The scale and associated costs of herbarium digitization make process automation appealing. One such process for many workflows is the association of specimen image files with barcode values stored with the specimen. Here, an innovation is presented that improves the speed and accuracy of decoding barcodes from specimen images.Geometric features common in barcodes are used to identify the regions of specimen images that are likely to contain a barcode. The proposed regions are then combined into a significantly reduced composite image that is decoded using traditional barcode reading libraries. Tested against existing solutions, this method demonstrated the highest success rate (96.5%) and the second fastest processing time (617 ms).This method was developed to support a larger effort to automate specimen image post‐processing in real‐time, highlighting the importance of execution time. Although initially designed for herbarium digitization, this method may be useful for other high‐resolution applications. It is estimated that the available records represent a relatively small proportion of the data that remain to be mobilized  . Common workflows for matching images to their records suggest naming image files according to a catalog number that is encoded on a barcode and stored with the specimen  to automate barcode decoding in a high‐throughput herbarium digitization system, and the natural history image processing software suite Inselect  called PyZbar . Each of these methods detect barcode patterns by “line scanning,” which evaluates sequential pixels along an image’s rows or columns to determine if the pattern of light and dark pixels corresponds to a barcode value. In the case of Zbar, images are evaluated both row‐wise and column‐wise to accommodate for unknown orientations . This accommodation comes at the cost of evaluating each pixel twice, once in the horizontal context and again in the vertical. Because line scanning is limited to these horizontal and vertical contexts, it is rotationally variant, meaning these methods fail when the entire barcode pattern does not lie along a single row or column. One workaround to this limitation is to repeatedly line scan while performing a series of rotations on the image until a barcode value is detected. This workaround has a number of drawbacks: (1) the transformations and additional line scanning incur additional computation time, (2) it may fail to detect multiple barcodes within an image if they are present at differing angles, and (3) this method delays identification when no barcode is detectable.Multiple solutions exist for decoding barcodes from images, and previous works have demonstrated their usefulness in natural history digitization. Sweeney et al.  used the Hudson, , which eThe limitations of traditional line scanning are typically negligible in other, more typical use cases where the barcode is the focal point of a relatively low‐resolution image. Herbarium images, on the other hand, are usually high resolution—often exceeding 24 million pixels—which increases the computation costs associated with line scanning. Furthermore, because barcodes on herbarium specimens only serve to facilitate curation, their size relative to the specimen has understandably been kept minimal. This makes traditional resolution reduction strategies infeasible as they discard much of the barcode pattern. Inspired by region proposal networks (RPNs), a novel protocol is presented that improves upon traditional line scanning by making it more computationally efficient and rotationally invariant. RPNs leverage faster, less precise methods to select specific regions of an image that are suitable for more intensive evaluation. The vector‐assisted region proposal (VARP) method presented here uses computer vision techniques to identify arbitrarily oriented lines  in an image that may contain barcode data, then organizes and evaluates only those regions through line scanning. This efficient, rotationally invariant method is demonstrated to be more performant at decoding barcodes than available alternatives.https://opencv.org) is used to convert images to grayscale and detect rectangle‐shaped contours  common in natural history collections. Fundamentally, these barcodes are composed of a series of rectangles with varying widths. OpenCV  of those pixels are then vertically concatenated into a matrix forming a new composite image Fig. . Organizhttps://github.com/CapPow/pyzbar). If no barcodes are initially decoded, multiple successive attempts are made while increasing the composite’s contrast and brightness. When performed on the much smaller composite image, these additional manipulations are computationally inexpensive, yet these fallback procedures do assume that the barcode is present in the composite.Barcodes are then decoded from the composite image using line scanning. To leverage this vertical orientation, the PyZbar library was modified to expose single‐axis decoding parameters, which are used to deactivate row‐wise line scanning (https://www.python.org) in a Jupyter notebook (https://jupyter.org). All evaluations were performed on a 3.6 GHz Intel Core i7‐3520M processor . Specimen images were gathered by randomly sampling 120 images from each of 10 herbaria  accessed through the SouthEast Regional Network of Expertise and Collections (SERNEC) data portal . The included herbaria were selected based on ability to provide high‐resolution images and the use of a uniform catalog number format. Image files were saved under collection code subdirectories and named according to the record’s universally unique identifier (UUID). Next, each image was inspected and those without a visible barcode were removed from the test data set. From the remaining images, 100 were randomly selected from each collection’s subdirectory and converted to grayscale.VARP’s performance was compared to alternatives using a test data set of 1000 herbarium records. Data gathering and evaluations were performed using Python 3.8  (“Gouda‐resize” and “Gouda‐roi”) and a method used by the Tennessee Herbaria Consortium (“THC‐rotation”). Similar to VARP, the Gouda‐roi method takes a proactive preprocessing approach using OpenCV to identify regions of interest. The Gouda‐roi approach differs from VARP in that it attempts to identify the entire barcode region through a series of thresholding, horizontal blurring, and vertical blurring. Gouda‐resize is a reactive method that attempts to repeatedly upscale the image until line scanning is successful. The THC‐rotate method is also a reactive strategy, which attempts to overcome rotational variance by repeatedly rotating the full‐scale image until line scanning is successful. Two of the methods evaluated use the ZXing library through the Python wrapper python‐zxing (https://github.com/dlenski/python‐zxing), which was evaluated with and without the “try_harder” parameter set . The ZXing method utilizes localized thresholding techniques that compare relative pixel intensity with neighboring pixels to convert the image to binary  before applying multiple format‐specific line‐scanning algorithms. The ZXing‐th method uses the same strategy as ZXing but with more computationally intensive thresholding and line scanning.Five alternative barcode decoding methods were evaluated alongside VARP for decoding success rate and execution time. Each method was tested against the entire test data set. Three of these methods used the Zbar library accessed through the unmodified PyZbar library. These methods included both strategies included in the Natural History Museum of London’s Gouda package  is likely attributable to how they handle failed attempts, with Gouda‐resize upscaling images after each failure while THC‐rotate rotated images. These results suggest that rotational variance plays a minimal role in overall success rates. Unsurprisingly, both of these reactive strategies  were significantly slower than their counterparts, exhibiting widely divergent execution times between successes and failures. The low success rates for both methods using the ZXing library  calls into question the accuracy of the implementation as well as the performance of the Python compatibility layer. Further testing of the ZXing methods outside of the Python environment may show improvements.Although developed, tested, and discussed from the perspective of herbarium digitization of vascular plants, it is possible the VARP method could similarly improve digitization workflows for other taxonomic groups. There may also be implications for VARP outside of natural history digitization, but there are two considerations worth noting before adapting VARP for other domains. The first consideration is the untested assumption that the benefits of VARP’s complex preprocessing are only realized when a barcode occupies a small portion of a high‐resolution image. The second consideration is that VARP’s processing disassociates positional relationships. Both decoding backends evaluated  can identify the position of barcodes within images, a feature some workflows may find useful. This positional information is currently being discarded when VARP constructs the composite image. It is feasible that VARP could be modified to support this feature by retaining vector coordinates relative to their position within the composite image, but as this has not been requested for natural history digitization, it has not been explored.https://github.com/CapPow/HerbASAP). Additionally, a simple‐to‐use command line interface (CLI) has been developed as a proof‐of‐concept. The CLI, as well as details concerning the test data set and performance evaluations, are available on GitHub . These materials have been provided under the open‐source MIT license and may therefore be freely inspected, used, or adapted for other works.VARP was developed in conjunction with other herbarium image analysis optimizations ; Investigation ; Methodology ; Software ; Validation ; Visualization ; Writing – original draft ; Writing – review & editing . Joey Shaw: Writing – review & editing .C.P. conceptualized and designed the work, performed analysis, produced figures and tables, and wrote the original draft. J.S. provided funding, feedback on content, and contributed to review and editing.https://forsbase.unil.ch.This article has earned an Open Data and Open Materials badges for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at


Boundaries between vegetation types, known as ecotones, can be dynamic in response to climatic changes. The North American Great Plains includes a forest‐grassland ecotone in the southcentral United States that has expanded and contracted in recent decades in response to historical periods of drought and pluvial conditions. This dynamic region also marks a western distributional limit for many passerine birds that typically breed in forests of the eastern United States. To better understand the influence that variability can exert on broad‐scale biodiversity, we explored historical longitudinal shifts in the western extent of breeding ranges of eastern forest songbirds in response to the variable climate of the southern Great Plains. We used climatic niche modeling to estimate current distributional limits of nine species of forest‐breeding passerines from 30‐year average climate conditions from 1980 to 2010. During this time, the southern Great Plains experienced an unprecedented wet period without periodic multi‐year droughts that characterized the region's long‐term climate from the early 1900s. Species’ climatic niche models were then projected onto two historical drought periods: 1952–1958 and 1966–1972. Threshold models for each of the three time periods revealed dramatic breeding range contraction and expansion along the forest‐grassland ecotone. Precipitation was the most important climate variable defining breeding ranges of these nine eastern forest songbirds. Range limits extended farther west into southern Great Plains during the more recent pluvial conditions of 1980–2010 and contracted during historical drought periods. An independent dataset from BBS was used to validate 1966–1972 range limit projections. Periods of lower precipitation in the forest‐grassland ecotone are likely responsible for limiting the western extent of eastern forest songbird breeding distributions. Projected increases in temperature and drought conditions in the southern Great Plains associated with climate change may reverse range expansions observed in the past 30 years. We evaluated distributional shifts of nine eastern forest songbirds at the western edge of their range extent along the transitional ecotone between eastern temperate forests and the central great plains of southcentral United States. Niche modeling of community science data revealed distributional limits of these species are sensitive to variable precipitation and periodic drought events. We selected presence data between 1980 and 2010 within the eastern temperate forest and central Great Plains ecoregions of the United States to match the extent and time frame of climate data used for training our models of species’ climatic niches. From the BBS, we obtained presence data from routes in which species were detected within the first 10 stops (8 km) of each route. Locations associated with BBS presence data were assigned to the starting point of each survey route. To match the effort and resolution of eBird presence data with BBS 10 stop data, we selected eBird presence data between June and July from checklists that covered less than 8 km in less than 60 min with fewer than 10 observers ; Data curation (lead); Formal analysis ; Investigation ; Methodology ; Writing‐original draft (lead); Writing‐review & editing . Timothy O'Connell: Conceptualization ; Funding acquisition (lead); Investigation ; Methodology ; Project administration ; Resources ; Software ; Supervision (lead); Validation ; Visualization ; Writing‐review & editing . Monica Papes: Conceptualization (supporting); Data curation (supporting); Formal analysis ; Investigation ; Methodology (lead); Software ; Supervision ; Validation ; Visualization ; Writing‐review & editing .https://doi.org/10.5061/dryad.dfn2z3525; http://biodiversityinformatics.amnh.org/open_source/maxent/, respectively.This article has earned an Open Data, Open Materials Badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at


Quercus petraea) dating back to 1680, in each of three different oak forests in France. The genetic covariances of allelic frequency changes increased between successive time periods, highlighting genome‐wide effects of linked selection. We found imprints of parallel linked selection in the three forests during the late LIA, and a shift of selection during more recent time periods of the Anthropocene. The changes in allelic covariances within and between forests mirrored the documented changes in the occurrence of extreme events (droughts and frosts) over the last 300 years. The genomic regions with the highest covariances were enriched in genes involved in plant responses to pathogens and abiotic stresses (temperature and drought). These responses are consistent with the reported sequence of frost (or drought) and disease damage ultimately leading to the oak dieback after extreme events. They provide support for adaptive evolution of long‐lived species during recent climatic changes. Although we acknowledge that other sources  may have contributed to temporal covariances of allelic frequency changes, the consistent and correlated response across the three forests lends support to the existence of a systematic driving force such as natural selection.The pace of tree microevolution during Anthropocene warming is largely unknown. We used a retrospective approach to monitor genomic changes in oak trees since the Little Ice Age (LIA). Allelic frequency changes were assessed from whole‐genome pooled sequences for four age‐structured cohorts of sessile oak ( A highly debated issue today in ecology is how fast evolutionary changes can occur, especially for long‐lived organisms, and if this pace is sufficient to cope with ongoing climatic changes. Climate warming started after the Little Ice Age (LIA), at the mid‐19th century; thus, multicentennial trees offer a unique opportunity to explore microevolution during documented climatic transitions. We implemented a retrospective approach in European white oak populations using whole genome sequences to track traces of selection. We found genome‐wide evidence of parallel linked selection during the late LIA, and a shift of selection during more recent time periods. Our results provide the first evidence that selection has operated on long‐lived trees over short time periods, and will likely continue to do so. Beyond oaks, other woody species sharing similar life history traits and attributes  may as well be prone to linked selection and allow retrospective tracking of evolutionary and adaptive pathways. Such retrospective approaches may improve our understanding of future responses to ongoing climatic changes.Our ability to forecast the response of species to climate change is limited by our lack of knowledge about the pace of adaptive evolution, particularly in long‐lived species, such as trees. Multicentennial trees in the Northern Hemisphere that are old enough to have experienced the transition from the cold Little Ice Age  to the warm Anthropocene (1850–today)  Liebl.) as the model species for this study, as the oldest forests in Europe contain sessile oak stands that came into existence in the middle of the LIA (near 1650), a time at which the French statesman Colbert implemented even‐aged management in French forests  indicated that the three forests were of natural origin , documentary records, ice core, and tree ring proxy data revealed clear temperature trends over the last 340 years in the three forests , allele frequency at 1850 is included in both terms).We filtered SNPs by removing sites with a depth below the number of alleles sampled for each cohort, and with a minor allele frequency below 0.02. Contigs were also filtered out when shorter than 200 kb long (excluding “N”s). Read depth and sample size were used to correct for bias in variance estimates :(2)GG estimates the contribution of linked selection to the variance of the allelic frequency changes from the starting point to the most recent time period. Buffalo and Coop  suggesteCov ΔiX–j, ΔiY–j) over the same period (i–j) or at different nonoverlapping time periods. The maintenance of a positive covariance would indicate similar responses and directions of selection  in the different forests, whereas negative values would provide evidence of differences in the direction of selection. The extension to different forests made it possible to estimate covariance over contemporary time periods, or disconnected time scales (adjacent or distant), thereby making it possible to determine whether parallel selection occurred over the same time period, or whether fluctuating selection occurred over different time periods.We extended this approach to the calculation of covariances between pairs of forests,  by selecting the best blast hit  to represent the oak gene model, and Arabidopsis GO terms from TAIR were used for annotation (https://www.arabidopsis.org/). For oak genes without orthologs in Arabidopsis, we performed eggNOG‐mapper version 2 functional annotation based on fast orthology assignments, using precomputed eggNOG version 5.0 clusters and phylogenies , Molecular Functions (MF), and Cellular Components (CC) were tested for GO terms enrichment with the “topGO” R package and the “weight” algorithm associated with a Fisher's exact test to select the most relevant terms . Levels of diversity were similar across cohorts and forests and consistent with previous estimates for oak  andCov) and distant time periods ). All proximal and distant covariances were significant in all three forests  were positive and significant in each forest  varied between the three forests, being positive in Bercé, slightly lower in Réno‐Valdieu, and negative in Tronçais  that were positive in all three forests, resulting in GΔ1680–1960 values of 9%, 18%, and 12%, respectively. We explored the genomic distribution of the temporal covariances between the two earliest adjacent periods by calculating the covariances for tiles of 100 Kb and plotting their distribution as Manhattan plots along chromosomes  larger than 0.01 in all three replicates were considered as outliers ) comprise SNPs that show temporal patterns of differentiation. Indeed, FST values of SNPs between proximal older time periods are larger than between proximal recent time periods, within and between forests, and they constantly decrease from older to recent time periods  indicate that covariances build up as a result of repeated numerous subtle allelic frequency changes over two successive time periods, rather than by a few recurrent extreme shifts , recombination distance, and linkage disequilibrium. We recently reported that Q. petraea forest stands display substantial heritable differences in reproductive success , and several pathogenic fungi  and root pathogens (Armillaria and Phytophthora)  Fig. . Many ofDelatour . In summDelatour , and, ulFST values usually lower than 2 %) throughout western lowland Europe .Click here for additional data file.Figure S4. Distribution of the temporal covariances between allelic frequency changes between the two oldest time periods )) and of Fst between the cohorts delimiting the time periods (Fst1680‐1850 and Fst1850‐1960).Click here for additional data file.Figure S6 Biological network showing the 15 selected Cell Processes and their 74 connected Arabidopsis genes (according to the pathway Studio™Plant database).Click here for additional data file.Table S1. Geographic coordinates of sampled forests and age structured cohortsClick here for additional data file.Table S2: Functional annotation of the 280 oak genes located in outlier tilesClick here for additional data file.Table S3: Significance of Biological Process GO terms according to topGO “weight” method and Fisher's exact test.Click here for additional data file.Table S4: Significance of Molecular Function GO terms according to topGO “weight” method and Fisher's exact test.Click here for additional data file.Table S5: Significance of Cellular Component GO terms according to topGO “weight” method and Fisher's exact test.Click here for additional data file.Table S6: List of the 74 Arabidopsis homologs known to participate to at least one cell process with their name used in Pathway Studio™ Plant, their locus name as defined in TAIR (https://www.arabidopsis.org) and alias commonly used in scientific publicationsClick here for additional data file.Supporting Information S1, S2, S3Click here for additional data file.


Ecological forecasting has vast potential to support environmental decision making with repeated, testable predictions across management‐relevant timescales and locations. Yet resource managers rarely use co‐designed forecasting systems or embed them in decision making. Although prediction of planned management outcomes is particularly important for biological invasions to optimize when and where resources should be allocated, spatial–temporal models of spread typically have not been openly shared, iteratively updated, or interactive to facilitate exploration of management actions. We describe a species‐agnostic, open‐source framework – called the Pest or Pathogen Spread (PoPS) Forecasting Platform – for co‐designing near‐term iterative forecasts of biological invasions. Two case studies are presented to demonstrate that iterative calibration yields higher forecast skill than using only the earliest‐available data to predict future spread. The PoPS framework is a primary example of an ecological forecasting system that has been both scientifically improved and optimized for real‐world decision making through sustained participation and use by management stakeholders. Ecological forecasting represents a paradigm shift in ecology focused on making repeated, testable predictions across management‐relevant timescales, geographies, and contexts to test scientific assumptions and predict near‐future conditions . Spatially explicit forecasting is particularly important for biological invasions, because . et al.  to itera  Forecasting Platform, which to the best of our knowledge is the first example of a flexible, open‐source platform for near‐term (≤ 5 years) iterative ecological forecasting that combines several key features typically missing in ecological models. For example, (1) PoPS was designed, and continues to be developed, through a collaborative, participatory process with government analysts and field operations personnel; (2) the system features interactive decision analytics for generating and testing management interventions; and (3) the underlying spread model is both dynamic (accounts for changes over time) and spatially explicit (incorporates relationships and changes across space). We developed PoPS specifically for use by management professionals who do not necessarily have modeling experience and tailored it for predicting the spread, containment, and control of biological invasions.Lycorma delicatula) and the other a pathogen , and show how iterative calibration with new data improves model skill as measured by accuracy assessments that capture spatial patterns.Here, we describe the PoPS Forecasting Platform and its use for controlling biological invasions. Our approach emphasizes the value of co‐designing an ecological forecasting system that is modifiable for widespread use by management professionals without modeling expertise. We illustrate the potential of PoPS for improving management outcomes using two case studies, one involving a pest  Animal and Plant Health Inspection Service (APHIS), which partnered with our research group at North Carolina State University to co‐develop a spatially explicit spread forecasting system to support their operations.https://popsmodel.org), and the decision analytics we designed can be built using the code available on GitHub (https://osf.io/q32p9)  and environmental conditions in a location (i) at a particular time (t) to determine reproduction. The dispersal kernel then determines where the new dispersing propagules go; dispersal distance (d) is a function of gamma (γ), which indicates how much dispersal is short‐distance  or long‐distance . The distance of each propagule is determined by drawing from a distribution using either α1 or α2, and its direction is drawn from a distribution that accounts for predominant wind direction (ω) and wind strength (κ). Once a propagule has landed in a new location, its establishment depends on the environmental conditions in that new location  and the availability of suitable hosts, calculated as the number of susceptible hosts (S) divided by the total number of potential hosts (N). Each application of PoPS uses a customized host map that provides the locations of target hosts in each grid cell across the modeled landscape. The value of Ψ  Forecasting Platform simulates reproduction, dispersal, and establishment of pests and pathogens through space and time. For every location in a landscape, at each time step, the model predicts the number of infested or infected hosts (Ψ). The model is conceptually driven, first, by the notion of beta  can be included or excluded from the model as necessary (via intuitive on–off switches on the interface) depending on the drivers that influence the species of interest. In addition, the model accounts for spatial relationships and movements between grid cells in a landscape, and forecasts across sequential time steps, which can be specified as daily, weekly, monthly, or yearly Panel .PoPS was developed, and continues to be refined, through an “iterative modeling cycle” Figure , a procePuccinia striiformis (wheat stripe rust), Phytophthora infestans (late blight), Lobesia botrana (European grapevine moth), Aphthae epizooticae (foot‐and‐mouth disease), Alphacoronavirus (porcine epidemic diarrhea virus), and Betaarterivirus suid 1 (porcine respiratory syndrome virus). Below, we describe the use of PoPS in two case studies, spotted lanternfly (SLF) and sudden oak death (SOD), focusing on the Calibration Loop and the value of new data supplied by the Field Observation and Scientific Feedback Loop.To date, we and our collaborators have been using PoPS and the iterative modeling cycle Figure  to forecet al. SLF and the pathogenic water mold that causes SOD are rapidly emerging threats to natural, economic, and cultural resources in the US, and have motivated the establishment of quarantine zones in highly affected areas. SLF, a nonnative invasive planthopper, has posed a threat to fruit crops in Pennsylvania and neighboring states since 2014 Urban . SOD is et al. 1, α2; see Panel In all applications of PoPS, the model is calibrated by comparing observed data to simulated data and setting thresholds so that the model iteratively approaches a best fit. Specifically, we use approximate Bayesian computation with a sequential Markov chain and a multivariate normal perturbation kernel ; we ran 100,000 simulations of the model  and compared model results with actual observations over the predicted time interval. For example, if we start with 2016 data and calibrate a model to forecast 2017 spread, we then use data collected in 2017 to both (a) forecast 2018 spread and (b) hindcast 2016 and 2017 spread, to see whether recalibration improved model skill. We repeated this process with each set of newly collected data.number of changed cells may predict very different spatial patterns of changed cells  that ecological forecasts be continuously challenged with new information, thereby improving predictions and decision making over management‐relevant timescales.The PoPS framework is a prime example of an ecological forecasting system that has been both scientifically improved and optimized for real‐world use through sustained participation of management stakeholders in both initial and ongoing development. We urge continued investment in ongoing data collection so that forecasts can be further validated and improved in the future. Such an approach represents a response to the request of Dietze et al.  and otheFig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.


Total hip arthroplasty (THA) is highly successful but some patients will require later revision surgery. This pilot study evaluates the effects of long‐term follow‐up for patients undergoing revision hip replacement.Consecutive patients undergoing aseptic revision of THA were recruited from a large orthopaedic unit to a single centre, observational study. Primary outcomes were changes in patient‐reported scores from pre‐revision to 12 months post‐surgery. Secondary outcomes were costs during hospital stay up to 6 months post‐revision. Participants were retrospectively allocated to two groups—those with regular orthopaedic review prior to revision (Planned revision) or those without (Unplanned revision).U test; none reached statistical significance. There was a significant difference for length of stay: Planned group 5 days (2–22), Unplanned 11 days (3–86) . No significant differences found for theatre time or component costs. Resource costs post‐revision surgery are presented.52 patients were recruited, 7 were unrevised, one incomplete baseline questionnaires. There were 25 planned and 19 unplanned revisions with no significant differences between groups at baseline. At 12 months, 34 complete data sets were available for analysis, 17 in each group. Change scores were analysed with Mann–Whitney This pilot study indicates that some change in methods would be required for future work. The results show that there may be some financial benefit from providing long‐term follow‐up of THA but a larger study is needed to explore these findings and to discuss the impact on recommended guidelines. This group was categorised as ‘Planned revision’. The comparator group were those patients scheduled for revision surgery following an emergency admission or general practitioner (GP) referral due to problems with the hip implant. They were considered as those with no orthopaedic long‐term follow‐up of their hip arthroplasty and/or an ‘Unplanned revision’. The assignment of each participant to one of these two groups was retrospectively completed using a variety of data sources and a decision algorithm The data gathered from the various sources and knowledge of participating orthopaedic units were incorporated in the decision algorithm to allocate participants to one of two groups—‘Planned revision’ or ‘Unplanned revision’. The ‘12‐month’ cut off from time from referral to revision surgery was used because of current practice at the time of this study. There was commonly a 22–24 weeks wait between GP referral to the orthopaedic service and the first orthopaedic appointment, and then further screening/results and waiting for a pre‐operative appointment, adding another 8–10 weeks. Surgery might take place as soon as 2–3 weeks after the pre‐operative assessment if no complications arose, hence a total of approximately nine months from referral to surgery. The choice of a 12‐month cut off was designed to differentiate between those participants who came to revision surgery without regular orthopaedic assessment (Unplanned revision) and those who were in a regular follow‐up programme or who were being monitored for progression of potentially damaging changes around the THA (Planned revision).2.4The participants in this study were recruited pre‐operatively and completed a set of patient‐reported outcome measures at that time. A self‐reported questionnaire was used to capture use of health resources at 6 months after revision surgery. The patient‐reported outcome measures were repeated 12 months after surgery. Peri‐operative data were collected from medical records.2.5The primary clinical outcomes were the difference in patient‐reported measures from the time of surgery to 12 months post‐surgery; the joint specific Oxford Hip Score . It has been shown to be valid and reliable for use in revision hip surgery  which comprises five dimensions  and a visual analogue scale Euroqol Visual Analogue Score (EQ‐VAS) which is a single mark on a scale of 0–100. The EQ‐5D‐5L dimensions were combined to give a single index value with reference to the United Kingdom value set, using the time trade‐off (TTO) method .2.6The secondary outcome measure was the cost of revision hip surgery and the early postoperative period, adopting a health and social care payer perspective for the economic evaluation.2.6.1Data were included for the initial inpatient stay for the revision THA , subsequent inpatient stays post discharge at any hospital and outpatient visits during the first 12 months post‐revision surgery. The data collected included location, duration and reason for visit.Volumes for all other resource use were collected using a patient‐reported questionnaire administered at 6 months post‐surgery based on a self‐completed resource use logbook. This included non‐hospital services, medication use, provision of equipment and adaptations made to the home, and use of social services. Non‐hospital services included contacts with GP, practice nurse and district nurse, and physiotherapy and occupational therapy at home or an outpatient clinic. Social services included home care, meal delivery and social worker contact. Participants were asked to include medication, equipment, adaptations, home care and meal provision that were self‐funded.2.6.2Volumes of resources and unit cost prices were treated separately. Resources used during the initial hospital stay were valued using unit costs obtained from the hospital finance department. Cost estimates for time spent in theatre and admissions to hospital wards included staff time, overheads, consumables and medications. Prosthesis costs were taken from consortium supply lists or from direct contact with the orthopaedic companies supplying components.For secondary care visits in the 6‐month post‐operative period, information on the reason for inpatient admission, duration of episode and clinical expert advice was used to derive healthcare resource group codes. Healthcare resource groups and outpatient appointment by clinical speciality were valued using Department of Health Reference Costs . The economic evaluation of health benefits is presented as a cost‐consequences table in view of the small sample size.This was a pilot study and adopted an exploratory approach to data analysis. Sample size was not predetermined but it was anticipated that 180 participants may be eligible, based on historic data from the orthopaedic unit. Several unanticipated problems led to a reduced number of eligible participants and consequently, there were insufficient primary outcome data for a linear mixed method model. Data are presented with descriptive statistics and tests for the difference between groups, parametric and non‐parametric as indicated by the data distribution  on 19 April 2016, reference number: 16/LO/0650. All participants gave written informed consent and the study was conducted in accordance with the Declaration of Helsinki.3Between 27 June 2016 and 30 June 2017, 110 patients were screened, 52 gave informed consent and were recruited for the study. The exclusions were as per protocol . There was no statistically significant difference between the median length of time taken for surgery in the two groups .The major costs associated with the revision hip replacement and hospital stay for all participants are summarised to give an average (median) participant value Table . A notabA total of 38 participants completed the resource use questionnaire. Some participants completed the 12‐month questionnaire but not resource use; they found it difficult to concentrate due to comorbidities or carer responsibilities. The data are presented with summary values and show that groups were similar apart from an extended hospital stay for one participant in the Unplanned revision group Table .4This pilot study indicates that changes would be needed in the delivery of any similar future study but offers insight into the effect of long‐term follow‐up services when revision hip arthroplasty is required. The sample was small but demonstrates some differences in patient reported quality of life and peri‐operative costs which favour the use of long‐term follow‐up in this patient population.The limitations of this work are the size and scope as it was a one‐centre, pilot study and the number of patients recruited were fewer than anticipated due to several factors including patient choice, operational changes, winter pressures and research staff availability. Maintaining the screening of potential participants and recruitment of eligible patients was inconsistent over the 12 months of the study due to staff shortages in research. Of those recruited, a proportion did not proceed to surgery as anticipated, and there was further loss to follow‐up through incomplete data. When compared with those who competed all scores, the only notable difference was a higher proportion of males in this group Tables . Future The retrospective allocation of participant to groups (Planned revision vs. Unplanned revision) employed an algorithm which was constructed with reference to clinical knowledge by the authors and orthopaedic colleagues. The variables included time from referral and frequency of appointments in secondary care. It did not differentiate the profession of the referrer, which may provide greater insight into the pre‐revision pathway. It did not attempt to establish if the participant was on a recommended follow‐up pathway, or if the participant had adhered to that pathway. Consequently, some participants may have been incorrectly assigned to a group. However, the allocation was seen to be correct in cases where pathways were known, and the principles incorporated in the algorithm facilitated a consistent approach that could be employed with larger data sets in subsequent work.p = 0.05). There are many reasons why this score may have shown a difference between groups, such as lower comorbidity scores or a shorter hospital stay. However, one of the possibilities is that the screening, preparation and planned pathway to revision provided by long‐term follow‐up services allows better management of the experience for the patient with a quicker return to health than those without such preparation. This would require further exploration in a larger study.The EQ‐VAS requires the patient to indicate on a scale how good or bad they consider their health to be on the day of score completion and provides well‐validated evidence for health quality (EuroQol Group, The cost consequences comparison table Table  shows soIn addition to the costs incurred by an increased length of stay, issues of hospital capacity and bed management are an ongoing, real‐world problem facing hospital management teams and based on this data, would be worsened by the prolonged occupancy of those without follow‐up. A proportion of the unplanned revision group received emergency surgery which may have contributed to the length of stay for reasons such as preparing the patient for surgery or planning for discharge. Emergency revision surgery will always be needed for some patients but there may be others for whom long‐term follow‐up of THA would lead to planned elective revision, thereby minimising the unpredictability of prolonged bed occupancy and its associated problems.The decision to undertake revision hip arthroplasty is made jointly by the orthopaedic surgeon and the patient. The threshold for a surgeon depends on many factors such as their own experience, the type of components and materials used in the primary arthroplasty, the extent of the damage in the THA and the threat of complete failure of the joint construct. In this study, the primary indication for revision in the Planned revision group was X‐ray change, although some may also have experienced pain Table . This raThe delivery of a service that takes place many years after the primary procedure raises questions about individual responsibility and personal attitude—should we be expecting those with a THA to initiate an orthopaedic assessment? There are divided views within the orthopaedic community with some suggesting that the burden of responsibility lies with care providers to keep track of patients and others suggest that it should be entirely patient‐driven with no action until symptoms are such that they re‐present in orthopaedics (Rose, Dures, & Smith, 5This pilot study has shown that modifications to the methods would be needed in any further study. The results suggest that there may be some financial benefit from providing patients with a follow‐up service in the long‐term after THA but the effect on patients is unclear from this small sample. A larger study is needed to explore these findings and to discuss the impact on current recommended guidelines.The authors report no conflicts of interest in this work.Shea Palmer, Jane Powell, Erik Lenguerrand and Lindsay K. Smith contributed to the design and implementation of the research, to the analysis of the results and to the writing of the manuscript. Emma Turner contributed to collection and analysis of health economic data, and to the writing of the manuscript.Supplementary MaterialClick here for additional data file.


A growing variety of biological macromolecules are in development for use as active ingredients in topical therapies and vaccines. Dermal delivery of biomacromolecules offers several advantages compared to other delivery methods, including improved targetability, reduced systemic toxicity, and decreased degradation of drugs. However, this route of delivery is hampered by the barrier function of the skin. Recently, a large body of research has been directed toward improving the delivery of macromolecules to the skin, ranging from nucleic acids (NAs) to antigens, using noninvasive means. In this review, we discuss the latest formulation‐based efforts to deliver antigens and NAs for vaccination and treatment of skin diseases. We provide a perspective of their advantages, limitations, and potential for clinical translation. The delivery platforms discussed in this review may provide formulation scientists and clinicians with a better vision of the alternatives for dermal delivery of biomacromolecules, which may facilitate the development of new patient‐friendly prophylactic and therapeutic medicines. The global sales of biologics accounted for over 70% of the worldwide revenue from the 10 top‐selling pharmaceutical drugs.However, the successful delivery of biomacromolecules has been challenging due to their complex structure and high molecular weight  in addition to stability issues encountered during manufacturing, storage, and administration.The main biological obstacle for efficient dermal delivery of biomacromolecules is the barrier function of the skin, mainly the stratum corneum (SC). The SC consists of flattened and tightly packed corneocytes embedded in a highly lipophilic matrix composed of ceramides, cholesterol, and fatty acids Figure . This laP = 1–3),The barrier properties of the SC limit the penetration of molecules that are less than 500 Da and that have moderate lipophilicity , tissue damage , and fibrotic skin conditions.22.1While significant effort is directed toward the development of new vaccines, the most common method for vaccine administration remains to be by subcutaneous or intramuscular injections using hypodermic needles. This makes vaccination painful; some individuals have trypanophobia, and it also requires trained personnel  offers a promising alternative to subcutaneous or intramuscular vaccine delivery owing to the large surface area of the skin and its connection to the rest of the body via a network of blood vessels and drain lymph nodes (LNs).In addition to vaccination against infectious diseases, noninvasive TCI is a promising approach for other conditions including allergies,2.22.2.1+ Foxp3− regulatory T cells (Tregs). In this case, generation of immune tolerance was dependent on uptake through the hair follicle.Viaskin® is an epicutaneous delivery system where the powdered antigen is electrosprayed on the surface of the patch. Under occlusive conditions, the antigen becomes solubilized the by natural transepidermal water loss due to emerging perspiration and is passively delivered to the SC. It has been extensively tested as a method for allergen desensitization of sensitized mice in preclinical studiesA recent placebo‐controlled double‐blind clinical trial used Viaskin® epidermal occlusive patches loaded with lyophilized food antigens for ECIT. This system showed inductions of tolerance to peanut allergies and CMA.2.2.2In a recent approach to enhance cytotoxic T‐lymphocytes (CTL) production, Kamei et al.2.2.3In solid‐in‐oil (S/O) systems, the hydrophilic proteins  are dispersed in an oil‐vehicle with the assistance of nonionic surfactants. They are prepared by freeze‐drying water‐in‐oil emulsions containing therapeutic cargo followed by dispersion of the surfactant–medicine complexes into an oil vehicle that has affinity to the SC.Recently, Kitaoka et al.Transcutaneous immunotherapy using S/O nanodispersions has also been implemented for the treatment of Japanese cedar pollinosis, which represents a major health concern in Japan. It is a type I allergic disease treated by subcutaneous or sublingual administration of whole antigens from the pollen extract. Kong and coworkers implemented S/O nanodispersions loaded with vaccine T cell epitope peptides derived from pollen allergen coated with hydrophobic surfactants for TCI in a pollinosis mouse model. This model showed suppression in serum antibody IgE and cytokine production as well as alleviated allergic symptoms compared to mice that received subcutaneous injections.In a further advancement, the same group conjugated the pollen extract to galactomannan (PE‐GM) to mask IgE‐binding epitopes in the PE and incorporated it in a S/O nanodispersion Figure . This syRecently, transcutaneous immunotherapy against cancer with a S/O system was demonstrated. This system successfully delivered OVA, a model antigen, through the skin and induce OVA‐specific antibodies. Mice vaccinated with OVA‐S/O were challenged with E.G7‐OVA thymoma cells, which express OVA. A significant inhibition of tumor growth was observed in the vaccinated mice.In a further advancement, Wakabayashi et al.2.2.4HA is a linear polysaccharide present in the extracellular matrix of the skin. It is known to promote KC proliferation as well as elasticity regeneration, and it is used in dermatologic clinics as dermal fillers.In the skin, HA is capable of releasing cargo by degrading into small fragments. The low molecular weight fragments are recognized in the body as damage‐associated molecular pattern molecules, which can potentiate an immune response against released antigens by activating Toll‐like receptors 4 (TLR4) and TLR2 and by stimulating the secretion of different cytokines.To confirm the migration of DCs to the draining LNs, where antigens are presented to the naïve T cells to initiate adaptive immune responses, the authors harvested the draining LNs of the back skin 2 days after topical administration. Fluorescence images showed a large number of HA‐OVA‐RhoB conjugates; however, no OVA‐RhoB and HA‐RhoB conjugates were detected in the LNs. The effectiveness of transdermal immunization was tested by applying the HA‐OVA to the back of BALB/c mice and measuring the anti‐OVA IgG antibody titers. HA‐OVA conjugates significantly increased both humoral and mucosal antibodies, with peak levels at 4 weeks. An OVA challenge at week eight elicited strong immune‐recall responses. The minimum OVA dose in HA‐OVA required to induce strong immune responses via the intact skin was 500 μg (25 mg/ kg), compared to 20 μg (1 mg/ kg) for intramuscular immunization. Pretreatment of the skin with non‐ablative fractional laser beams as adjuvant before the application of HA‐OVA elicited an immune response that was comparable to intramuscular injection of HA‐OVA (1 mg/kg) and higher than that produced by intramuscular injection of OVA alone. Moreover, the transdermal immunization induced both systemic (lgG) and mucosal (lgA) immune responses, whereas intramuscular injection did not elicit the mucosal immune response.HA‐conjugates can be sterilized by filtration and stored in lyophilized powder forms vaccines to be reconstituted before use. The dissolved HA vaccine can be easily applied to the skin as lotion or skin toner or easily incorporated in skin patches facilitating self‐administration, which may of particular use in countries or regions with limited access to healthcare.2.2.5Polymeric nanocapsules (NCs) are one of the nanoscale carriers that have been widely employed for vaccination. Having a size similar to pathogens, NCs can be efficiently recognized by the immune system, leading to a potent immune response.Chitosan (CS) is a natural polymer that is known to enhance skin penetration besides its effect as an adjuvant.Ex vivo studies using porcine skin showed that CS‐NCs enhanced the penetration of OVA by 15 times compared to OVA in solution. This effect was primarily attributed to their nano‐size (100 nm) that allows for a higher penetration, as the antigen diffuses through different routes, including transcellular and intercellular pathways as well as through hair follicles.In another study, HA‐NCs were prepared with an oily core of α‐tocopherol stabilized by benzalkonium chloride and Lipocol® HCO‐40 (LP‐HCO).HA‐NCs produced immune complement activation over a range of concentrations, a property similar to vaccine adjuvants like aluminum salts. Studying their penetration across pig skin revealed that the association of OVA to HA‐NCs resulted in 22 times higher and 33 times higher penetration and retention compared to OVA in solution, respectively. The authors proposed that the possible mechanisms by which these glycosaminoglycan‐coated NCs overcame the SC to deliver OVA could be mediated by HA receptors widely expressed on KCs and fibroblasts as well as the hydration effect that forms paths for transport of OVA across the SC to aid in antigen retention within the hydrated epidermal layers.2.2.6Phospholipid vesicles, including liposomes, transfersomes, and ethosomes, have emerged as popular nanocarriers for dermal delivery of encapsulated vaccines owning to their physiochemical and structural resemblance to biological membranes.Since induction of protective T cell immunity by cancer vaccines requires both tumor‐associated antigens and adjuvants to be delivered specifically and efficiently to the epidermal LCs, Wamhoff et al.In a subsequent study, the uptake and intracellular routing of liposomes was studied in model cell lines by confocal and live cell imaging as well as by flow cytometry. The liposomes were made from a mixture of 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC), cholesterol and 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine (DSPE)‐polyethylene glycol (PEG), and they were made to encapsulate protein antigens. The liposomes were internalized into early endosomal compartments and accumulated in late endosomes and lysosomes of primary human LCs followed by a release of the protein antigens. These data further support the applicability of the targeted liposomal particles for protein vaccine applications.Transfersomes are elastic liposomes containing edge activators  to form ultra‐deformable vesicles. Their ability to squeeze through the intercellular regions of the intact SC gives them greater potential for dermal antigen transport to APCs compared to liposomes.3 as adjuvant. A negative control was also included, which received topical OVA and saponin. Ethosomes showed the highest serum antibody titers, despite having the lowest antigen encapsulation efficiency, suggesting that the high loading of antigen into the formulation is less important compared to the optimal interaction with lipids in the SC. Both elasticity (deformability) and size distribution are key parameters that affect dermal penetration and cellular uptake,Ethosomes consist of phospholipid bilayers with high ethanol content that increases the lipid fluidization of the vesicles and of the skin lipids by reducing the melting point of polar head groups in the skin.Despite this positive data, there are two challenges for the clinical translation of ethosomes. One is physical instability wherein the vesicles tend to fuse into larger vesicles, leading to breakage and leakage of encapsulated vaccine molecules. The other limitation is the low viscosity of the ethosomal dispersion, which constrains its dermal use. To address this point, Zhang and coworkers Archaeosomes are modified liposomes prepared with the polar lipids found in Archaea and display superior chemical and colloidal stability.Leishmania braziliensis promastigotes with sodium cholate (dsLp) and formulated them within UDA and ultra‐deformable liposomes (UDL). In vitro, the UDA was extensively taken up by macrophages and induced rapid cytokine secretion. Topical application on BALB/c mice twice per week on consecutive days for 7 weeks showed that, despite the immunostimulatory effects of dsLp on macrophages in vitro, it raised no measurable in vivo response unless associated to UDL or UDA. The UDA elicited the highest systemic response, comparable to alum. Although both UDL and UDA acted as penetration enhancers, only UDA succeeded as a topical adjuvant due to its high uptake by APCs. Based on these findings, choosing materials with potent immunostimulatory properties is an important consideration when designing a system for passive dermal immunization.Higa et al.2.2.7Virus‐like particles (VLPs) are inert, empty capsids of viruses that retain the virus structure without DNA/RNA from the virus itself. VLPs can be engineered to display antigens on their surfaces.In another study, Runcan et al.2.2.8Star‐shaped, or STAR, particles are millimeter‐scale particles with micron‐scale projections that increase skin permeability and are made of biocompatible materials as aluminum oxide that can be incorporated invisibly into topical formulations similar to conventional skin products. They can be engineered to have different geometries and variable number of arms. Upon rubbing on the skin, their microscopic projections painlessly create micron‐scale pores in the SC, increasing the permeability of topical compounds independent of their physicochemical properties Figure . They prRecently, topical delivery of tetanus toxoid vaccine to mice using STAR particles generated immune responses that were similar to intramuscular vaccine injection. Moreover, application of STAR particles to the skin of human participants indicated they were well‐tolerated and effective in creating skin pores. Star particles could widen the range of compounds that can be topically applied for a variety of skin diseases.2.2.9Ionic liquids (ILs) have gained significant attention for topical and transdermal delivery due to their potential benefits, including a broad safety profile and their ability to tune the physiological properties of active pharmaceutical ingredients.In a recent study, Ukidve et al. synthesized ILs composed of two body metabolites, choline and lactic acid (ChoLa). ChoLa maintained OVA antigen integrity and enhanced immune infiltration at the injection site when administered subcutaneously, leading to a potent immune response against the antigen. This IL was suggested as a promising safe vaccine adjuvant. Moreover, it showed the ability to maintain the stability of SARS‐CoV‐2 spike protein. Although not yet investigated, combining the properties of ILs as skin penetration enhancers with their ability to stabilize antigens may open the door for this class of compounds to serve as bioactive and noninvasive TCI vehicles. More effort should be directed toward the screening of different components for synthesizing ILs that can synergistically enhance immune activity and promote dermal permeation.33.1Topical delivery of NAs is emerging as an effective strategy to treat inflammatory skin disorders such as psoriasis,3.2Atopic dermatitis (AD) is an inflammatory disease that affects 10–20% of childrenPsoriasis is another inflammatory skin condition characterized by itchy and scaly patches where several genes that encode for proinflammatory cytokines are upregulated in the plaque area.Several studies have shown the effectiveness of different siRNAs in the treatment of psoriasis in preclinical models. Frequently targeted genes include IL‐23, IL‐17, IL‐36, and TNF‐α, as these genes play a key role in pathogenesis of psoriasis.Generally, the skin barrier function of the SC is disrupted in psoriasis and AD due to the altered function of ceramide and the sebaceous gland. Accordingly, the major barrier of siRNA delivery to AD‐affected skin are the cell–cell tight junctions (TJs) that control the paracellular pathway in the granular layer of skin.3.2.1Peptides that promote the transport of NAs into the skin and elicit a therapeutic response have been implemented as carriers for siRNA and gene delivery. TD‐1 is a short synthetic peptide that enabled the successful delivery of anti‐glyceraldehyde‐3‐phosphate‐dehydrogenase (GAPDH) siRNA through non‐follicular rat skin.Arginine‐rich peptides such as transcriptional activator (TAT) derived peptides have been used to increase siRNA stability against RNase A and promote its transdermal delivery.In another study, Ibaraki et al.The use of peptides for noninvasive dermal delivery has also been extended to deliver plasmid DNA. Amphipathic peptide Mgpe9 induced reversible modulation of TJ proteins in the skin, facilitating the successful delivery of plasmid DNA and resulting in efficient gene expression in the basal skin layer without adverse effects.3.2.2Several recent reports suggest the use of amphiphilic lipid nanocarriers (LNCs) as a vehicle for topical drug delivery of siRNAs. These LNCs, typically positively charged, can interact favorably with the negatively charged siRNA. Their cationic nature also improves cellular uptake and allows endosomal escape of the siRNA. LNCs overcome the significant barrier posed by the SC by temporary fluidization of the lipid layers in the SC, facilitating delivery of NAs into the deeper layers of the skin where they form a reservoir for sustained release.Lee et al.Another LNC, consisting of a glycerol stearate and oleic acid core coated with polyethylenimine (PEI), was reported to deliver a combination of tacrolimus and siRNA against TNF‐α through the skin. This system generated a synergistic effect by reducing the severity of psoriatic plaques in an imiquimod‐induced psoriasis mouse model compared to treatment with tacrolimus alone or TNF‐α siRNA‐loaded nanocarriers alone.3.2.3The combination of liposomes and functional peptides has been also studied. The incorporation of SPACE peptides in cationic ethosomes was shown to enhance dermal siRNA delivery, resulting in effective gene silencing in vivo. This was achieved either by conjugating the SPACE peptide to the siRNA, conjugating it to the surface of the ethosomal particle or incorporating it directly into the formulation.In another recent study, liposomes encapsulating siRelA were prepared and designed to have a pH‐sensitive responsiveness. It formed a stable lamella at physiological pH. Under acidic endosomal conditions, the lipid became protonated and resulted in structural changes from the lamellar phase to a reverse hexagonal layer, resulting in collapse of the liposome. To improve the efficacy of the siRNA, AT1002 peptides were incorporated in the formulation to open the TJ of the granular layer by phosphorylating the TJ structural protein ZO‐1, facilitating the penetration of the SiRNA via the paracellular pathway. Studies on RAW264.7 murine macrophages indicated higher cellular uptake and reduced toxicity of siRNA‐encapsulated liposomes compared to naked siRNA. Further, siRelA liposomes with AT1002 showed superior efficacy in alleviating the symptoms of AD and reducing the concentration of inflammatory cytokines in a mouse model.3.2.4Desai et al. designed a biodegradable, cyclic cationic head lipid‐polymer hybrid NC (CyLiPn) composed of poly(lactic‐co‐glycolic acid) (PLGA) and cationic amphiphiles.3.2.5ILs have emerged as a versatile class of materials for dermal and transdermal delivery of a variety of macromolecules including hormones like insulin,3.2.6Lyotropic liquid crystalline dispersions have been shown to reversibly reduce the skin barrier properties, facilitating the penetration of siRNA and other macromolecules.3.2.7Spherical nucleic acids (SNAs) are three‐dimensional nanocomplexes of ordered oligonucleotides on a spherical core nanoparticle. SNAs prepared with lipid‐based nanoparticles such as liposomes possess the ability to overcome the barriers to transdermal drug delivery and silence gene expression in the epidermis. Liu et al. reported the use of SNAs to silence the expression of IL‐17 receptor A (IL‐17RA) in mice with imiquimod‐induced psoriasis.Nemati et al.3.2.8Polyamidoamine (PAMAM) dendrimers possess an amine‐terminated surface that can be easily functionalized, providing a positive charge for interaction with siRNA.While siRNA remains the focus of the majority of topical NA‐based therapies for psoriasis in recent years, other types of NAs are being studied for similar purposes as well. Examples of such NAs include aptamers and self‐amplifying RNAs (saRNAs).Aptamers are oligonucleotide‐based macromolecules that can bind to specific epitopes on cellular targets such as proteins. Lenn et al.saRNAs are derived from the alphavirus genome that encode for alphaviral replicase and the gene of interest. Alphaviral replicase enables the amplification of the gene of interest in the cytosolic region of the host cells, which leads to increased and prolonged in the expression of the proteins. Blakney et al. demonstrated the use of mannosylated PEI copolymers as vehicles for enhanced uptake of intradermally delivered saRNAs in human skin explants.3.3Skin fibrosis such as hypertrophic scars and keloids affect millions of people worldwide. Fibrotic tissue is like a scar showing a bumpy, irregular and thick surface due to the excessive accumulation of protein under the skin.3.3.1Recently, Kang et al.3.4Chronic wound healing remains a serious problem worldwide, causing a significant public health burden.Therapeutic strategies for wound healing include small molecule drugs, growth factors, stem cells, fibroblasts and RNA interference (RNAi) therapies such as siRNA and microRNA (miRNA).3.4.1While cationic lipids are widely used as topical siRNA delivery carriers,3.4.2Inorganic nanoparticles, especially gold nanoparticles, are a popular nanocarrier for NA delivery due to their low toxicity and immunogenicity combined with ease of surface functionalization.3.4.3Polymeric nanoparticles offer tunability combined with biocompatibility and protection of cargo degradation. Their charge and size can be optimized for use as oligonucleotide vehicles.The introduction of an external stimuli‐responsive moiety in these polymeric carriers can enable temporal control of siRNA and miRNA release. Blersch et al.All of the aforementioned studies use an aqueous carrier solution for topical delivery. Particularly for wound healing, this is insufficient from a translational perspective. The mechanical stresses endured by wounds demand a more robust formulation accompanied by a protective dressing. Moving forward, RNAi‐based carriers for wounds should focus on formulation and application strategies to render these strategies more favorable for clinical translation.3.4.4Hammond et al.LbL assemblies have also been used for scar therapy. The process of wound healing often results in thick, collagen‐enriched tissue called scar tissue, which can negatively impact quality of life.3.4.5Lactobacilli were delivered topically to a wound site, resulting in accelerated wound healing mediated by C‐X‐C motif chemokine 12 (CXCL12) production from the bacteria.Apart from RNAi, other biological therapeutics such as cytokines, cells and bacteria have been delivered topically to enhance wound healing. He et al. demonstrated the efficacy of topically delivered IL‐33 in accelerating wound closure.3.5Cutaneous malignancies such as melanoma and nonmelanoma skin cancers represent a significant burden on global health.3.5.1The favorable penetration of lipid‐based nanocarriers into the skin and their ability to effectively deliver NAs to the endosome has encouraged their use in treating cutaneous malignancies. Dorrani et al.3.5.2Skin and cell‐penetrating peptides (SCPs) are short peptides with 5–30 amino acid residues that can target cell surfaces specifically. They can be screened for their affinity to certain receptors on the cell surface and subsequently used for targeted delivery.SCPs have also been combined with polymers to form targeted nanocarriers. Wang et al.Escherichia coli to topically deliver indocyanine green (I‐P‐OMVs) for a combination photo‐TNF‐related apoptosis‐inducing ligand (TRAIL) therapy against melanoma.vb3 integrin‐targeting peptide to specifically target melanoma cells. The combination of TRAIL and phototherapy showed significantly higher in vivo efficacy compared to individual therapy and combination therapy without the OMV carrier  derived from transgenic r Figure .3.5.3l‐lysine (PLL) to render a net cationic charge. Topical administration had sustained siRNA concentration within the tumor compared to intravenous or intratumoral administration and proved efficacy in suppressing tumor growth  in liposomes for complexing NAs, the clinical translation of such systems has been limited due to carrier‐related toxicity arising from the highly positive charges. In some cases, such lipids can inhibit the gene silencing process.For polymeric nanoparticles, PEI and its derivatives are the most commonly used polymers for treating skin diseases with NAs. To overcome PEI‐associated toxicity, the polymer can be copolymerized with more biocompatible moieties such as cyclodextrinILs are a relatively recently studied drug delivery system whose physicochemical properties can be tuned to enhance the transport and stability of biological molecules. They are designer materials that can be tailored to attain the desired concentration and adequate viscosity desired for topical application without the need for thickening agents. They can also be formulated with cation and anion counterparts that possess a desirable pharmacological effect, acting as bioactive delivery system to augment the efficacy of the loaded bioactive cargo. They offer a promising tool for noninvasive dermal delivery of antigens and NAs. Recently, the use of ILs has moved to into clinical trials. Ko and coworkers reported the clinical translation of CAGE for the treatment of rosacea, which is an inflammatory skin disease.5COX2) gene. This drug has proceeded to Phase II clinical trials in patients with hypertrophic scars.Dermal delivery of NAs can have a potential impact in the clinic due to the existence of many skin diseases with NA therapeutic targets accounting for their pathogenesis.Apart from intradermal injection, other current clinical trials are based on physical disruption methods (mainly microneedles and microjets) with exception for a few transdermal patches that are used for the dermal delivery of some peptide therapeutics. Helix BioPharma developed a cream containing IFNα‐2b encapsulated in liposomes that was used in Phase II human trials for treating low‐grade squamous intraepithelial lesions.It is worth pointing out that combining noninvasive active delivery techniques with the discussed formulation approaches can synergistically promote dermal delivery and achieve better therapeutic outcomes. While not discussed in detail in this review, iontophoresis and sonophoresis have shown promising outcomes in this regard. For example, iontophoresis has been employed to deliver cetuximab, an anti‐EGFR monoclonal antibody, to the deep layers of skin.While sonophoresis has been reported to improve the delivery of a variety of macromolecules,Iontophoretic patches and sonophoretic devices can be used for self‐administration. However, use of such combinations should be carefully considered  to ensure that delivery is directed to target layers of skin while avoiding further penetration into systemic circulation. The combinatorial use of active techniques with rationally designed formulations may help the performance of therapeutic moieties at lower doses, while using physical methods at lower strengths to ensure safety and potentially enable a take‐home therapy.6Despite the recent efforts to advance noninvasive dermal delivery of vaccines and topical NA therapies (summarized in Table The use of peptides and dendrimers may be constrained to some NAs with a certain size as SiRNA and oligonucleotides as opposed to DNA. Lipid nanoparticles and cationic polymers, despite their efficiency for cytosolic delivery, still suffer from some toxicity and instability issues. Chemical conjugations, despite being tunable to provide cellular targetability and controlled release rates, suffer from time‐consuming and labor‐intensive processes.The stability of the biological macromolecules during manufacturing and storage is of critical importance. Care should be taken during the manufacturing process to avoid excessive agitation or shear, as well as drastic changes in pH, ionic strength, or temperature, as those factors can affect the molecular structure and result in a loss of biological activity.When designing lipid or polymer‐based nanocarriers, the formulation parameters such as the choice of excipients and the loading method should be optimized based on the individual properties of the macromolecule and its possible interaction with any of the formulation ingredients. What works with a particular cargo might adversely affect the properties and transport of others.Another important aspect is the translatability of the experimental results obtained with animal models to humans. With exception to studies performed on human skin explants, the majority of the studies have shown success in small animal models. One should take into consideration the biological and structural differences before extrapolating findings to humans since the human skin is thicker, has fewer hair follicles and is less permeable than rodent skin.7Looking at the clinical landscape for vaccines and cutaneous disorders reveals the critical need for noninvasive approaches to deliver biological macromolecules. Recent efforts have been made to overcome the barrier function of the skin by implementing a variety of smart delivery systems. To further advance this field, we need a better understanding and practice of how to deliver various macromolecules safely and efficiently to target cells. Translational value of preclinical work can be increased by using human‐relevant models such as human skin explants, tissue‐engineered skin models, and human‐based tissues grafted onto mice. Addressing these aspects and combining them with a deeper understanding of cell biology can lead to the improved design of bioengineered materials to navigate the skin and improve prophylactic and therapeutic outcomes.Marwa Sallam: Conceptualization; data curation; methodology; writing‐original draft. Supriya Prakash: Data curation; writing‐original draft. Ninad Kumbhojkar: Data curation; writing‐original draft. Wyatt Shields: Writing‐original draft. Samir Mitragotri: Conceptualization; writing‐review & editing.


We compared the response to resource enhancement of a simple empirical model of intra‐guild predation (IGP) to the predictions of published, simple mathematical models of asymmetric IGP . The empirical model was a food‐web module created by pooling species abundances across many families in a speciose community of soil micro‐arthropods into three categories: IG Predator (large predatory mites), IG Prey , and a shared Resource (fungivorous mites and springtails). By pooling abundances of species belonging to broadly defined functional groups, we tested the hypothesis that IGP is a dominant organizing principle in this community. Simple mathematical models of asymmetric IGP predict that increased input of nutrients and energy to the shared Resource will increase the equilibrium density of Resource and IG Predator, but will decrease that of IG Prey. In a field experiment, we observed how the three categories of the empirical model responded to two rates of addition of artificial detritus, which enhanced the food of fungivores, the Resource of the IGP module. By the experiment's end, fungivore densities had increased ~1.5× (ratio of pooled fungivore densities in the higher‐input treatment to plots with no addition of detritus), and densities of IG Predators had increased ~4×. Contrary to the prediction of mathematical models, IG Prey had not decreased, but instead had increased ~1.5×. We discuss possible reasons for the failure of the empirical model to agree with IGP theory. We then explore analogies between the behavior of the empirical model and another mathematical model of trophic interactions as one way to gain insights into the trophic connections in this community. We also propose one way forward for reporting comparisons of simple empirical and mathematical models. We compared the response to increased input of detrital resources of a simplified trophic grouping of many species of soil micro‐arthropods—the empirical model—to the behavior of published simple mathematical models of intra‐guild predation (IGP). IGP theory did not predict the overall behavior of the empirical model. We discuss how the failure of two simple models to agree could lead to a deeper understanding of the trophic connections in this speciose community. Consumers that feed extensively on more than one trophic level present a challenge to this simplification; ecologists have retained the abstract concept of trophic chains by labelling this feeding pattern trophic‐level omnivory : the direct and indirect interactions between two predators (at least one of which feeds on the other) and a shared non‐predaceous prey. This module  that describe the dynamics of the three IGP elements in terms of consumer–resource interactions, with the condition that the Resource grows to its carrying capacity in the absence of both consumers  micro‐arthropods such as mites and springtails are abundant in leaf litter and underlying soil horizons  soi Hopkin,  yet are  Hopkin, , increas Hopkin, ; (d) num Hopkin, . We thenn Figure .22.12 plots were located within a 1‐ha area of forest at the Morton Arboretum in Lisle, Illinois, USA. One hundred and fifty plots, selected at random, were fenced with low aluminum flashing; the remaining 50 plots were left open as the reference (REF) treatment. Although there were no obvious gradients across the study site, plots were distributed among five blocks to account for possible effects of undetected gradients in soil properties. Three levels of detrital supplementation  were randomly assigned to the fenced plots. The added detritus was flakes of fruitfly‐culture medium mixed with chopped mushrooms and potatoes. The amount added per square meter every 2 weeks in the Low (1X) treatment was similar to that employed in an earlier experiment with unfenced plots in which densities of mites (Acarina) and springtails (Collembola) had increased after ~4 weeks  and 3 months later. Animals were sampled again in the fenced plots in early April the following year, just prior to resumption of detrital supplementation in the Low and High treatments, and were sampled in all plots at the end of the experiment in mid‐August 2015 . Separate samples of leaf litter and the underlying organic soil horizon were taken to the laboratory where animals were extracted into 70% ethanol with a modified Berlese/Tullgren funnel  and springtails (Order Collembola), the two most abundant taxonomic groupings of micro‐arthropods in our samples. Adults were classified as IG Predators (10 families), IG Prey (7 families), or Fungivores (23 families) based upon published field and laboratory studies of gut analyses or what individuals in each family have been directly observed to consume. Our category “Fungivores” includes organisms that not only graze fungal hyphae but also consume animal and plant detrital materials—hence their occasional designation as “microbi‐detritivores”  at the end of both field seasons.Evidence for a response to detrital supplementation in the three fenced treatments was the presence of a Treatment × Time interaction in a repeated‐measures, mixed‐effects linear or generalized linear model. Whether to retain Block as a factor, and the impact of extreme outliers on model behavior, was evaluated by whether ∆AIC > 2 and by testing the underlying mathematical assumptions of the simplified model. Because repeated‐measures models revealed treatment effects, differences between the None, Low, and High treatments were evaluated at each sampling period following the initial, pre‐treatment sample, with log as a covariate. Results are presented as model‐fitted effect sizes expressed as ratios  with 95% confidence intervals (CIs). Details appear in Appendix 32 areas of the forest floor, suggesting that our fenced plots enclosed a relatively intact, natural community. We then describe how densities responded to detrital addition in the fenced experimental units—the behavior of our empirical model.We first present the results of model selection and simplification, followed by evidence that fencing did not adversely affect densities of micro‐arthropods within 1‐m3.1The initial models that adequately satisfied model assumptions were linear models with a log transformation for counts of Fungivores, and for counts of IG Prey and IG Predators, generalized linear models with a log link and the negative binomial family Appendix .1. Block3.2Fencing the plots had no discernable impact upon densities of any of the IGP categories in plots that had not received a detrital supplement Figure 4. Althou3.3P = 0.0001, 0.022, and 0.006, respectively; Appendix Initial inspection of mean abundances per sample Figure  reveals Does this pattern portend an eventual trajectory of densities of IG Prey in the High treatment becoming less than those in the None treatment, as predicted by simple mathematical models Figure ? Unfortu3.4The temporal pattern of model‐fitted effects of the Low and High detrital enhancements leads to a different conclusion. The system's response was not congruent with the behavior predicted by simple mathematical IGP models when effect sizes are examined Figure .Effect size for the Low treatment (Low/None) for all IGP compartments at the end of each field season was ~1.5 or less Figure  but not IG Predators displayed a consistently large response to High rates of supplementation, exhibiting densities ~4× higher than those in the None treatment at the end of both field seasons Figure , Month 9One way to discern where the system may have been heading is to examine the overall pattern of concurrent changes in the High‐treatment effect Figure .4Deciding how trophic groups relate to modularity in the simplification of food‐web structure is a recurring theme in food‐web ecology  that are linked by a top generalist predator  the hypothesized agreement between empirical model and mathematical model —and how this agreement will be tested statistically. Then, publish and interpret results of the empirical test of the model no matter what the discovered level of agreement.Ways exist to navigate around this whirlpool. We suggest the following research tack as one solution. Before the empirical model is investigated, publish in some readily accessible form ; data curation (supporting); formal analysis (lead); funding acquisition ; investigation (supporting); methodology ; project administration (supporting); resources ; software ; supervision ; validation ; visualization (lead); writing‐original draft (lead); writing‐review and editing (Lead). Monica A. Farfan: Conceptualization (supporting); data curation (lead); formal analysis (supporting); funding acquisition ; investigation (lead); methodology ; project administration (lead); resources ; software ; supervision ; validation ; visualization (supporting); writing‐original draft (supporting); writing‐review and editing (supporting).Appendix S1‐S3Click here for additional data file.


Determination of pleural dissemination of lung cancer helps define the treatment strategy. Positron emission tomography‐computed tomography imaging could be false‐negative for dry pleural dissemination of lung cancer. Clinicians should consider preoperative thoracoscopy in affected patients showing limited pleural effusion, interlobar fine granular shadows, and no metastasis on high‐resolution computed tomography. Aggressive thoracoscopy can help clinicians diagnose dry pleural dissemination, which leads to the appropriate treatment in lung cancer cases with interlobar granular shadows in HRCT even if PET‐CT shows negative uptake in pleural dissemination. Computed tomography (CT) performed with 1.25‐mm slices showed a spiculated 12‐mm nodule in the right upper lobe with limited pleural effusion Figure  and indiLocal anesthetic thoracoscopy is usually considered in patients with cytology‐negative pleural effusion.An aggressive thoracoscopy may contribute to the diagnosis of dry pleural dissemination of lung malignancy in patients showing no PET‐CT uptake except for that by the primary tumor.None.AN contributed to the clinical management of the patient, was involved in study conception, acquisition and analysis of the data, and drafting of the manuscript. HT was involved in study conception, acquisition and analysis of the data, and drafting of the manuscript. MT was involved in supervision of the drafting of the manuscript and critical revision of the manuscript. All authors reviewed the final draft of the manuscript and approved its submission.This study was approved by the institutional review board and ethics committee of the Japanese Red Cross Ise Hospital (permission number: ER2021‐33).Written informed consent was obtained from the patient for the publication of this case report and accompanying images. A copy of the written consent is available for review on request by the Editor‐in‐Chief of this journal.


The coronavirus disease 2019 (COVID‐19) is currently a major pandemic challenge, and cancer patients are at a heightened risk of severity and mortality from this infection. In recent years, immune checkpoint inhibitor (ICI) use to treat multiple cancers has increased in oncology, but equally has raised the question of whether ICI therapy and its side‐effects is harmful or beneficial during this pandemic.A combination of published literature in PubMed between January 2010 and December 2020, recommended guidelines in non‐cancer patients, and clinical experience was utilized to outline recommendations on glucocorticoid timing and dosing regimens in ICI‐treated patients presenting with AI during this COVID‐19 pandemic.The potential immune interaction between ICIs and COVID‐19 require major consideration because these agents act at the intersection between effective cancer immunotherapy and increasing patient susceptibility, severity and complications from the SARS‐CoV‐2 sepsis. Furthermore, ICI use can induce autoimmune adrenal insufficiency (AI) that further increases infection susceptibility. Thus, ICI‐treated cancer patients with AI may be at greater risk of COVID‐19 infection. Glucocorticoids are the cornerstone for replacement therapy, and for treatment and mitigation of adrenal crisis and relief of mass effects in ICI‐related hypophysitis. High‐dose glucocorticoids have also been used with cytotoxic chemotherapy as part of cancer treatment, and iatrogenic AI may arise after glucocorticoid discontinuation that increases the risk of adrenal crisis. Furthermore, in patients who develop the “long COVID‐19” syndrome, when to discontinue glucocorticoid therapy becomes crucial to avoid unnecessary prolongation of therapy and the development of iatrogenic hypercortisolemia.During the COVID‐19 pandemic, much of cancer care have been impacted and an important clinical question is how to optimally manage ICI‐related AI during these unprecedented times. Herein, we suggest practical recommendations on the timing and dosing regimens of glucocorticoids in different clinical scenarios of ICI‐treated cancer patients presenting with AI during this COVID‐19 pandemic. Cancer patients treated with immune checkpoint inhibitors (ICIs) who develop adrenal insufficiency may be at a greater risk of severity and mortality from the COVID‐19 infection. The effects of ICIs and the appropriate use of glucocorticoids is crucial in optimally managing these patients and preventing unnecessary hypercortisolemia and worsening of symptoms of some patients, including those with long COVID‐19. We suggest practical recommendations on the timing and dosing regimens of glucocorticoids in different clinical scenarios of ICI‐treated cancer patients with AI during this COVID‐19 pandemic. We utilized a combination of published literature, recommended guidelines in non‐cancer patients, and clinical experience to help outline our proposed recommendations on the timing and dosing regimens of GCs in different clinical scenarios of ICI‐treated cancer patients presenting with AI during this COVID‐19 pandemic.33.1Cancer patients are more susceptible to infections due to co‐existing chronic diseases, overall poor health status, and systemic immunosuppressive states caused by the cancer and anti‐cancer treatments.3.2Immune checkpoint inhibitors are monoclonal antibodies that target immune checkpoints (PD1 and PD‐L1), and by virtue of restoring the antitumor immunity through the reversal of immune escape or evasion have led to significant antitumor activity. Because of their mechanism of action in enhancing immune response, specific immune‐related endocrinopathies are increasingly apparent ranging from moderate to severe and life‐threatening ones,3.33.3.1Hypophysitis is one of the most common endocrine irAEs associated with ICI therapy.The clinical manifestations of hypophysitis can range from having none to one or more features of pituitary hormone deficits  to the acute onset of AI  and mass effect symptoms .Previous studies have demonstrated that non‐cancer patients with AI have a twofold to eightfold higher risk of infection.3.3.2Primary AI due to ICI therapy is uncommon and has been associated primarily with ipilimumab and rarely in combination with other ICIs.3.3.3Cancer patients may develop iatrogenic AI following use of high‐dose GCs as part of chemotherapy or targeted towards suppression of an inflammatory response for other chronic conditions.4During the COVID‐19 pandemic, it is important that ICI‐related endocrinopathies are not missed. Prior to initiation of ICI therapy, baseline measurements of basal pituitary hormones , fasting glucose and electrolytes should be obtained , or if the patient cannot eat due to nausea or vomiting, hospitalization is mandatory and close monitoring for features of acute AI  is recommended to avoid adrenal crisis.A unique feature of ICI‐treated patients is that they may present with pituitary mass effect symptoms  that could also precipitate adrenal crisis. In these circumstances, high‐dose GC therapy  is recommended treat the mass effectIt is also important to consider the possibility of patients with AI presenting with adrenal crisis with COVID‐19 being the precipitating factor, and yet display no COVID‐19 symptoms. Thus, in any patient that presents with clinical signs of AI, we recommend performing COVID‐19 testing in these patients, even in the absence of typical COVID‐19 symptoms. Additionally, COVID‐19Finally, the syndrome of ‘long COVID‐19’ has recently been recognized to refer to patients being ill for more than 4 weeks.7This review presents a summary of research, clinical guidance and practical recommendations on the management of ICI‐related AI during the COVID‐19 pandemic. As ICI therapy is a promising therapeutic agent in oncology, its use is increasing but unwanted immune system activation against the endocrine system is an unfortunate reality. In the ongoing COVID‐19 pandemic, one important question is whether cancer patients—already vulnerable to COVID‐19—may be at significantly greater risk of severe COVID‐19 illness if their cancer therapy included ICIs and concurrent AI. The current thinking is that, at least for now, halting or modifying ICI‐related treatment decisions is not justified, and close surveillance with SARS‐CoV‐2 testing is recommended for patients who are on or about to start ICI therapy. Furthermore, knowledge of ICI‐related AI and its potential mass effects due to hypophysitis necessitates GC therapy. However, because symptoms of AI and COVID‐19 are not specific and may be confused with those caused by the cancer, clinicians treating patients with ICI‐related AI should promptly recognize the need to optimize GC therapy in a variety of clinical scenarios. However, GCs is a double‐edged sword in the COVID‐19 setting; hence, they need to be used carefully, considering the risk‐benefit ratio, such as a short treatment course  in a select group of COVID‐19 patients for whom survival benefit has been reported. Although some of the symptoms of the ‘long COVID‐19’ syndrome may overlap with those of AI, for example, fatigue and joint pains, there is currently no evidence supporting long‐term GC use to prevent potential adverse sequelae such as pulmonary fibrosis. In this review, we have highlighted several management strategies based on current best practice, but acknowledge the gaps in knowledge of COVID‐19 and the need for more research. It is inevitable that as the prevailing circumstances of the COVID‐19 pandemic continue to change, we will learn more about SARS‐CoV‐2 and that the recommendations for GC therapy will evolve based on developing evidence.KCJY, MJM, AA, EF and JWF have no conflicts of interest to declare.KCJY conceived and presented the idea for review, and drafted the manuscript. MJM, AA, EF and JWF helped to focus the concept and direction of the review, were involved in reviewing and revising the manuscript critically, and approved the final version of the manuscript.Since this review summarizes and informs already published studies, ethical approval is not applicable.