuscript in response to this comment:No changes were made in response to this comment.Reviewer #3 (Recommendations for the authors):In this article, the authors monitored the spatiotemporal dynamics of dopamine release in dendritic processes of dopaminergic neurons using synthesized DopaFilm nanosensors. The strength of this work is included in the "Public Review" section and won't be repeated here. Overall, this is a high-quality study and the experiments were systematically carried out. I support the publication of this work with revisions. Below are some questions and comments that shall be addressed.1. During the co-culture of dopaminergic neurons with hippocampal and cortical neurons, are the density well controlled such that these neurons mostly form single-layer on the DopaFilm such that all dopamine release measured is from the actual dopaminergic neurons in direct contact with the film?We would like to thank the reviewer for this important comment. Controlling the density of neurons in dish is very important in the protocol. While we did note this importance in the original text, we acknowledge that is should be emphasized more.Changes to manuscript in response to this comment:1. We now have the following sentences in the manuscript:+, ensuring that detected activity can be assigned to single identifiable processes with minimal crosstalk.”“During seeding, we optimized density of cells on DopaFilm such that mature neurons formed a monolayer of cells on the substrate. Optimized seeding density allowed us to record activities arising from isolated dopamine neurons where no other neurons in the vicinity of the neuron of interest were TH2. In Figure 3, the authors showed the localized dopamine release after stimulation. Would the time interval between stimulation events affect the localized signal?We addressed this point as a response to comments from reviewer 2. We will note that for multiple simulations applied within one imaging session, some short-term depression can be seen . However, with rest periods of 2-3 minutes between imaging sessions, we see a robust response across stimulations .Changes to manuscript in response to this comment:1. New Figure 3—figure supplement 1A-B addresses this comment.3. Following up on the previous point, the authors also showed some spots with no dopamine release. What is the percentage of these 'silent' sites compared to all observed dopamine release events?This is the same comment (#1 above) that is repeated here. It has been addressed.4. The authors mentioned the dendritic release of dopamine is less diffusive than axon release. However, if the diffusive coefficient of dopamine is constant within the DopaFilm, then what is the biophysics of this observation? Would this have anything to do with, for example, the effective surface area/numbers/spatial distribution of the release sits? More comments are needed here.We thank the reviewer for this interesting question, and we agree that some discussion related to this is warranted. We think that these differences in spatial propagation of dopamine released in axons and dendrites are likely driven by biological differences. One key difference is the density of release sites. Axonal arbors of dopamine neurons are highly ramified and produce a large number of release sites (boutons) per unit area. On the other hand, dendritic processes tend to be less dense and their ramifications are not as elaborate. Therefore, there is a notable difference in the “release site density” that explains some of these differences. Additionally, even on a per-release site bases, axon terminals appear to be more release capable per stimulation . It is possible that more release-ready vesicles are available in axons than dendrites. The nature of vesicles in dendrites of dopamine neurons is not well understood and it will be interesting to examine dendritic release site ultrastructure with electron microscopy and compare that with axons. These will be explored in future studies.Changes to manuscript in response to this comment:1. We added the following statement in the Discussion section:“These differences may be driven by the dense axonal arbors of dopamine neurons, which give rise to higher number of release sites per unit area or could be driven by intrinsic differences between axonal and dendritic release sites.”5. The authors mentioned DopaFilm transients clearly localized to MAP2-positive and TH-GFP- processes, but later showed dopamine release is more directly related to Bassoon puncta. More clarification between dopamine release and these processes is needed.We regret this potential source of confusion. MAP2 is a protein that we used to distinguish between axons and dendrites. MAP2 is absent in axons but it is enriched in dendrites. It is generally expressed throughout the cytoplasm of dendritic processes. On the hand, Bassoon is a protein that is selectively enriched at neurotransmitter release sites. Bassoon labeling is punctate. Therefore, it is possible to have both MAP2 and Bassoon signals at a given location. In our study, we used MAP2 to confirm that a neuronal process is dendritic, and then looked for Bassoon puncta in areas where we see hotspots of activity.Changes to manuscript in response to this comment:1. We now add the following clarifying sentence in the Results section:40; therefore, MAP2 immunoreactivity was used to distinguish between the dendritic vs. axonal nature of observed transients.”“MAP2 is enriched in dendrites and is absent in axons


On 18 October 2022, the German national public health institute, Robert Koch Institute (RKI), convened a meeting to launch a new World Health Organization (WHO) Collaborating Centre (CC) for AMR, Antimicrobial Consumption (AMC) and Health Care-Associated Infections (HAI) and to bring together international experts for a panel discussion on the critical global gaps and existing opportunities to leverage for improved AMR prevention and control.In 2019, an estimated 4.95 million deaths were associated with bacterial antimicrobial resistance (AMR), making it one of the leading causes of death worldwide [The World Health Organization (WHO) coordinates a network consisting of more than 25 collaborating centres (CC) from 17 countries across the globe, and since 2019 RKI supports the network coordination .The WHO CC AMR launch event was held following the World Health Summit (WHS) in Berlin, Germany, in the same week, during which political leaders and speakers stressed the fundamental need to “act together and effectively to ensure a healthy life for all on a healthy planet.” The WHS included a session on AMR as a global challenge and the task of national public health institutes in combatting its threat . In thisThe event was opened by Sara Tomczyk  and welcome remarks were given by Carmem Pessoa-Silva , Benedetta Allegranzi , and Dagmar Reitenbach . Under the moderation of Johanna Hanefeld , a panel discussion was then held with Hanan H. Balkhy , Ruth Schumacher (German Development Agency (GIZ), Berlin, Germany), Sabiha Essack  and Muna Abu Sin . Both in-person and digital participation from the broader international community working on AMR prevention and control was offered.Throughout the remarks and discussions, five key themes emerged including the importance of: (i) a multisectoral approach with societal and political engagement; (ii) a patient-centred focus; (iii) universal health coverage for diagnostics, treatment and infection prevention and control (IPC); (iv) strengthened antimicrobial stewardship; and (v) an effective role of national public health institutes and surveillance systems.The coronavirus disease COVID-19) pandemic exposed the gaps and dysfunctionalities in global health governance and local systems. Speakers underlined the need for actors across sectors to leverage these lessons and work together in more meaningful and actionable ways on the prevention and control of AMR. It was agreed that this should include initiatives to improve health literacy on AMR and make the economic case for its prevention and control. Policymakers, practitioners and civil society should work together to develop consistent, accurate and tailored messages to encourage societal and political ownership on preventing and controlling AMR, taking local circumstances into account. Examples given included: (i) building on the ‘lived experience’ of the health, social, economic and political impacts of the COVID-19 pandemic but framing AMR as a One Health issue with even more far-reaching impacts as demonstrated by data such as the recent global AMR burden study [9 pandemiAs it relates to healthcare service delivery and public health measures for the prevention and control of AMR, speakers agreed that the individual patient needs to be more explicitly at the centre of planning and implementation. AMR prevention and control measures should be patient- or people-centred with a need for equitable access to diagnostics, treatment and immunisation. This could also include collaboration with patient organisations and consideration of patient needs in activities such as AMR, AMC and HAI surveillance and data collection which can lead to better patient outcomes. WHO is currently working on implementing a people-centred approach to address AMR by supporting countries with national action plans, technical programmes, advocacy, community engagement and further efforts. An urgent need for support of local and national clinical networks as well as support of a global microbiological laboratory network to improve diagnostics was emphasised in this context. Improving diagnostics for patients with timely results that allow for improved patient management and surveillance quality should be among the first steps.There was common acknowledgement among speakers that affordable procurement of diagnostics and antimicrobial therapy and reliable supply chains continues to be a notable challenge in low- and middle-income countries, hindering improved AMR prevention and control. Long-term support of development of diagnostic capacities which would feed the surveillance data in those countries is needed. Diagnostic material such as blood culture bottles remain expensive, and in many countries, patients must pay for diagnostics out of pocket. These issues should be prominently included on universal health coverage agendas, and advocacy and mitigation could include ideas such as intensified public-private engagement and leveraging of combined purchasing power of countries for better access to diagnostic and antimicrobial products among other ideas.Speakers also highlighted the importance of strengthening antimicrobial stewardship efforts. Globally, this necessitates balancing three important aspects: (i) equitable access to antimicrobial therapy in the context of universal health coverage; (ii) conservation to make the best use of current antimicrobial effectiveness by reducing demand, e.g. through critical use of antibiotics, infection prevention and control, vaccination, provider and public education, and restriction to ‘last-resort’ antibiotics; and (iii) innovation to improve the efficacy of current therapy and develop new antimicrobial agents through research and development (R & D) schemes.The COVID-19 pandemic also underscored the important role that effective national public health institutes and timely surveillance data play in the prevention and control of infectious diseases. Speakers emphasised that these institutions and activities play the same critical role for the AMR pandemic. In the case of AMR, this should extend to integrated One Health surveillance systems across human, animal and environmental domains to more effectively mitigate AMR. Speakers also addressed the importance of implementation research. Many of the evidence-based interventions addressing AMR have been based on data from high-income countries and such evidence cannot always be directly translated to low-resource settings. Thus, more (representative) data are needed in low-resource settings to tailor context-specific, cost-effective and sustainable interventions and facilitate their uptake into routine practice for the prevention and control of AMR in these settings. To carry out effective AMR surveillance and implementation research, critical gaps particularly affecting low-resource settings need to be addressed, such as lack of well-maintained laboratory equipment and consumables for bacteriology and quality control, trained personnel, reliable water, sanitation and hygiene systems in healthcare institutions, and information systems which can capture, manage and analyse data in a timely way. Generated surveillance data on AMR and AMC should be consequently used for political decision-making, infection prevention and control programmes as well as for advocacy.The discussions and conclusions on the five key themes in the meeting will be reflected in the new WHO CC at the RKI and fed by it into the ongoing planning and development of the WHO AMR Surveillance and Quality Assessment Collaborating Centres Network’s activities. More information on the Network and RKI’s role can be found under the following links:https://www.who.int/initiatives/glass/networkhttps://www.rki.de/EN/Content/Institute/International/WHO_CC_EIBT/Coordinator-WHO-Antimicrobial-Resistance-Network.html


Vaccination can reduce antibiotic use by decreasing bacterial and viral infections and vaccines are highlighted in the WHO Global Action Plan on Antimicrobial Resistance (AMR) as an infection prevention measure to reduce AMR. Our study aimed to analyze whether WHO Member States have developed AMR national action plans that are aligned with the Global Action Plan regarding objectives on vaccination.We reviewed 77 out of 90 AMR national action plans available in the WHO library that were written after publication of the Global Action Plan in 2015. Each plan was analyzed using content analysis, with a focus on vaccination and key components as defined by WHO , II. Operational plan, III. Monitoring and Evaluation plan).n = 13/13, the Eastern Mediterranean: n = 15/16, Europe: n = 10/14, the Americas: n = 8/8, South-East Asia: n = 8/11, and the Western Pacific: n = 13/15). Pneumococcal and influenza vaccination were most frequently highlighted (n = 12 and n = 11). We found indications that vaccination objectives are more often included in AMR plans from lower income countries, while higher income countries more often include specific vaccines. The key WHO components of national action plans were frequently not covered . In total, 33 countries (43%) included indicators (e.g. strategic objectives) to capture the role of vaccines against AMR.Vaccination was included in 67 of 77 AMR plans (87%) across all WHO Regions (Africa: While vaccination to reduce AMR is seen as an important global public health issue by WHO, there appears to be a gap in its adoption in national AMR plans. Country income levels seem to influence the progress, implementation and focus of national action plans, guided by a lack of funding and prioritization in developing countries. To better align the global response to AMR, our review suggests there is a need to update national action plans to include objectives on vaccination with more focus on specific vaccines that impact antibiotic use.The online version contains supplementary material available at 10.1186/s12992-022-00878-6. Antimicrobial resistance (AMR) remains an increasing threat to global health and it has been projected that in 2050, 10 million deaths a year will be attributable to drug-resistant bacteria –6.vaccination, where appropriate as an infection prevention measure, should be encouraged.” Vaccination is highlighted in Objective 3 of the WHO GAP-AMR [The WHO 2015) was one of the first global actors to acknowledge that “015 was oHaemophilus Influenzae type B (Hib) vaccines should be prioritized for its impact on antibiotic use and AMR. Solid evidence suggests that influenza and pneumococcal vaccination can reduce antibiotic use in different risk groups  [Streptococcus pneumoniae [S. pneumoniae was responsible for almost 600.000 deaths in 2019 and one in five deaths attributable to bacterial AMR occurred in children < 5 years [More recently, WHO (2020) has published an Action Framework ‘Leveraging Vaccines to Reduce Antibiotic Use and Prevent AMR’ in which they state that the increased uptake of Influenza, Pneumococcal vaccines (PCV), Typhoid vaccines (TCV) and ditions) , 8 and teumoniae , 9, 10.  5 years . Researc 5 years  and aver 5 years . It was  5 years , but als 5 years . These fTo our knowledge , it is uOur study aims to analyze whether AMR national action plans are aligned with the Global Action Plan on AMR and include strategic objectives on vaccination. It presents an overview of vaccination in national action plans on AMR, focusing on PCV, TCV, Hib vaccines, influenza vaccines, rotavirus and measles vaccines.We included national action plans on AMR that were available online on 22 July 2021 in the WHO library of AMR national action plans [The AMR national action plans were reviewed based on the information on vaccination and the operational structure. The 2016 manual for developing AMR national action plans  was usedInformation was extracted from the national action plans by three researchers . Each plan was analyzed using content analysis based on two main factors: 1) information on vaccination, with a focus on promoting vaccination, optimizing vaccination coverage and/or strengthening vaccination programs targeting the six selected vaccines, and (2) the WHO key components of national action plans. The analysis also focused on similarities with the GAP-AMR (as defined by Munkholm and Rubin ), extens informatThe association between income and vaccination (focus on objectives related to vaccination and the inclusion of specific vaccines) was explored using multi-level logistic regression analysis, including WHO region as a random effect (countries are nested in six regions) and income level as a fixed effect. The significance threshold was set at .05.n = 13), EMR (n = 16), EUR (n = 14), RAM (n = 8), SEAR (n = 11), and WPR (n = 15). The income level of these countries covered: LIC (n = 9), LMIC (n = 24), UMIC (n = 20), and HIC (n = 24). Most national action plans were published in 2017 (n = 32), following 2018 (n = 15) and 2019 (n = 15). The Supplementary File This study included 77 national action plans on AMR from the WHO Member States, available in the WHO library of AMR national action plans. This selection included countries from every WHO region: AFR n = 3, EMR (nn = 4), SEAR (n = 3), WPR (n = 2), and EMR (n = 1), did not mention (human) vaccination at all in their national action plans, while all countries in the WHO African Region (n = 13) and American Region (n = 8) have included vaccination. Seventeen countries reported information on the effect of specific vaccines on AMR, with 10 national action plans covering multiple vaccines as shown in Table n = 9) include vaccination, however only one LIC country (Afghanistan) also mentions specific vaccines in its national action plan. One LIC (11%), five LMIC (21%), two UMIC (10%), and nine HIC (38%) include specific vaccines.Vaccination was mentioned in 67 out of 77 national action plans (87%) across all WHO regions, see Fig.  WPR n = , and EMROf the 67 national action plans that highlighted vaccination, 33 countries (49%) developed specific indicators to promote vaccination  while 34 countries (51%) merely mentioned vaccination as an example. Table We noticed that AMR national action plans overlap  with other plans across WHO regions and income groups, focusing on objectives on vaccination and specific vaccines included in the plans . Countries include more objectives on vaccination (43%) compared to specific vaccines (22%). Table Figure p = .11; 95% CI .90-2.81). We found a weaker association, non-significant as well, in the opposite direction between income and the probability of including vaccination objectives in action plans; here an increase in income is accompanied by a slightly lower probability . The multilevel model shows that regional variation is larger when it comes to the inclusion of specific vaccines in national action plans compared to objectives on vaccination  included no key components and 19 (27%) included all key components (with 10 out of 19 countries in WHO EMR). Most countries include an operational plan (57%), followed by a strategic plan (47%) and M&E plan (40%). Table n = 33; 47%) included a strategic plan. The other 37 plans presented the objectives or strategies to tackle AMR throughout the text, focusing mainly on awareness, surveillance, infection prevention and control, antibiotic stewardship, and research and innovation. These main objectives are similar to the Global Action Plan on AMR, with additional country-specific objectives.The strategic objectives in national action plans are not always specified in the strategic plan, as less than half of the 70 AMR plans  increase national awareness and understanding of AMR, (2) enhance national surveillance of AMR, (3) reduce the incidence of infections and contain the spread of antimicrobial-resistant organisms through effective IPC (e.g. sanitation and hygiene), (4) optimize the appropriate use of antimicrobials in humans, animals and agriculture, and (5) increase investment in research and development (R&D) for new antibiotics, vaccines, tools and other interventions.Vaccination was often referred to in objectives related to infection prevention and control  and research and innovation . Out of the 33 strategic plans, 11 plans (33%) included the promotion of vaccination or strengthening of vaccination programs as a strategic objective.An implementation and operational plan was included in 40 out of 70 national action plans 57%). For each activity and sub-activity (what), these plans include a responsible entity (who), a timeline (when), and indicators, milestones or targets (how). Most operational plans are included in WHO EMR and AFR . The costs or estimated budget often, but not always, included a source of funding and are described for each objective and activity. Costs and/or funding is described for five objectives and activities specifically related to promoting vaccination, included in the national action plans of Eritrea, Libya, Nigeria, Tanzania and Tunisia (WHO EMR and AFR).Twenty-eight countries (40%) included a M&E plan. For each activity and sub-activity, the plan describes the indicator, method and targets (by year). Only seven national action plans (10%) described monitoring and evaluation of objectives related to promoting or strengthening vaccination coverage: Eritrea, Nigeria, Pakistan, Sierra Leone, Tanzania, Tunisia and Zimbabwe (WHO EMR and AFR).Globally, in a total of 77 AMR national action plans, vaccination is frequently mentioned (87%) but is not always recognized as a main objective in these plans (43%). Only five countries (6%) included all of the WHO key components (with a focus on promoting or strengthening vaccination) in their national action plan; these include, a strategic plan, an operational plan with costs and funding, and a monitoring and evaluation plan. We identified a clear variation in themes and objectives discussed in national AMR plans. At this moment, awareness to the potential of vaccination to reduce antibiotic use is low and a confirmed effective strategy is not utilized to its full potential in all WHO Member States. When updating AMR national action plans, countries should include strategic objectives to increase and promote vaccination and focus on vaccination programs that target specific vaccines.This review shows that vaccination is not highlighted in every AMR action plan, while there is considerable evidence on the effect of vaccination on antibiotic use (and AMR). Two systematic reviews by Buckley  and DoheA lack of implementation of available action plans is one of the main issues with realizing effective vaccination strategies against AMR. Two main barriers for implementation are a lack of funding and a lack of  prioritization of AMR, especially in LMIC . Global At the moment, the actions and objectives included in AMR national action plans differ between countries, while WHO directs all plans to be aligned with global frameworks like the GAP-AMR and to focus on five strategic AMR objectives. Research by Munkholm and Rubin (2020) shows that there is strong alignment with the objectives and corresponding actions outlined in the Global Action Plan, but not in the actual implemented policies . ExpertsThe differences in the quality of the action plans and their implementation can lead to inequity between countries in the fight against AMR. The SARS-CoV-2 pandemic has reaffirmed the importance of vaccination against infectious diseases. While AMR is often referred to as a ‘silent pandemic’, it can affect as many people as, and be influenced by, the COVID-19 pandemic , 29. Henn = 77) of AMR plans from countries with diverse income levels from different regions and we belief that this sample is representative of the income and regional diversity of all WHO Member States. The sample size does require considerations with the interpretation of  country comparisons and the small effect size identified through our data analysis. It is possible that a larger sample could result in a significant association between income and specific vaccines, and it would be therefore valuable to perform a more detailed country comparison in the future also as the WHO library is continuously updated when new plans are available.One limitation of this study is the use of the WHO database to select AMR national action plans. The library collects existing, publicly available, officially approved plans, with 83 plans available on October 2021 . We cannThis study gives an overview of countries that express attention to vaccination in their AMR national action plan. Future studies should also review country specific vaccination programs (e.g. influenza vaccination programs) or AMR policies  that relate to this topic. Differences in existing vaccination policies and vaccination coverage rates between WHO regions  might exIn short, we recommend that the progress of AMR national action plans with regards to the inclusion of vaccination is reviewed every 5 years  complementary to tracking national progress in the implementation of the plans, as it will take some time until the gap between objectives and practice is bridged. This means the implementation of AMR policies should not only be monitored by country self-assessment (e.g. TrACSS ), but alThis review is a first step to study the inclusion of vaccination in (inter)national policies. Extensive research has shown that vaccination can reduce antibiotic use and AMR, hence there is a benefit to include vaccination in global AMR policies and national action plans. Vaccination is frequently discussed in national AMR plans, however this topic is often not included in strategic objectives or outlined in detail in implementation and evaluation plans. Countries have paid most attention to pneumococcal and influenza vaccination and the attention to these and other specific vaccines appears to be linked to cultural, political and socioeconomic country characteristics that warrant further inquiry. The present lack of detail and awareness regarding the implementation of the national AMR plans and interventions related to vaccination are points of concern and require new, effective governance strategies to increase the global attention to vaccination. Thus, to better align the global response to AMR and national activities, our review suggests there is a need to update national action plans on AMR to include objectives on vaccination and specify vaccines of interest for priority action.Additional file 1.Additional file 2.


Anopheles subpictus complex (A and B) against DDT and pyrethroids, the occurrence of knockdown resistance (kdr) mutations in these forms, and the association of kdr mutations with insecticide resistance.To investigate the differential insecticide-susceptibility of two molecular forms of An. subpictus s.l., collected from coastal and inland areas of mainland India, were performed against DDT, permethrin and deltamethrin using the WHO standard insecticide susceptibility test kit. The mosquitoes were characterized for molecular forms using a diagnostic PCR developed in this study. Representative samples of An. subpictus molecular forms A and B were sequenced for a genomic region encompassing the IIS4-5 linker to the IIS6 segments of the voltage-gated sodium channel to identify kdr mutations. A common PIRA-PCR was developed for identifying L1014F-kdr mutation and used for genotyping in both molecular forms of An. subpictus.Insecticide susceptibility tests of An. subpictus was resistant to all three insecticides, i.e., DDT, Permethrin and deltamethrin, whereas Form B was categorized as ‘possibly resistant’ to these insecticides. Significantly higher mortalities in WHO insecticide susceptibility tests were recorded in Form B compared to Form A in sympatric populations. Molecular characterization of the IIS4-5 linker to IIS-6 segments of the voltage-gated sodium channel revealed the presence of two alternative nucleotide transversions at L1014 residue in Form A, both leading to the same amino acid change, i.e., Leu-to-Phe; however, such mutations could not be observed in Form B. PIRA-PCR-based kdr-genotyping of field populations revealed high frequencies of L1014F-kdr mutations in Form A and the absence of this mutation in Form B. The proportion of L1014F mutation was significantly higher in resistant mosquitoes following insecticide-bioassay with DDT (p<0.0001), permethrin (p<0.001) and deltamethrin (p<0.01) as compared to their susceptible counterparts.Molecular form A of An. subpictus complex in sympatric populations. The L1014F-kdr mutation was observed in Form A only, which was found to be associated with DDT, permethrin and deltamethrin resistance.Significant differences in insecticide susceptibility were found between two molecular forms of  Anopheles subpictus s.l. is widely distributed in the Oriental Region and some parts of the Australasian region [An. subpictus dominates [An. subpictus s.l. is highly complex. In India, Suguna et al. [An. subpictus from the Indian subcontinent revealed the presence of three molecular forms, namely Form A, B and C [An. sundaicus s.l. Wilai et al. [An. subpictus in Thailand, and more than one species in Indonesia based on ITS2  and cytochrome oxidase-1  sequences but none of them is identical to any molecular forms present in the Indian subcontinent but is closely related to molecular form B.n region –3. It han region –4. In Inn region , but repn region –7, whereominates –11. The a et al.  identifia et al.  also est B and C . Form A  B and C  and Sind B and C , therefo B and C  have desi et al.  have recAn. subpictus is poorly studied in the Indian population [An. subpictus in India. In this study, we report the differential susceptibility of two molecular forms prevalent in India (A and B) against DDT and pyrethroids, as well as the presence of knockdown resistance (kdr) mutations that are known to confer reduced sensitivity to these insecticides.Synthetic pyrethroids are the main insecticides being used in India for Indoor Residual Spraying (IRS) and for the Long Lasting Insecticidal Nets (LLINs) in addition to DDT and malathion. Carbamate has never been used in India. The insecticide susceptibility of pulation  and therAn. subpictus as molecular forms A and B following Sindhania et al. [In this study, we classified and described the molecularly identified sibling species of a et al.  in absenAn. subpictus, two coastal areas were selected where molecular forms A and B are present in sympatric association based on the previous study by Sindhania et al. [2 in Odisha state (eastern India), and (ii) villages near Puducherry (non-contiguous enclaves). The villages from where mosquitoes were collected near Chilka lake were Panasapada, Satapada, Brahmgiri, Sipakuda, Minsa, Siara, Gambhari  and near Puducherry were Kaliankuppam, Munjalkuppam, Sedarapet and Pillayarkuppam . In addition, mosquitoes were also collected from inland areas in northern India  where molecular form A is allopatric. Female An. subpictus s.l. were collected from their resting habitats (human dwellings and cattle sheds) in the morning between 6:00 to 8:00 AM with the help of a mouth aspirator and flash torch and brought alive in a field laboratory, where mosquitoes were maintained in an insect cage with access to cotton pads soaked with 10% glucose and water. Once the mosquitoes had attained a gravid stage, individual mosquitoes were morphologically identified in a test tube under a stereo microscope. Morphologically identified An. subpictus s.l. were pooled in a mosquito cage to lay their eggs. F1 progeny was obtained from a pool of 25–150 mosquitoes from each collection. The progenies were reared till their emergence into adults following the method described by Sharma et al [For determining the differential susceptibility of molecular forms of a et al. : (i) vilAn. subpictus s.l. mosquitoes were transferred in each holding tube provided with the kit which were subsequently transferred to an exposure tube lined with the insecticide paper impregnated with diagnostic doses  along with appropriate controls. Mosquitoes were exposed to insecticide-impregnated paper for one hour and transferred back to the holding tubes and placed in a field laboratory maintained at a temperature of 25±1°C. A cotton pad soaked in sugar solution was placed on top of the tube during recovery to maintain adequate humidity. Dead and alive mosquitoes after 24 hours of insecticide exposure were re-identified for species under a dissecting microscope. The corrected mortalities of An. subpictus s.l. were estimated. The alive and dead mosquitoes tested against each insecticide were preserved in microfuge tubes containing isopropanol for molecular studies.Insecticide susceptibility assays were performed using the WHO adult mosquito insecticide susceptibility test on three-to-four-days-old and sugar-fed adult female mosquitoes (F1). Up to 25 DNA was isolated from individual mosquitoes by the method of Black and Duteau An. subpictus s.l. was performed following keys by Christophers [An. subpictus, a new PCR assay was developed due to the limitations of previously designed methods for this purpose. The earlier molecular methods developed by Surendran et al. [An. stephensi and An. vagus, respectively, both of which can be mistaken as An. subpictus s.l. morphologically in some cases where specimens have lost their scales. Two new species-specific primers, SubA1R and SubB1R, were designed which were specific to molecular form A and B, respectively and used with the universal primer SubF designed by Surendran et al. [An. stephensi, An. vagus and An. sundaicus which may be misidentified as An. subpictus in case of lost hair and scales.Morphological identification of stophers . For then et al.  and Sindn et al.  had shown et al. . Similarkdr mutations in the VGSC of An. subpictus, primers were designed for the amplification of partial VGSC covering a region of IIS4-5 linker to IIS6 segments, which included two kdr locus M914 and L1014 residues (amino acid positions based on housefly sequence) based on sequences available for An. epiroticus, which is a closely related species of An. subpictus [kdr loci M918T (super-kdr) and L1014F/C/S (kdr). PCR amplification was carried out in a reaction mixture (20 μl) using DreamTaq Green PCR Master Mix (2X) (ThermoFisher Scientific) with 0.5 μM of primer Sub1F, and Sub2R. The PCR conditions were: initial denaturation at 95°C for 5 min followed by 35 cycles each with 95°C for 30 sec, 52°C for 30 sec, 72°C for 90 sec and a final extension at 72°C for 7 min. 3 μL of the PCR product was electrophoresed on 1.5% agarose gel, visualized under UV, and the remaining PCR products were purified using Exo-Sap IT (Thermo Fisher Scientific) and subjected to sequence termination reaction using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem). Since initial sequencing using these two primers often failed due to indel present in the intron between two exons containing the above-mentioned kdr mutations, each PCR product was subjected to four sequencing termination reactions using two additional internal primers Sub1R and Sub2F (For the identification of nd Sub2F  in additApoI/XapI (5′-R|AATTY-3´) in the PCR amplicon in case of presence of Phe codon (TTT or TTC) at L1014 residue. This primer a tail of 20 bp was introduced at the 5´ end to increase the size of the PCR-RFLP product for better resolution on the agarose gel. Two reverse primers SubA_kdR and SubB_kdR were designed based on sequences of molecular forms A and B, respectively. The primer SubA_kdR is identical to SubR [ApoI/XapI restriction site already present in the sequence  is highly diverse among two molecular forms, this needs the development of multiple ASPCR (for two mutations and two molecular forms). We, therefore, designed a single PIRA-PCR that could detect both mutations in each molecular form. Three primers i.e., Sub_kdF , SubA_kdR (specific to Form A) and SubB_kdR (specific to Form B), were designed for PIRA-PCR. The universal primer Sub_kdF is a modification of primer SubF designed by Singh et al.  wherein  to SubR  and has sequence . The expApoI restriction enzyme (New England Biolabs Inc.). The reaction mixture was incubated at 50°C for 4 hours or overnight at room temperature. The product was electrophoresed on 2.5% agarose gel and visualized under UV (The PCR amplification for the PIRA-PCR was carried out in a reaction mixture of 20 μl using GoTaq Green Master Mix 2X (Promega) and primers Sub_kdrF, SubA_kdr and SubB_kdr (0.25 μM each). The PCR conditions were: Initial denaturation at 95°C for 3 min followed by 35 cycles each with 95°C for 30 sec, 47°C for 30 sec, 72°C for 30 sec and a final extension at 72°C for 7 min. Five μl of PCR product was subjected to restriction digestion in a reaction mixture of 20 μl containing 2X CutSmart Buffer and 5 units of under UV .kdr allele with insecticide resistance phenotype, the odd ratio (OR) and Fisher exact test were used.Pearson chi-square test was performed to compare differences in percent mortalities observed during bioassay using Microsoft Office-Excel. To establish the association of the The corrected percent mortalities in mosquitoes after 1-hour of exposure to diagnostic doses of insecticides along with the 95% confidence interval have been shown in A), i.e., A>T and A>C, in molecular Form A, both leading to the same amino acid substitution, i.e., Leu→Phe , permethrin  and deltamethrin .The proportions of L1014 and 1014F alleles in the dead and alive mosquitoes following WHO’s standard insecticide susceptibility test against DDT, deltamethrin and permethrin have been shown in kdr mutations that confer resistance against DDT and pyrethroids. These two molecular forms are highly diverged to the extent that Form B is phylogenetically closer to the sundaicus complex; thus various authors [The importance of recognition and identification of sibling species has been realized for decision-making process in vector control programme owing to differences in biological characteristics, such as host preferences, breeding behaviour, malaria vectorial competence, resting behaviour (outdoor/indoor) and responses to the insecticides. Significant differences have been reported in host preferences , role in authors , 12–13 hAn. subpictus populations [An. sundaicus s.l., i.e., species B of the An. subpictus, collected in Kilinochchi were completely susceptible to 0.05% deltamethrin and 5% malathion and resistant to 4% DDT, whereas those from Jaffna were relatively susceptible to all three insecticides [An. subpictus s.l. against DDT, deltamethrin and permethrin [kdr in molecular Form A also varied in different populations which were found to be higher in Nuh (51%) followed by Puducherry and Chilka with 45% and 40%, respectively. In another study [kdr mutation in Form B. There are two reports of the presence of L1014F kdr mutation in Sri Lanka, one reporting TTA>TTC substitution in Jaffna city [kdr mutation. The sequence analysis of DNA sequences generated through these two studies and submitted to GenBank revealed that the mosquitoes with kdr mutation belong to molecular Form A based on intron sequence. In this study, we recorded two alternative kdr mutations in all three populations of Form A but not in Form B. Though both molecular forms of subpictus in the coastal region might have experienced identical insecticide selection pressure, insecticide resistance and two independent kdr mutations evolved only in Form A. The possible explanation for this is the widespread presence of Form A in the Indian subcontinent which has a higher chance of selection of new genetic changes/mutations as compared to Form B which is restricted to coastal areas only.Not much information is available about insecticide resistance in Indian ulations . In Sri ulations  have repcticides . Such dicticides . In our rmethrin  which maer study  in the mfna city , and anofna city , both leAn. subpictus revealed the presence of only two molecular forms based on 28S and ITS2 sequences which have been described as molecular forms A and B by Sindhania et al. [An. stephensi and An. vagus respectively, which are often misidentified as An. subpictus s.l. in specimens with lost hairs/scales. Therefore, in this study, we designed a new method to differentiate two molecular forms of An. subpictus. This method can be used for the large-scale identification of molecular forms. The molecular tools developed for the identification of molecular forms can be used in the vector control programme for vector surveillance which will aid to understand the seasonal dynamics and geographical distribution of different molecular forms of An. subpictus. Earlier studies have indicated the prevalence of two molecular forms/species of An. subpictus varies in different seasons [The identification of sibling species is challenging in field studies. The chromosomal method is difficult to carry out due to various reasons, mainly, due to the requirement of live adult female mosquitoes with semi-gravid conditions which constitute a small proportion of a population and a difficult procedure that required a highly skilled technician to read polytene bands. The chromosomal technique has never been utilized in field studies for sibling species identification after Suguna  and Sugua et al.  which caa et al.  and Sinda et al. , both of seasons , 8, 10. kdr, was absent in both forms. We developed a common PIRA-PCR assay for the identification of L1014F mutation raised from two alternative transversions in both molecular forms. PIRA-PCR is advantageous over allele-specific PCR (AS-PCR) because it is highly specific due to the high specificity of the recognition site of specific restriction enzyme, whereas primers designed based on a single SNP are prone to non-specific extension [The identification of genetic markers associated with resistance was included in the priorities of the WHO Global Plan for Insecticide Resistance Management . In thisxtension , 33.An. gambiae which showed resistance against DDT (>24-fold) and pyrethroids [kdr mutation along with other mechanisms of metabolic resistance is important in the vector control programme for effective insecticide resistance management.In this study, we showed that L1014F mutation is associated with resistance against DDT and pyrethroids. We observed that this mutation conferred significant protection against all three insecticides tested, i.e., DDT, permethrin and deltamethrin. Similar results were also observed during functional validation of this mutation in CRISPR/Cas9 modified ethroids . In thisethroids  which isethroids . Thus moOne limitation of this study is that the number of mosquitoes tested for insecticide bioassay in one of the two coastal areas  is not adequate as per WHO norms. However, the susceptibility status of molecular Form A and B as determined in this area is at par with the results obtained in another coastal area, i.e., Puducherry, and serves as a supplementary result.An. subpictus against DDT and pyrethroids in addition to the presence or absence of L1014F-kdr mutation in sympatric populations which may have experienced a similar degree of insecticide exposure. Such information has an important bearing in vector management, especially of species B which is considered to be an important malaria vector in India.In conclusion, the current study provides information on the differential susceptibility of molecular forms of S1 Table(PDF)Click here for additional data file.


Topic modeling is an active research area with several unanswered questions. The focus of recent research in this area is on the use of a vector embedding representation of the input text with both generative and evolutionary topic modeling techniques. Unfortunately, it is hard to compare different techniques when the underlying data and preprocessing steps that were used to develop the models are not available. This paper presents two secondary datasets that can help address this gap. These datasets are derived from two primary datasets. The first consists of 8145 posts from the r/Cancer health forum and the second consists of 18,294 messages submitted to 20 different news groups. The same preprocessing procedure is applied to both datasets by removing punctuation, stop words and high frequency words. Each dataset is then clustered using three different topic modeling techniques: pPSO, ETM and NVDM and three topic numbers: 10, 20, 30. In addition, for pPSO two text embeddings representation are considered: sBERT and Skipgram. The secondary datasets were originally developed in support of a comparative analysis of the aforementioned topic modeling techniques in a study titled “Comparing PSO-based Clustering over Contextual Vector Embeddings to Modern Topic Modeling” submitted to the Journal of Information Processing and Management. The present paper provides a detailed description of the two secondary datasets including the unique identifier that can be used to retrieve the original documents, the pre-processing scripts, the topic keywords generated by the three topic modeling techniques with varying topic numbers and embedding representations. As such, the datasets allow direct comparison with other topic modeling techniques. To further facilitate this process, the algorithm underlying the evolutionary topic modeling technique, pPSO, proposed by the authors is also provided. Value of the Data•Public sharing of the fully labeled data rather than a subset of the data generated by the topic models allows the replication and validation of the results as well as enables direct comparison with competing topic modeling techniques.•Topic modeling and health science researchers can derive benefit from this data.•A well-defined benchmark from two different domains  is an opportunity for a shared baseline for various NLP applications. It also allows for the exclusion of the text pre-processing techniques as a source of difference between various studies. The current benchmark can also be combined with other benchmarks, such as the one offered in •The labeled documents can help promote retrospective studies that investigate topics important to the r/Cancer subscribers.1The data release consists of two types of files: document and keyword files. The document file includes two columns: the document ID which uniquely identifies the raw document in the source archive, and the corresponding topic cluster ID assigned by pPSO. This file is only available for pPSO because this topic modeling technique is a hard classifier that assigns each document to a single topic. The other two techniques  generate a probabilistic distribution of each document across all topics.The second type of file is a keyword file which is provided for all topic modeling techniques. The file is organized by topic and includes the top ten topic keywords for each topic. An example keyword file is shown in For each dataset and topic modeling technique, the number of topics was varied from 10, 20 to 30 topics. While the topics identified by each topic modeling technique for a given dataset may differ, the main themes are usually consistent. 2•Data cleaning: This step includes the removal of stop words, high frequency words, and punctuation from raw documents. The text is also forced to lower case.•Vector embedding: In this step, the initial embedding vector for each document produced in the previous step is generated from the pretrained language model sBERT •Topic modeling: The source code for topic modeling using pPSO on the reduced embedded vectors allows for the clustering of the documents into distinct topics. The source code for ETM and NVDM can be found in ,5, respe•Data analysis: This is the final step in the pipeline. The topic keywords are extracted for each topic cluster and the topic coherence and diversity are computed.The protocol used to generate the topic models is provided under methods in the Repository. This protocol defines a data processing pipeline that is comprised of multiple steps:The raw r/Cancer data are extracted from a public domain archive Samuel Miles: Methodology, Software, Formal analysis, Investigation, Data curation, Writing – original draft. Lixia Yao: Conceptualization, Supervision. Weilin Meng: Conceptualization, Investigation, Supervision, Project administration. Christopher M. Black: Supervision, Project administration. Zina Ben-Miled: Conceptualization, Validation, Investigation, Writing – review & editing, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.