Seattle protocol biopsies for Barrett's Esophagus (BE) surveillance are labour intensive with low compliance. Dysplasia detection rates vary, leading to missed lesions. This can potentially be offset with computer aided detection. We have developed convolutional neural networks (CNNs) to identify areas of dysplasia and where to target biopsy.i‐scan  imaging in patients with dysplastic and non‐dysplastic BE (NDBE). We trained an indirectly supervised CNN to classify images as dysplastic/non‐dysplastic using whole video annotations to minimise selection bias and maximise accuracy. The CNN was trained using 148,936 video frames , validated on 25,161 images from 11 patient videos and tested on 264 iscan‐1 images from 28 dysplastic and 16 NDBE patients which included expert delineations. To localise targeted biopsies/delineations, a second directly supervised CNN was generated based on expert delineations of 94 dysplastic images from 30 patients. This was tested on 86 i‐scan one images from 28 dysplastic patients.119 Videos were collected in high‐definition white light and optical chromoendoscopy with The indirectly supervised CNN achieved a per image sensitivity in the test set of 91%, specificity 79%, area under receiver operator curve of 93% to detect dysplasia. Per‐lesion sensitivity was 100%. Mean assessment speed was 48 frames per second (fps). 97% of targeted biopsy predictions matched expert and histological assessment at 56 fps. The artificial intelligence system performed better than six endoscopists.Our CNNs classify and localise dysplastic Barrett's Esophagus potentially supporting endoscopists during surveillance. Summarise the established knowledge on this subjectBarrett's esophagus is associated with an increased risk of progression to esophageal cancer.Despite advances in endoscopic imaging early cancer in BE is still being missed.Studies have investigated neural networks for dysplasia detection using Fuji and Olympus systems. No studies have done so using Pentax imaging.What are the significant and/or new findings of this study?Demonstrate a computer aided detection (CAD) system that is able to detect BE dysplasia with high accuracy on a per image and patient level and localise areas of interest with targeted biopsies with high sensitivities.This is the first study demonstrating an Artificial intelligence (AI) system in BE using the I‐scan/Pentax imaging system.We demonstrate and compare different approaches to development of algorithms for detection/localisation of BE to help identify the best approach to training these systems.Barrett's Esophagus (BE) is associated with increased progression risk to oesophageal cancer (EAC), progressing from non‐dysplastic Barrett's Esophagus (non‐dysplastic BE (NDBE)) to low grade dysplasia (LGD), high grade dysplasia (HGD) and then EAC. The overall 5‐year survival rate of EAC is less than 20%The current standard of care for BE surveillance is to take biopsies every 2 cm as part of the Seattle protocol. This is time consuming and may suffer from sampling errorThere is growing interest in the use of computer aided detection (CAD) of early lesions in the gastrointestinal tract. To date, this has focused on detection of colonic polypsWith advances in endoscopic optical technology, classification protocols have been developed based on vascular architecture and mucosal pit pattern to improve dysplasia detection.I‐scan  is a virtual chromoendoscopy technique that uses post processing technology to provide contrast and surface enhancement. There are 3 modes – i‐scan 1 (surface enhancement), i‐scan‐2 (contrast enhancement), i‐scan 3 (tone enhancement). I‐scan 1 has become the default equivalent of high‐definition white light (WL) with the Pentax system. It is the best imaging mode for lesion detection . We defined these as endoscopists with more than 5 years' experience of BE endotherapy and who perform endotherapy procedures weekly in BE expert centres as defined by the European Society of Gastrointestinal Endoscopy guidelines.i‐scan one imaging modes in patients with dysplastic lesions in BE (LGD/HGD/EAC) and patients with NDBE. I‐scan 1 was the default imaging modality in the collected data in all centres. ‘Pull throughs’ were also collected in WL for most patients.Videos were prospectively collected using the Pentax endoscopy system . Mucus lining the Esophagus was removed using a simeticone and water solution. The endoscopist then performed a ‘pull through’ withdrawal of the endoscope from the gastroesophageal junction to the maximal extent of BE. Procedures were recorded in high‐definition WL and BE with no suspicion for dysplasia was biopsied as per the Seattle protocol. Areas suspicious for dysplasia either were target biopsied or resected by endoscopic mucosal resection (EMR). Histology was reviewed by expert BE histopathologists with more than 10 years' experience. Cases of suspected dysplasia were reviewed by two different BE histopathologists in each expert centre.A computer vision annotation tool  was used to annotate a sequence of video frames. Annotation confirmed that dysplasia was present within individual frames without defining the position. Gold standard was determined from histology of EMR specimens or biopsies and the annotation segments matched these areas on the videos. In NDBE patients, all the frames from the Esophagus, including squamous mucosa, were included. The dysplastic and non‐dysplastic frames were used to train and validate a classification convolutional neural network  and 1 (dysplasia present). Further algorithm development details can be found in the supplementary section.A directly supervised learning classification network is trained on BE images which have been individually delineated for areas of dysplasia (methodology for training model 2). We derived heat maps for areas of interest from the classification CNN using an indirectly supervised learning approach. This relies on informing the model whether an image contains dysplasia (Methodology for training model 1). The network works out what constitutes an abnormality for itself.i‐scan one images and six WL images were randomly selected per patient. Two expert delineations per image on 86 dysplastic images from 28 patients helped test the reliability of the computer‐generated heat map outputs. Refer to supplementary Table One hundred eighteen different patients were included. Lesions were randomly split (computer generated) into training, validation and testing sets with no overlap of data. Each set was stratified to ensure consistent proportions of patients Figure . The netWe trained a model with a FCNResnet50 architecture to classify pixels as dysplastic or non‐dysplastic using still images with an expert delineation of an area of dysplasia. The backbone of the model was trained from an external data set .One hundred ninety‐two images containing BE dysplasia from 64 different patients were each delineated by two experts for dysplastic areas. These images were randomly selected. Further model details are in Figure Sixty one i‐scan one images from the testing set were randomly selected . Six non expert endoscopists with greater than 3 years of endoscopic experience assessed for the presence/absence of dysplasia on each image. The performance of the CNN in detecting dysplasia was compared to endoscopists.Descriptive statistics consisted of the mean (+/− standard deviation). We measured the sensitivity and specificity at a per‐image and per patient level. The area under the receiver operator curve (AUC) was calculated. Further details are in the supplementary section.i‐scan 1 images with an AUC of 93%, sensitivity of 91% and specificity of 79%. The AUC was 10% greater than on WL  and a per image specificity of 79% in the 16 patients with no dysplasia.i‐scan 1 images, where at least 2 of the 6 images presented for each patient were correctly diagnosed as dysplastic, the CNN achieved a per patient sensitivity of 100%. Using a majority voting approach where at least 4 of 6 images were correctly diagnosed, the sensitivity for dysplasia detection was 89.3%.Using a minority voting approach on Four different scenarios for localisation of points of interests were generated Table . A targeExpert consensus determined that the optimal scenario would be to recommend two points of interests for biopsies. This balances the number of necessary biopsies with high performance. Based on this, the system recommended targeted biopsies in areas of the union of expert delineations with a sensitivity of 91% in the model two test set of 28 dysplastic patients and 78% in the intersection of expert delineations Figure , Table 2The delineation outline prediction for an area of dysplasia generated by the segmentation model overlapped with at least one expert delineation in 98% of images with a minimum of one pixel of overlap Figure . This suThe mean (+/− standard deviation) time for the classifier analysis of each image was 0.021 s per image (+/− 0.008), or 48 frames per second (fps). The mean time for the segmentation network to delineate dysplasia on each image was 0.018 s (+/− 0.006) or 56 fps.On a subset of testing set images 6 non expert endoscopists detected dysplasia with a mean sensitivity of 79% and specificity of 49%. The CNN classified dysplasia with a sensitivity of 96% and specificity of 88% on the same images Table .i‐scan 1 imaging. The heat map generated by the indirectly supervised learning algorithm, once thresholded, overlaps with at least one expert endoscopist in 98% of images with a minimum of one pixel of overlap. We have developed a second, directly supervised, CNN which can localise points of interest to detect dysplasia with a sensitivity of up to 97%.We demonstrate for the first time a CAD system which can accurately detect early neoplasia in BE using Pentax endoscopes. It has a per image sensitivity and specificity of 91% and 79%, respectively, on In current practice endoscopists perform ‘pull throughs’ in the esophagus during BE surveillance and take targeted biopsies. An indirectly supervised machine learning approach, can potentially be a promising methodology to identify an area of interest in BE with heat map overlays. It works fast, within 20 milliseconds, without the need for specialised expert delineations of every frame as in previous studies.We chose to use an indirect learning approach to generate heat map outputs from the classifier to assess whether they would help endoscopists to identify areas of interest in BE without the need of expert delineations to train, to provide insight into the classifier's predictions, and to compare to the segmentation output using a directly supervised learning approach to assess the potential implications on the way we train and process data when developing BE CAD systems. The outputs suggest that the model focuses on important dysplastic features.n = 64) each with a video assessment. We also trained on still images like in previous studies but use multiple frames per video to maximise the training data set and provide more valuable information for the CNN's. The CNN achieved high sensitivities in detecting EAC and HGD but also LGD. Previous studies did not include LGD. We felt it was important to include this subgroup in our data as these lesions are often upstaged on EMR to HGD or EAC.Van der Sommen et al developed a computer algorithm using 100 still images. The system identified early BE dysplasia with a sensitivity and specificity of 83%.I‐scan one is often the default imaging modality on these systems as this provides the natural colour tone of WL and added advantage of surface enhancement. Therefore, the training set was predominantly composed of I‐scan 1 as opposed to WL. Our results hint towards improved performance of the classifier model on i‐scan 1 versus WL. However, of note the data set for i‐scan 1 was larger than that of WL and matched studies are needed to directly compare the performance of the two.Previous studies trained and tested networks on Olympus and Fujinon endoscopy systems for the detection of BE.Seattle protocol biopsies on a surveillance endoscopy will sample a small surface area of a BE segment.Studies have developed systems which are able to detect dysplasia in BE at varying speeds. De Groof et al.i‐scan one images.Six images per case were selected for the testing set to simulate the clinical workflow, where relevant endoscopic images would be taken. A previous pilot study tested the performance of a CAD system during live endoscopy assessment of BE on freeze frames. They used a majority and minority voting approach to label an area of BE as dysplastic.The CAD system in our study performed better than all 6 non expert endoscopists with a range of 3–11 years of endoscopic experience. This is the cohort for whom a BE CAD system would be most beneficial. A smaller subset of images was randomly selected for this experiment as it will mean endoscopists will be more likely to complete the task. The AI system performed better on this subset however when comparing the endoscopists performance to the AI system performance on the whole data set it is still weaker.There are limitations in this current study. We developed a model which is trained using videos from a single endoscopic system. Ideally, to allow for more generalisation, we would incorporate data from other systems. The current CNN is tested on higher quality still images. However, our training strategy and data selection would allow us now to develop networks which could potentially work better on whole video data in real time. Another limitation is the threshold for neoplasia detection was based on the test set. However, the performace can be compared independently of the threshold selection by looking at the AUC scores. It is important to test the threshold selection on an independent, hold out, data set, which was not available at the time. Another limitation is the testing set in ‘model 2’ (segmentation) was 86 images. Ideally, we would have selected a larger test set however in this model these images were all required to be delineated by experts. For the purposes of the available time of experts and to complete the task a smaller data set of images was selected. This problem could be rectified in future by selecting a broader range of experts. The data set for i‐scan 1 was larger than that of WL. This should be kept in mind when looking at the improved performance on i‐scan 1 versus WL in the classification of dysplasia. In future we will perform matched studies comparing the two light modalities where both cohorts are matched in terms of number of images, histology and Paris classification of lesions. Another potential limitation is the segmentation model was trained based on the delineations of one expert. In future to help improve our results further we will aim to train the model using the intersection of expert delineations to improve the localisation ability of the CAD system.We demonstrate a CAD system which is able to detect BE dysplasia with high accuracy on a per image and per patient level. It localises areas of interest with targeted biopsies with high sensitivities. Our next step is allowing this to work in real time with whole video predictions.MH: Speaker fees . JGP,DL, DT and PM: Employees at Odin Vision. DS: Share holder Odin vision and Digital Surgery Ltd. LBL: Consultancy and minor share holder Odin Vision. RH: Educational grants to support research infrastructure from Medtronic ltd. Cook endoscopy (fellowship support), Pentax Europe, C2 therapeutics, Beamline diagnostic, Fractyl Ltd.Supplementary MaterialClick here for additional data file.


The number of species shared by two or more sites is a fundamental measure of spatial variation in species composition. As more sites are included in the comparison of species composition, the average number of species shared across them declines, with a rate increasingly dependent on only the most widespread species. In over 80% of empirical communities, models of decline in shared species across multiple sites (multisite similarity decline) follow one of two distinct forms. An exponential form is assumed to reflect stochastic assembly and a power law form niche‐based sorting, yet these explanations are largely untested, and little is known of how the two forms arise in nature. Using simulations, we first show that the distribution of the most widespread species largely differentiates the two forms, with the power law increasingly favored where such species occupy more than ~75% of sites. We reasoned the less cosmopolitan distribution of widespread species within exponential communities would manifest as differences in community biodiversity properties, specifically more aggregated within‐species distributions, less even relative abundance distributions, and weaker between‐species spatial associations. We tested and largely confirmed these relationships using 80 empirical datasets, suggesting that the form of multisite similarity decline offers a basis to predict how landscape‐scale loss or gain of widespread species is reflected in different local‐scale community structures. Such understanding could, for example, be used to predict changes in local‐scale competitive interactions following shifts in widespread species' distributions. We propose multiple explanations for the origin of exponential decline, including high among‐site abiotic variation, sampling of highly specialized (narrow niche width) taxa, and strong dispersal limitation. We recommend these are evaluated as alternative hypotheses to stochastic assembly. Comparison of diversity patterns for the two dominant forms of multisite similarity decline (a) decline in similarity (b) species accumulation. Given the important role of widespread species biodiversity patterns  assemblage structure (defined using community biodiversity properties) and compositional (among‐sample) patterns offers a powerful means to predict how changes either within or among samples will manifest in the other  site‐by‐species matrix, where marginal totals quantify basic diversity properties of the community; row sums quantify the species richness of each site, and column sums reflect how widely, or narrowly, distributed each species is. Variation in species composition among multiple sites can be quantified using various measures of similarity, or its complement, dissimilarity, depending on the research question Arita, . If the 2.3increase in species number as sites are combined, an analogous curve can be built that quantifies the average decline in the number of species shared , then an exponential model of decline takes the form: ζi=a·exp−b·i and power decline ζi=c·i−d, where a, b, c, and d are model parameters  a; Ni is the total abundance of species i in total area A; ki is a parameter controlling conspecific aggregation ; m is the number of samples (zeta order); and Γ(n) = (n − 1)! is the gamma function. For a given α (= a/A), individuals of each species are assumed to follow density‐dependent conspecific aggregation ki=kNiα, where k is a scaling factor used to model scale dependency in the strength of conspecific spatial aggregation of the community , conspecific aggregation , and species abundance distribution (Ni) to calculate zeta diversity. Observed multisite similarity decline was then fitted to the two different parametric forms and the most likely form for each set of conditions identified (see Post‐processing of simulations). All abundance‐based simulations were based on the 50‐ha Barro Colorado Island stem‐mapped forest plot .Simulations varied the sampling grain  toward random placement (k = 10). We repeated this for each of the three abundance distributions and each scenario we fit the two forms of decline (see Post‐processing of simulations).From the results of the abundance‐based simulations . While a common assumption in biodiversity models McGill, , this doRather than pursuing an exhaustive list of possible structures, the aim here was to test the most likely form of decline as the interspecific association among species became more negative . As the abundance‐based simulations demonstrated a strong influence of sampling grain and therefore matrix fill Section , all matFive different matrix simulations are presented in the main text .A purely random matrix  will have an exponential form of decline. Having simulated matrices with an imposed structure, we then asked two questions: what is the form of decline in the unperturbed matrix structure, and for structures favoring a power law form, how much perturbation in the structure is required to change the best‐supported form from power to exponential? The perturbation algorithm proceeded at each iteration by selecting one row (site) of the matrix randomly. Within each selected site, a swap was performed between a presence and an absence of species. Swaps were constrained to species with at least two occupancies in the matrix to maintain total species number. The number of species at each site and total matrix fill were thereby preserved, but the sites occupied by species (other than species at a single site) were free to vary. With each of 100 iterations of the randomized perturbation algorithm, support for the two forms of multisite decline was calculated (see the following section). Based on the results of the simulations, we present the change in relative support for the exponential form as a function of the corresponding 2.4.3wAIC). To calculate wAIC, the relative likelihood of each model is calculated as exp−0.5·∆AIC, where ∆AIC is the difference in AIC from the top‐ranked model. The Akaike weight for a model is this value divided by the sum of these values across all models, thus giving the relative likelihood of each model in the candidate set. A value of 1 for wAIC indicates no support for the alternative model  and a value of 0.5 represents equal support for both models. Our main interest in simulations was in identifying which form of decline was favored for a given combination of community biodiversity properties or imposed matrix structure, and in the latter case, how disturbed  these structures would need to become to follow this  form.In all analyses of simulations , we fit the exponential and power law forms using the linearized form of the exponential and power law models , and abundance scores (n = 9) were used directly, while percentage cover (n = 6) or other metrics  were back‐transformed where appropriate and then rounded to integer values  database , then multisite decline can be modeled using all sites; if p1 < 1, then the maximum number of orders should be the number of sites occupied by the most widespread species or ln0.05/lnp1, whichever value is smaller , and using a higher number of orders was limited by the available range of sites in the data. For example, using 20 orders would have precluded 25% of datasets. We manually inspected each fit to ensure the better‐supported form provided a reliable qualitative description of the data. Note that the classification of the communities was unchanged if all orders where zeta diversity remained above 1 were used, provided at least 10 orders were used to fit the models. When the minimum number of orders was reduced to five, three communities were reclassified  but the results for the community biodiversity properties remained qualitatively the same Appendix .Having classified the 80 empirical communities as either exponential or power law, we tested for differences in community biodiversity properties Table  between For interspecific and conspecific spatial patterns, our main interest was in comparing community biodiversity properties among the most widespread species in each dataset because of their influence on the observed form of decline Appendix . Given tSES=Obs−mean(Exp)/SDExp, where the mean and standard deviation of the expected (Exp) values were calculated from 999 matrices created using an appropriate null model.To further control for differences in matrix dimensions in the communities, we calculated a standardized effect size in addition to the raw metrics  in measuring the strength of both positive and negative interspecific association and in requiring no data transformation to meet distributional assumptions  that preserves row and column marginal totals , realm , level of anthropogenic impact, and kingdom . All correlates were tested for influence on the observed form of decline using the same approach as for community biodiversity properties. We include other supporting results, including pairwise scatterplots of the relationships among the community biodiversity properties in the Supporting Information , with a random term included to control for non‐independence of multiple datasets from individual studies. We also tested for the importance of the number of sites and total number of species along with ecological correlates provided in the CESTES database: study extent , increasingly even relative abundance distributions increased the probability of observing exponential decline . Stronger conspecific aggregation  also increased the probability of an exponential form  decreased . Further, because they contain fewer widespread cosmopolitan species , and a smaller fraction of total species number in each site , this should allow larger local (within‐site) subpopulations to establish. This would be evident in empirical data as exponential communities have more aggregated conspecific spatial patterns, consistent with the abundance‐based simulations. Further, any such localized conspecific aggregation in exponential communities should result in less even relative abundance  within sites. Conversely, having fewer numerically dominant cosmopolitan species, we anticipated pooled abundance distributions in exponential communities would be more even, as found in the abundance‐based simulations.3.4z = 0.02, p = .6), total number of species , broad taxonomic grouping , ecological realm , or level of anthropogenic impact , suggesting limited influence of ecological context on the form of decline. There was marginal evidence that, despite fitting the model to a constant number of orders, communities represented by more sites favored exponential decline .Of the 80 empirical communities assigned to a form of multisite similarity decline, 27 (34%) supported an exponential form, and the remainder (66%) a power law form , providing general support for this hypothesis of stronger conspecific aggregation in exponential than power law cases.Interspecific spatial patterns among the four most abundant species in communities following exponential decline showed weaker interspecific association  than communities with power law decline. This was evident both as weaker correlation in their abundances  interspecific associations and conspecific aggregation and relative abundance distributions. From an applied perspective, these results show that a simple stochastic versus niche‐based dichotomy for explaining the two forms are not adequate.Non‐mutually exclusive conditions that could foster an exponential form include Table  (i) high4.1Exponential decline has to date been assumed to reflect a dominance of stochastic processes in community assembly  only cannot explain exponential decline at a realistic sampling grain. This suggests that the exponential form is not compatible with an assumption that individuals are positioned at random with respect to the individuals of other species , or in the presence of barriers limiting dispersal of widespread species across the study extent. Moving forward, more fruitful use of multisite similarity decline in empirical studies can be achieved by posing a priori hypotheses on the form it will take based on the known or anticipated strength of ecological processes, particularly abiotic sorting and dispersal among sites. Such an approach will also aid researchers in designing studies that are able to differentiate the most important influences on multisite similarity decline.David C. Deane: Conceptualization ; data curation (lead); formal analysis (lead); methodology (lead); visualization (lead); writing – original draft (lead). Cang Hui: Conceptualization (supporting); formal analysis (supporting); investigation (supporting); methodology (supporting); visualization (supporting); writing – original draft (supporting). Melodie McGeoch: Conceptualization (supporting); formal analysis (supporting); funding acquisition (lead); investigation (supporting); methodology (supporting); project administration (lead); resources (lead); supervision (lead); visualization (supporting); writing – original draft (supporting).The authors thank the handling editor and two anonymous reviewers for insightful comments.This research was supported by the Australian Research Council Discovery Project (DP200101680), and CH is supported by the National Research Foundation of South Africa (grants 89967).Appendix S1Click here for additional data file.


Terpenoids are a highly diverse group of natural products with considerable industrial interest. Increasingly, engineered microbes are used for the production of terpenoids to replace natural extracts and chemical synthesis. Terpene synthases (TSs) show a high level of functional plasticity and are responsible for the vast structural diversity observed in natural terpenoids. Their relatively inert active sites guide intrinsically reactive linear carbocation intermediates along one of many cyclisation paths via exertion of subtle steric and electrostatic control. Due to the absence of a strong protein interaction with these intermediates, there is a remarkable lack of sequence‐function relationship within the TS family, making product‐outcome predictions from sequences alone challenging. This, in combination with the fact that many TSs produce multiple products from a single substrate hampers the design and use of TSs in the biomanufacturing of terpenoids. This review highlights recent advances in genome mining, computational modelling, high‐throughput screening, and machine‐learning that will allow more predictive engineering of these fascinating enzymes in the near future. There is a remarkable lack of sequence‐function relationship within the terpene synthase family making product‐outcome predictions from sequences alone challenging. Engineering of designer terpene synthases is enabled via integration of multidisciplinary approaches, including generation of sequence diversity, genome mining, high‐throughput product profiling, modelling and computational chemistry, and data‐driven computational tools. DMAPP and IPP are produced from acetyl‐coenzyme A via the mevalonate (MVA) pathway in eukaryotes, or from pyruvate and glyceraldehyde‐3‐phosphate via the methylerythritol (MEP) pathway in most bacteria and plant plastids. Alternative pathways, including additional entry points into the MVA and MEP pathways, archaeal MVA pathways, and artificial pathways have also been identified at the sequence level.10), farnesyl pyrophosphate , geranylgeranyl pyrophosphate  and geranyl‐farnesyl pyrophosphate , the pre‐cursors for mono‐, sesqui‐, di‐ and sesterterpenoids, respectively. These prenyl‐PP substrates are converted into structurally diverse terpene scaffolds by terpene synthases (TSs).Terpenoid biosynthesis is highly modular and all terpenoids are derived from the CMany examples exist of microbial production of terpenoids using engineered yeast or bacteria, including drug precursors,The highly branched reaction pathways of TSs is a major cause of the observed terpenoid product diversity. Two classes of TSs exist in nature, each using a different initiation reaction resulting in highly reactive carbocation intermediates. Class I TSs catalyse the metal‐dependent ionisation of the prenyl‐PP substrate, and class II TSs initiate the reaction via electrophilic activation of the substrate.Distantly related class I TSs from plants, fungi and bacteria all contain a strongly conserved class I TS fold containing the highly conserved metal‐binding motifs DDXXD and the NSE/DTE triad [DXXXE)],2Streptomyces species, have long been known for the production of the terpene derivatives 2‐methylisoborneol and geosmin, which are responsible for the characteristic earthy odours of moist soil,epi‐cubenolThe broad spectrum of plant TS activity was first identified in crude plant extracts. TS encoding genes were subsequently isolated from plant material by exploiting the high levels of sequence similarity in amino acid sequences and gene organisation between TSs from the same or related plant species.Streptomyces and other Actinomycetes.S. clavuligerus.S. bungoensis,Amycolatopsis benzoatilytica,S. lincolnensis.Streptomyces sp. (CL12‐4),Chryseobacterium polytrichastri and C. wanjuense,Catenulispora acidiphilaS. venezuelae, with the latter producing a unique 5‐5‐6‐7 tetracyclic skeleton.S. mobaraensis,S. xinghaiensis with promiscuous sesqui‐, di‐ and sesterterpene synthase activity.Rubrobacter radiotolerance, a 10‐epi‐cubebol synthase and aromandendrene synthase from Sorangium cellulosum, as well as a previously identified geosmin synthase from S. coelicolor A3, suggesting that bacteria may be an unexplored rich source for further TS activity. The use of bacterial TSs over enzymes from plant sources is advantageous, as the former often express better in heterologous microbial hosts.With the wide availability of whole genome information, bacterial genomes have become a new source for mining TS activity. However, despite the overall conserved fold, the amino acid sequences of microbial TSs show no significant sequence similarities to related enzymes from plant origin. As such, conventional BLAST searches may not recognise sequences with overall low sequence similarity. Profile Hidden Markov Models containing the two signature metal binding motifs of class I TSs (PF03936) are effective in finding potential TSs in bacterial genomes, as they use statistical descriptions of consensus sequences for a given functional domain. This approach was first applied on bacterial genomes for the identification of 2‐methylisoborneol synthases in Actinomycetes.3Streptomyces UC5319epi‐aristolochene synthase from tobacco (TEAS),S)‐(−)‐limonene synthase (SLimS) from spearmint,The first crystal structures of TSs, the STSs pentalenene synthase from The class I TS fold consists of an α‐helical fold that forms the hydrophobic active site which is closed off from the bulk solvent by flexible loops located on the surface of the protein. Binding of the three metal ions and the pyrophosphate (PP) moiety of the substrate trigger a conformational change resulting in the closure of the active site offering protection from bulk solvent.R)‐(+)‐limonene synthaseS)‐(−)‐α‐terpineol intermediate is observed in bacterial cineole synthase (bCinS),[R)‐(+)‐α‐terpineol intermediate is accumulated in cineole synthase from sage (SfCinS),Another mechanism employed by TSs to avoid by‐product formation is the creation of an active site contour that forces the substrate to bind in a product‐like conformation. This is observed for many TSs, and is particularly striking when the active sites of SLimS (bCinS),51, 564Early domain swapping studies on chimeric enzymes confirmed TS activity is confined to the C‐terminal class I TS domain in plant TSs. These studies also revealed that a limited set of amino acids, located both in, and distant from, the active site, are responsible for product outcome.When several mutational studies on MTSs from plant origin are compared, patterns start to emerge. A residue just upstream of the DDXXD motif, although not conserved, was shown to be important in product outcome in several different enzymes. The presence of a polar residue Asn345) at this position was shown to be essential for the formation of limonene in SLimS.5 at thisepi‐isozizaene synthase resulted in variants that exhibited an expanded product chemo‐diversity.Recent mutational studies on several bacterial TSs yielded valuable insights into how subtle remoulding of the active site contour, often involving aromatic residues, can lead to the accumulation of alternative products. The substitution of aromatic residues with polar residues in the active site of the STS More systematic mutagenesis approaches yielded insights into the mutability landscape of TSs. The reciprocal inter‐conversion of catalytic specificity between two distinct, but evolutionary related STSs, TEAS and premnaspirodiene synthase, allowed the mapping of structural features responsible for functional attributes. It was shown that catalytic specificity indeed relies on active site contour, but crucially, the importance of supporting layers of residues that surround the active site was exposed. The latter may affect catalysis via altering the position or dynamic properties of the active site residues relative to the carbocation intermediates.These systematic mutagenesis studies demonstrate the underlying evolvability of the class I TS fold. Interestingly, the identified plasticity residues generally play a shared role in the catalytic outcome, i. e. the mutations introduced were effectively additive, increasing the likelihood that rational design ideas can be applied to class I TS provided that enough information about the fitness landscape is available5The use of computational chemistry methods can provide valuable insights in TS catalysed reactions that are not accessible experimentally. Quantum mechanical (QM) calculations are used to study gas phase carbocation chemistry, modelling and bound states are studied using molecular dynamics (MD), and in‐enzyme reactions are studied using multiscale methods such as quantum mechanics/molecular mechanics (QM/MM), and account for the effects of enzyme environment, cofactors, solvent and salt.Computational prediction of the correct fold, and therefore shape of the active site in the absence of a crystal structure is challenging, although this is enabled by recent advances in structure predictions such as AlphaFold.Computational studies have revealed crucial insights into the strategies used by TSs to control reactive carbocation inter‐mediates. QM/MM free‐energy simulations have shown that TSs gain chemical control over the reactive carbocation intermediates by raising the energy barriers of branch‐point intermediates to by‐pass the formation of unwanted side‐products. This is in contrast to most other enzymes that reduce the free‐energy barriers of the transition state to achieve enhanced reaction rates.Aspergillus terreus. QM/MM MD simulations revealed that the carbocations are guided at each step via strict conformational restraints or cation‐π interactions from additional aromatic residues that are not present in TEAS, the latter being a much more promiscuous enzyme. The absence of cation‐π interactions in TEAS resulted in higher reaction barriers and less heat release in the latter stages of the cyclisation cascade.Ever since the first crystal structures became available for TSs the important role of bulky aromatic residues in active site contouring and carbocation stabilisation has been recognised. However, the number of aromatic residues in the active site of different TSs varies. bCinS contains four Phe residues restricting the active site volume of the enzyme, which provides a rationale for the fact it does not accept FPP as a substrate unlike the related enzyme bLinS.Computational chemistry has also been applied to study the quenching mechanisms of TS catalysed reactions. Deprotonation is the most common termination mechanism, but the catalytic base for proton abstraction is not always obvious. Computational approaches supplied further evidence for the involvement of PP,6So far the methods discussed largely gained insights into the reaction mechanism of individual enzymes. Data‐driven approaches such as machine‐learning (ML) can produce models that give additional insights into the reaction chemistry of the class I TS enzyme family as a whole. However, ML algorithms are highly “data‐hungry” often requiring a large number of sequence‐function observations to reach acceptable performance levels. This sequence‐function data can be obtained from natural occurring sequence diversity or generated artificially via directed evolution (DE), the latter being heavily reliant on the availability of efficient high‐throughput (HTP) screening assays.in vivo in engineered microbes, where terpene formation competes with other artificial pathways for a shared isoprenoid substrate pool,in vivo in engineered Escherichia coli.The development of suitable HTP screening assays for TS activity is particularly challenging due to the fact that multiple volatile products may be produced from a single substrate. Several screening assays for the detection of enhanced TS activity have been developed, such as colorimetric detection of PP by‐product release using malachite green,7As mentioned previously, TS sequences are highly diverse, and sequence similarity amongst plant TSs is strongly influenced by phylogeny,An early example of the application of computer algorithms to predict combinations of mutations in a TS was performed on γ‐humulene synthase, a promiscuous STS generating >50 products. The enzyme was first subjected to active‐site saturation mutagenesis to identify plasticity residues, which were then systematically recombined using a mathematical model that predicts a set of mutations based on how much the product distribution moved towards the desired product for a given combination of mutations. This approach yielded several variant synthases with narrower product specificities than the native enzyme.A recent study combined different computational approaches, including homology modelling, ML and co‐evolutionary analysis, to predict precursor cation specificity for plant STSs.8TSs are intriguing enzymes that catalyse the high‐energy cyclisation of reactive carbocations in highly branched cyclisation cascades. These reactions often result in the formation of multiple terpenoid products which hampers the use of TSs in biotechnological applications for the production of industrially desirable terpenoid products. To prevent enzyme inactivation, TSs have evolved to ‘manage’ the reactive carbocations in relatively inert, hydrophobic active sites. This lack of direct interaction with the reaction intermediates has resulted in an overall lack of sequence‐function correlation amongst TSs, and product predictions from the amino acid sequence alone remains elusive to date.Despite their overall low sequence similarity, through the use of clever data‐mining tools and the growing availability of genome sequence data the discovery of novel TS activities has accelerated in recent years. Structural analysis revealed a highly conserved fold for all class I TSs. The availability of structural and mutagenesis data has resulted in the identification of conserved areas of plasticity collectively responsible for product outcome. The critical involvement of aromatic residues in guiding the reactive intermediates via placing steric constraints and cation‐π interactions is a common feature in TSs. In fact, the number of aromatic residues in the active site may be used to predict if an enzyme is likely to be promiscuous or not, with high product fidelity seemingly being correlated with a larger number of aromatic residues. Furthermore, crystallographic studies have shown that substrates bind in product‐like conformations. Computational docking tools have been developed that allow the prediction of substrate and carbocation binding modes, including stereo‐chemical preferences, paving the way for the prediction of product properties from protein sequences, provided that tools such as the recently developed AlphaFold for the highly accurate prediction of protein structures, allow precise prediction of the active site contour.The continuing development of HTP assays for the detection of diverse volatile terpenoids, will allow the screening of larger TS variant libraries increasing the chance of finding TSs with desired properties in DE experiments. In addition, these experiments will lead to large sequence‐function data‐sets which, along with the wealth of putative TS sequences stored in public databases can be used in data‐driven ML approaches, ultimately leading to designer TS activities. It is important to note that HTP screening can only detect known compounds, so classical isolation experiments will also continue to play an important role in the discovery of new TS activities.In conclusion, through recent advances in the availability of genome information and mining tools, efficient DE protocols and the development of HTP screening technologies, including laboratory automation, as well as sophisticated computational tools, it is increasingly possible to acquire a holistic picture of the TS enzyme family and TS chemistry. And by combining the wealth of available information the prediction of function from sequence is becoming a reality Figure .The authors declare no conflict of interest.Nicole Gerharda Henrica Leferink received her BSc in Biochemistry and Biotechnology from the Saxion University of Applied Sciences and her MSc and PhD in Biochemistry from Wageningen University. Prior to joining the Future Biomanufacturing Research Hub as Research Fellow, she conducted postdoctoral research in molecular enzymology at the Manchester Institute of Biotechnology. Her main research interests are rational engineering and directed evolution towards the development of designer enzymes for applications in biomanufacturing.Nigel Shaun Scrutton is Professor of Molecular Enzymology and Biophysical Chemistry at the University of Manchester. He received his BSc from King's College, University of London, and his PhD and ScD degrees from the University of Cambridge. His research interests are focused on enzyme catalysis from structural, mechanistic and kinetic perspectives, synthetic biology and the biomanufacturing of chemicals, materials and synthetic fuels. He is Director of the UK Future Biomanufacturing Research Hub and the Synthetic Biology Research Centre SYNBIOCHEM. He co‐founded the fuels from biology company C3 BIOTECH in 2015.


To estimate the effects of state‐level changes in the minimum wage on marriage and divorce among low‐wage earners.Proponents of raising the minimum wage highlight the potential benefits of increased earnings for low‐income families, yet to date research on the effects of raising the minimum wage has focused almost exclusively on economic outcomes. No research has yet documented whether these changes actually affect marriage and divorce.Using the Current Population Survey and the American Community Survey, this project applied a quasi‐experimental difference‐in‐difference method to exploit similarities between states that have, and have not, raised their minimum wage.Across data sources, among men and women earning low wages, a one‐dollar increase in the state minimum wage predicts a 3%–6% decline in marriage entry and a 7%–15% decline in divorce one and 2 years later.Both changes are likely to strengthen low‐income families, and are substantially larger effects than those obtained by federal policies directly targeting interpersonal dynamics within low‐income couples.Government policies that reduce stress on couples and facilitate their access to resources may improve family outcomes, invisibly and without making additional demands on the time of couples who are already strained. ACSAmerican Community SurveyCPSCurrent Population SurveyFPLFederal Poverty LineGDPGross Domestic ProductOLSOrdinary Least SquaresSESSocioeconomic StatusAlthough individuals with low incomes aspire to marriage and stable partnerships as much as more affluent individuals  and ensuring parallel trends before the first wage change. Second, multiple changes per state allow us to estimate a separate state‐specific time trend after the first minimum wage change . Finally, this method lends itself to straightforward estimators that are robust and easy to evaluate. Since the now‐classic Card and Krueger  is a primarily telephone‐based survey of approximately 60,000 households in populous areas, designed to be representative of the labor force; the American Community Survey (ACS) is a primarily mail‐based survey of approximately 300,000 households in all geographic areas, designed to be representative of the broader population. As the analyses focused on family formations among potential minimum wage‐earning adults, only individuals aged 18–35, who comprise the majority of minimum wage earners  by state, year, and gender, using survey‐provided individual weights to ensure state and national representativeness. For example, we calculated the fraction of males ages 18 to 35 who had ever been married in Ohio in 2005, and treated that as one observation in the resulting aggregate panel. Each regression specification had a sample size of 51 states and districts providing data annually over 11 years. As we did not expect immediate translation of minimum wage increases, we explored the impacts of policy changes on marriage and divorce at 1‐ and 2‐year lags. We did not examine further lags to avoid any chance that estimates of the effects of one adjustment to the minimum wage are affected by a second adjustment within the same state  would have threatened our ability to identify precisely who is affected by minimum wage changes. Second, indirect classification avoids introducing the endogeneity of being a minimum wage earner—a minimum wage increase may encourage entry into the labor force, into employment, and into minimum wage employment specifically. Instead, following prior work , and would suffer from any endogeneity related to the timing of the minimum wage change. Pooling all of the states together across many years allows each state to serve as its own control, permitting state‐level adjustment for parallel trends in addition to a posttreatment time trend. Thus, we used a quasi‐experimental difference‐in‐difference methodology, estimating both the difference within‐state (over time) and the difference between states that differ in their timing of minimum wage increases.before each state's first observed minimum wage change. We also controlled for the logarithm of state GDP and recessionary periods to account for other time‐varying economic impacts on marriage and divorce.Our state‐level approach rests on two key assumptions. The first is that trends in control states and in states changing their minimum wages would have followed parallel trajectories in the absence of a minimum wage increase. To confirm that this assumption was satisfied in our model, we detrended data for each state‐gender panel, using only observations The second assumption is that effects in the post‐change period can be attributed only to the minimum wage change. An external factor affecting the number of marriages or divorces in a state while or shortly after a minimum wage change occurred would bias our estimates by incidentally absorbing that impact. We thus estimated a second trend during the period after the first minimum wage change, which was feasible because nearly all states with a minimum wage change in our analysis frame had enacted multiple changes between 2003 and 2015  rates as:ϕst is the predicted outcome of interest in a state‐specific, gender‐specific regression with a linear time trend and no other controls, estimated using only the pretreatment observations for each state. premst is the amount by which a given state's minimum wage exceeds the federal minimum wage in a given year. Thus, a state with a state minimum wage equal to the federal minimum wage has a premium of zero.We estimated the effect of states' minimum wage premia—defined as the amount that each state's minimum wage exceeded the federal minimum wage ‐ on the detrended state proportion of the low‐wage population married  denoted by β1 gives the average change in family formation  in response to a one‐dollar increase in the state minimum wage in excess of any federal requirements. We estimate each model in the CPS and ACS separately to evaluate whether our results replicate within each independent dataset.The use of pretreatment and posttreatment state‐specific time trends permit us to interpret minimum wage premia as minimum wage changes while controlling for the state context that may have led to the change. Thus, positive effects on earnings and no effect on hours worked for at‐risk men and women both 1 and 2 years after the minimum wage increase , firms may hire fewer workers or cut hours, thereby reducing household earnings. To evaluate this possibility, we used our same difference‐in‐difference approach to examine wages within low‐wage households likely to be sensitive to minimum wage changes, defined as those in which at least one worker was earning $20 per hour or less. Our estimates found no evidence for a negative effect of minimum wage increases on earnings among this population. In fact, state‐level minimum wage increases had significant ease see . BecauseTable Table To evaluate the robustness of our results, we conducted several additional tests. First, we estimated a series of placebo treatment regressions where states were randomly assigned the minimum wage history of another state, and then re‐estimated our baseline regressions. We conducted this test 1000 times and found that the minimum wage effect was never statistically correlated with marriage or divorce outcomes in these randomization tests see . This efWe also explored different low‐wage definitions  and a specification which pooled men and women together. All of these results see  provide To date, debates over the effects of raising the minimum wage have focused narrowly on their economic impact (Cengiz et al., A preliminary result of this analysis is to confirm that, when states increase their minimum wage, incomes among low‐wage households do in fact rise significantly without increasing hours worked. Even as debates about the minimum wage continue to invoke concerns about the potential for disemployment and thus income reductions as a consequence of higher labor costs (e.g., Machin & Manning, Commensurate with these effects, we find robust evidence that state‐level increases in the minimum wage predict lower rates of marriage and divorce 1 and 2 years following such changes. Although it remains possible that rising incomes spurred marriages for low‐wage earners who were already coupled, the net decline in marriage among unmarried individuals supports Life History perspectives (Giudice et al., Because later marriages are on average less likely to end in divorce Rotz, , a conseEstimated effects on marriage and divorce were strikingly similar across the CPS and the ACS, despite the differences in scope and methods of the two surveys. Nevertheless, several factors constrain interpretation of these results. First, although we compared results across 1‐ and 2‐year lags, other effects may emerge at longer lags. These analyses could not determine whether declines in rates of marriage and divorce represented transitions deferred or decisions to forgo these transitions entirely. Second, our analyses focused on the younger workers who represent over half of minimum wage earners (U. S. Bureau of Labor Statistics, In sum, during the same years that federal policymakers spent nearly a billion dollars on educational programs that had negligible effects on marriage and divorce, states that simply raised their minimum wages delayed marriages and reduced divorce rates significantly among low‐wage earners. Future efforts to strengthen low‐income families should therefore explore additional policy levers, such as expanding the earned income tax credit (Michelmore, The authors declare that they have no conflict of interest.Appendix S1. Supporting InformationClick here for additional data file.


Cutaneous angiosarcoma (AS) of the head and neck is a rare highly aggressive tumor, often associated with difficult local control of the disease and poor prognosis. This article describes a case of multifocal cutaneous AS of the scalp, mainly addressing its difficult surgical management and challenging reconstruction and concludes with a review of the literature.A 70‐year‐old Caucasian male was referred to our hospital with a growing scalp lesion initially suspected to be benign, but later diagnosed with AS.The patient had tumor recurrence and a difficult reconstruction for which dermal substitutes proved very useful.AS can mimic a benign lesion in its early stages. Skin substitutes, namely dermal templates, can be useful to meet the complex needs of reconstruction and oncological surveillance of patients with AS. ASangiosarcomaAJCCamerican joint committee on cancerCAScutaneous angiosarcomaCTcomputerized tomographyDRTdermal regeneration templatesENDelective lymph node dissectionFNACfine needle aspiration cytologyFNCLCCfédération nationale des centres de lutte contre le cancerHNCAShead and neck cutaneous angiosarcomaHNSAhead and neck sarcomasLNMlymph node metastasisMDTmultidisciplinary head and neck oncology teamNPWTnegative pressure wound therapyOSoverall survivalPETpositron emission tomographyPFSprogression‐free survivalVEGFvascular endothelial growth factor1Angiosarcoma (AS) is a rare, aggressive, malignant tumor, originating from vascular endothelial cells that can arise at any anatomical location.armamentarium.This article describes a case of multifocal cutaneous AS of the scalp, reporting not only the early delay in the definitive diagnosis, but also the difficult surgical management secondary to its large defect and anatomic location, that was managed with dermal substitutes, an emerging add‐on to the reconstructive surgeon 2A 70‐year‐old retired male patient with good overall performance status was referred to the Head and Neck Surgery department of the Portuguese Institute of Oncology of Lisbon, due to a large violaceous scalp lesion with 10 months of progressive growth and associated spontaneous bleeding.He had a personal history of cervical lymph node tuberculosis, hepatitis B virus infection, hypertension and dyslipidaemia, but no previous hospital admission record. He denied smoking or high alcohol intake. He reported a family history of unspecified cancer (brother) and leukemia (grandson).On physical examination, the patient displayed a wide ulcerated red lesion of the scalp at the right occipital region, 15 × 11 cm wide, associated with cutaneous inflammation. A smaller lesion was observed close to the vertex where the patient exhibited a small area of alopecia. Also, subcutaneous lumps at left parietal and right occipital areas were palpated. Remaining neck and general examination was unremarkable  scan, that suggested multifocal cutaneous and subcutaneous lesions in the scalp adherent to the skull bone. No lymph node involvement or distant metastasis were identified on CT scans of neck, thorax, abdomen and pelvis.At our hospital, cytology of the palpated subcutaneous satellite lesions confirmed AS.The case was discussed at the multidisciplinary head and neck oncology team (MDT) and the decision was for surgical treatment.An experienced team of head and neck surgeons performed the excision of the cutaneous lesions to the level of the pericranium and reconstruction of the resulting defect with galea rotation flap with full‐thickness skin graft collected from the left thigh. No complications occurred in the immediate post‐operative time and the patient was discharged after 4 days of hospital stay. The histologic result was of epithelioid AS of high degree of malignity (G3) – Fédération Nationale des Centres De Lutte Contre Le Cancer (FNCLCC) histologic grading system – with perineural spread, vascular invasion and positive deep resection margins Figure .en bloc with the grafted skin, galea aponeurosis, and outer layer of skull bone. The large scalp defect was reconstructed with heterologous acellular dermal matrices—Integra® double layer and vacuum dressing. The histologic analysis of the surgical specimen demonstrated positive deep surgical margin and microscopic lymph node tumor invasion  scan reported right occipital and left iliac crest lesions. The first one was confirmed to be a lymph node metastasis (LNM) after fine needle aspiration cytology; the second was submitted to a CT guided biopsy and was negative for neoplastic cells.Due to the start of COVID‐19 pandemic, the patient missed immediate clinical and wound care follow‐up after hospital stay. At 4 weeks after the second intervention, one‐third of dermal template was lost to wound infection with The case was revaluated at the multidisciplinary decision clinic, and it was decided reoperation followed by adjuvant radiochemotherapy. Thus, the patient underwent right type III modified radical neck dissection, excision of the frontal lesion with superficial ostectomy of the skull, surgical debridement of the exposed area of scalp and reconstruction of both defects with acellular skin substitute Integra® single layer and split‐thickness skin graft Figure . The patAt 4 weeks of follow‐up from previous intervention, the patient presented with favorable wound healing of the scalp Figure . FollowiFour months after latest surgery, the patient had no symptoms, maintained good wound healing of the scalp and a clear neck physical examination after surgery. For the next 4 months the patient decided to go living far away to his countryside home because he was afraid of current pandemic situation, so that he had to be follow‐up by phone. Although he reported no suspicious symptoms or physical signs by teleconsultation, a new follow‐up PET scan reported local and regional progression of the disease, confirmed by biopsy. This time, the decision was for adjuvant chemotherapy with paclitaxel, which the patient had to delay for 2 weeks because he contracted COVID infection and needed hospitalization.Currently, at 9 months of follow‐up since last surgical procedure and about 12 months after evaluation at our hospital, the patient is undergoing chemotherapy with good tolerance, and displays no signs of further disease progression at clinical examination Figures  and 8.3armamentarium—dermal substitutes—with a high degree of success. In line with what is most frequently described in the literature, the present report occurred in a male Caucasian patient, in the 60–70 years age group, located in the head and neck  of all AS is reported between 6% and 13.5%.Distant metastasis may occur in up to 50%.There are no stablished guidelines for the treatment of cutaneous AS of the head and neck.Our case also brings awareness to the lymph node management in angiossarcomas because cervical metastasis was identified in our patient, so an elective neck dissection (END) was performed. Usually, END is not indicated because reported LNM is low.Our case report is an example of the difficulty of local control of head and neck sarcomas (HNSA).Also present in this case report is the reconstructive challenge of large scalp defects, particularly when adjuvant radiotherapy was considered.The skull was exposed, and the dermal substitutes covered a wide area, so it was decided to associate negative pressure wound therapy (NPWT), which is thought to promote tissue granulation and decrease wound volume by debriding devitalized tissue, decreasing bacterial colonization, promoting blood flow, and removing excess serous fluid that might inhibit wound healing.Microssurgical free tissue transfer also plays an important role in scalp reconstruction because large defects with bone exposure as well as the need for adjuvant therapy are frequent.Ultimately the appropriate reconstructive approach to the scalp is a case‐by‐case decision that depends on the wound characteristics, patient factors and available technical expertise.There is insufficient published data of an advantage in progression‐free survival or overall survival (OS) with the use of systemic chemotherapy in localized AS.A recent metanalyses of AS of the scalp and face showed a 5‐year survival rate of 33.5%.Prognosis is also influenced by AS etiology—radiation induced (secondary) AS have poorer prognosis than sporadic types—and treatment modality—patients who undergo both radical surgery and adjuvant radiotherapy have 5‐year survival of 67%, better than those who undergo either chemotherapy plus radiotherapy or surgery alone.There is a trend toward improved outcomes in HNCAS in recent years, which may stem from greater awareness toward the disease, new radiotherapy modalities, and improved reconstruction of larger defects, namely with free tissue transfer.The SARS‐CoV‐2 pandemic interfered at least in the follow‐up of the second and last surgical interventions of our patient, the last one motivated by fear of infection and angst of potential lethal complications due to his oncologic health status. Indeed, there is evidence that the COVID‐19 pandemic, significantly impaired emotional wellbeing in patients with cancer.In conclusion, it is important to be aware of AS in the differential diagnosis of head and neck cutaneous lesions because it may mimic a benign lesion at early stages and is often associated with difficult disease control, as it was described in our patient. Surgery is the cornerstone of treatment, frequently followed by chemoradiotherapy. Skin substitutes are a potential solution with proven efficacy in the present case to the complex reconstruction of large scalp defects caused by aggressive tumors such as AS, with the potential benefit of local surveillance, usually not suitable with other options, such as free tissue transfer.Future studies are needed to assess whether Covid‐19 pandemic implications to follow‐up routine or delayed start of chemotherapy influence prognosis in AS patients.Henrique Messias: Conceptualization (supporting); data curation ; formal analysis ; investigation (lead); methodology ; project administration ; resources ; validation ; visualization ; writing – original draft (lead); writing – review and editing (lead). Mariluz Martins: Conceptualization (lead); resources ; supervision (lead); validation (lead); visualization (lead); writing – original draft (supporting); writing – review and editing (supporting). Carlos Zagalo: Conceptualization ; data curation ; formal analysis ; supervision ; validation ; visualization . Pedro Gomes: Investigation ; project administration (lead); resources ; supervision ; validation ; visualization .The authors have stated explicitly that there are no conflicts of interest in connection with this article.Ethical approval was obtained from and in accordance with the local institutional ethics committee.


To (i) describe the prevalence of malnutrition among a cohort of central Australian, predominantly Indigenous, haemodialysis patients and (ii) determine the sensitivity and specificity of the Patient Generated Subjective Global Assessment total score for identification of malnutrition in these patients.Cross‐sectional observational study of all patients attending haemodialysis units within the Central Australia Health Service. Patients were assessed using the Patient‐Generated Subjective Global Assessment. Chi‐Square tests were used to determine the association between nutritional status and location, age and gender. Receiver Operator Characteristic curves were used to ascertain the predictive validity for malnutrition of the total score.n = 249/253). One third were male, and 72% were aged between 30 and 59 years. Approximately 29% (74/253) were malnourished, and 93% (69/74) had a total score ≥ 4. The most frequently reported problems that kept malnourished patients from eating were early satiety (32%), no appetite (31%), diarrhoea (26%) and dental problems (24%). Money problems were reported by 32%, as were transport (20%) and depression (19%). The traditional tool cut off score of ≥9 had low sensitivity (50%) for detecting malnutrition. Instead, a score ≥ 3 is suggested due to a higher sensitivity (96%) and specificity (45%).Indigenous patients comprised 98% of study haemodialysis patients (Malnutrition was found to be common, and we suggest using a Patient‐Generated Subjective Global Assessment total score of ≥3 to improve the identification of malnourished individuals in this cohort of predominantly Indigenous haemodialysis patients. This will significantly increase referrals for dietetic intervention. In the most recent census of haemodialysis in Australia (2020), there were approximately 10 916 people undertaking in‐centre haemodialysis.Few studies in Australia have examined the nutritional status of Indigenous Australians who undertake dialysis. Of the single published study of 25 South Australian Aboriginal and 51 non‐Aboriginal South Australian haemodialysis patients in two satellite units, it was found that 35% of Aboriginal patients were malnourished.There are also few studies that have examined the predictive validity of the PG‐SGA in the Australian dialysis population, or more specifically the Australian Indigenous dialysis population. Campbell et al.Given these evidence gaps, and the important health and financial implications of malnutrition among dialysis patients,2All maintenance haemodialysis patients attending the acute or satellite haemodialysis units in the Central Australia Health Service were approached to participate in the study. Exclusion criteria included patients with known cognitive impairment, or inability of the carer to assist with completion of the PG‐SGA; or unavailability of an interpreter/Cultural Liaison Officer to assist with communication and completion of the assessment.Ethical approval was obtained from the Central Australia Human Research Ethics Committee . Patients were approached by either a dietitian or student dietitian and asked to participate in the study, which included a full nutritional assessment completed by the dietitian, and a physical exam using the PG‐SGA tool. The study was conducted in each unit over the course of 1 week in October 2016. Verbal informed consent was obtained from all participants or their guardian.Dietitians completed the PG‐SGA alongside patients. To ensure consistency between assessors, three team training sessions were conducted prior to undertaking the audit. In addition, all assessments were completed in pairs (with a more senior staff member paired with a less experienced staff member) to ensure accuracy of assessments. A script was also provided to dietitians to ensure questioning was consistent during the assessment.Section three of the PG‐SGA (i.e. ‘symptoms that have kept me from eating enough during the past 2 weeks’) included one additional prompt. This prompt was used when exploring ‘other’ factors impacting intake and explored if transport impacted access to food. This is widely known to impact the nutritional status in this population group.Demographic and anthropometric data including dry weight, height, and weight history were obtained from the medical records by the renal dietitian. Where dry weight did not correspond to the weight history recorded on the dialysis charts, clarification was sought from the nephrology team or discussion was held between the renal dietitians known to the patient and the Nurse Unit Manager to determine a more appropriate dry weight to record. Responses from patients regarding the presence of wounds was cross checked with medical record files and data on patients under the care of the wound management team. Ethnicity as reported in the medical file was recorded. Dialysis locations were recorded as one of five dialysis units located within the Alice Springs region. Age was retrieved from the medical record, and for the purposes of this study were categorised as 18–29; 30–59; 60–64; 65–74 and ≥75 years. Self‐reported residence was noted and verified by assessors by cross checking against the renal transport bus list . BMI was categorised according to the recommendations from the KDOQI 2020 Clinical Practice Guidelines for Nutrition in Chronic Kidney Disease (CKD).U was used to explore the relationship between total PG‐SGA score, anthropometric variables  and nutritional status. To ascertain the sensitivity and specificity, and optimal PG‐SGA total cut off score for identifying malnourished haemodialysis patients, the receiver operating characteristics (ROC) curve was used. An area under the curve (AUC) of 0.9–1 indicates the PG‐SGA score was an excellent measure for detecting malnutrition; 0.8–0.89 a good test; and 0.70–0.79 a fair test.p value of 0.05. Reporting of this study is consistent with the STROBE statement for cross sectional studies.All statistical analysis was conducted using SPSS software . Normality of continuous data was determined using the Shapiro–Wilk test. Descriptive data is reported as median and interquartile range or number and proportions. Chi Square tests were used to explore the association between malnutrition status and categorical variables such as gender, place of residence, and dialysis location. The Kruskal–Wallis test or Mann–Whitney 3Over the study period, a total of 253 patients consented to participate in the study (equating to a response rate of 76%). The study sample was around one third male , gender (p = 0.78), dialysis location  or a patient's place of residence (p = 0.38). There was also no significant difference in malnutrition rate between the Indigenous and non‐Indigenous participants (p = 0.62).The overall prevalence of malnutrition for the Central Australia Health Service haemodialysis population was 29% . The most frequently reported problem that kept malnourished haemodialysis patients from eating enough were: money problems (Table n = 24/74); early satiety ; dental problems ; diarrhoea ; and no appetite . Transport was reported as a problem impacting the ability to eat enough by 20% (15/74) of malnourished haemodialysis patients. The proportion of patients overall with a total score ≥ 4 indicating that referral to a dietitian was required was 52% (132/253).There were significant differences in the total PG‐SGA numerical score between nutritional status categories Table . Those wp < 0.0001); depression ; diarrhoea ; early satiety ; money problems ; nausea ; no appetite ; swallowing problems ; smells bothering them ; taste changes ; vomiting ; transport issues ; and a higher proportion with a total PG‐SGA score of ≥4  among those who were malnourished.When examining symptoms that impacted eating in the prior 2 weeks, there were many important differences between malnourished and well‐nourished groups. This included a higher proportion of dental problems .The ROC analysis is shown in Figure 4This study provides a comprehensive overview of the nutritional status of a large cohort of predominantly Indigenous haemodialysis patients in the Central Australia Health Service. This represented nearly one fifth (16%) of the national Indigenous haemodialysis population in 2016.The rates of malnutrition in this study are comparable to those previously documented in Australian dialysis populations. A meta‐analysis of the global prevalence of protein energy wasting estimated 17.9% of the Australian dialysis population were malnourished.The 7‐point Subjective Global Assessment is now recommended as the preferred tool for nutrition assessment due to the robust evidence base, particularly for assessment of body composition in dialysis cohorts.Strategies to address malnutrition in the Indigenous dialysis population are urgently needed. This includes strategies to identify those at risk. Development of a culturally appropriate, specific malnutrition assessment tool for Indigenous Australians may be useful. Recent work on malnutrition in the central Australian inpatient setting identified that a mid‐upper arm circumference of <23 cm demonstrated a strong positive predictive value for malnutrition (96% 95% CI: 89–99.2).The critical importance of involving Indigenous consumers in any strategies designed to reduce malnutrition also cannot be understated. A 2016 report identified that more than 75% of all new dialysis patients were required to relocate from a remote community to commence dialysis.There are several strengths to this work. Researchers assisted patients to complete the PG‐SGA and ensured training among assessors occurred in the week prior to assessment to reduce intra‐assessor variability. Assessors were also dietitians well known to the patient cohort and this may have increased participation and trust among patients. However, there are several limitations. The study was not able to examine relationships between nutritional status and dialysis vintage, dialysis clearances  or any biochemical markers. The findings may not be generalisable to other Australian dialysis units as the proportion of Indigenous patients in this study was significantly higher.To conclude, this study has found that malnutrition appears to be present in about one in three haemodialysis patients from the Central Australia Health Service. The symptom burden among these patients is also high. Future work should explore symptom clusters to help to develop and triage new models of care, in addition to interventions to assist with financial and social factors impacting nutritional status. In the absence of specific nutritional assessment tools, the authors suggest use of the PG‐SGA using a total score of ≥3 to improve the identification of malnourished individuals in this cohort of predominantly Indigenous haemodialysis patients. This will significantly increase referrals for dietetic intervention.LC and LN designed and conducted the research; KL analysed the data; LC, LN and KL wrote and revised the paper. LC and LN had primary responsibility for the final contents. All authors read and approved the final manuscript. The authors acknowledge Margo Bell and Clare Brown for assistance with data collection.The authors declare no conflicts of interest.


We review here the use of container molecules known as cavitands for performing organic reactions in water. Central to these endeavors are binding forces found in water, and among the strongest of these is the hydrophobic effect. We describe how the hydrophobic effect can be used to drive organic molecule guests into the confined space of cavitand hosts. Other forces participating in guest binding include cation−π interactions, chalcogen bonding and even hydrogen bonding to water involved in the host structure. The reactions of guests take advantage of their contortions in the limited space of the cavitands which enhance macrocyclic and site‐selective processes. The cavitands are applied to the removal of organic pollutants from water and to the separation of isomeric guests. Progress is described on maneuvering the containers from stoichiometric participation to roles as catalysts. Calix[4]resorcinarene‐based water‐soluble deep cavitands and their applications in molecular recognition, isomers separation, sensing and removal of organic pollutants and reaction vessels are summarized and discussed. Very recently, we showed that metallo‐cavitand H7−2Pd showed a good shape and size selectivity for o‐difluorobenzene over its isomers. Competitive experiments concerning the binding of difluorobenzene isomers to cavitand H7−2Pd used 19F NMR spectroscopy and revealed their stabilities decreased in the following order: o‐difluorobenzene>p‐difluorobenzene ≫ m‐difluorobenzene  – up to 6.32×104 m−1 for the complexation of THF and 1.09×104 m−1 for 1,4‐dioxane. Quantum chemical calculations were also employed to optimize the host−guest geometries and showed that, in all cases, no hydrogen bonds could be formed directly between the guest and the NH groups of the benzimidazole. Instead, the oxygen atoms of the guests were engaged in one or two hydrogen bonds, with either water molecules at the seam  or water molecules hydrogen‐bonded to them  experiments showed high association constants , the reduction of alkyl dihalide guests with trialkylsilanes (R3SiH) was performed in a water‐soluble metallo‐cavitand H6−2Pd  and good selectivity. Unlike conventional templates with convex structures that become guests inside their assembled hosts, cavitands with concave surfaces reverse the roles and resemble the situation in biological catalysis – the templates are hosts for guests undergoing the assisted reaction processes.End‐to‐end intramolecular cyclization of long‐chain linear precursors is generally hard to achieve and often unpredictable because of unfavorable entropic problems and competitive intermolecular reactions. Beyond the mono‐functionalizations described above, template effects are also featured in complexes where intramolecular reactions can occur through control of guest conformation.56, 574In summary, this mini‐review focuses on the most recent advances of resorcin[4]arene‐derived water‐soluble deep cavitands. It includes their applications in molecular recognition, isomeric guest separations, sensing and removal of organic pollutants and their use as reaction vessels. Molecular recognition is the basis for distinguishing the subtle differences between isomers, determining the relative strength of cation−π and hydrophobic eﬀects in water, sequestering small hydrophilic molecules and separating complex mixtures. Some fundamental principles of noncovalent interactions were also determined from the study of these synthetic host‐guest systems, which enhanced our understanding of both chemical and biological molecular recognition phenomena. Guests confined in the limited spaces of synthetic containers can adopt unusual orientations, which leads to unexpected reactivity and changes in their chemical and physical properties. Mono‐functionalization and macrocyclization were successfully reported, in which water‐soluble cavitand hosts act as protecting groups, templates and reaction vessels. While these examples constitute encouraging applications of synthetic molecular containers, some problems still need more attention. Future cavitand research should address applications of these molecules as catalysts rather than as recyclable stoichiometric agents. Additionally, the development of resorcin[4]arene‐based cavitands as molecular sensors outside laboratory settings remains a challenge.The authors declare no conflict of interest.5Yujie Zhu obtained her Ph.D. degree in Chemistry from Nankai University in 2018 under the supervision of Professor Zhijin Fan. In 2018, she joined the team of Professor Julius Rebek, Jr. and Professor Yang Yu at Shanghai University, focusing her research on the synthesis of new water‐soluble funcionalized deep cavitands, moleculer recognition and reactivity in confined cavity. Since 2021, she joined the faculty of the department of chemistry at Shanghai University.Mingkai Zhao received his BS degree in chemistry from Hunan University in 2018. Currently, he is a master student in the group of Professor Julius Rebek Jr. and Yang Yu at Shanghai University. His research interests are supramolecular chemistry based on organic synthesis.Yang Yu received her Ph.D. in 2013 from Heidelberg University working under the direction of Professor Stephen Hashmi and then moved to UT Southwestern Medical Center as a post‐doctoral researcher with Professor Uttam Tambar. In 2015, she joined the group of Professor Julius Rebek, Jr., at The Scripps Research Institute as a research associate then started her independent career as a professor in Shanghai University in 2017. Her current research interests lie in molecular recognition and catalysis.Julius Rebek, Jr., is the Director Emeritus of the Skaggs Institute for Chemical Biology and at the Scripps Research Institute and special professor at Shanghai University since 2021. He received his Ph.D. degree in chemistry from MIT and has held professorships at UCLA, the University of Pittsburgh, and MIT. He is a member of the U.S. National Academy of Sciences, and honorary member of the Hungarian Academy of Sciences and the Royal Swedish Academy. His current research interests include self‐assembling systems and molecular behavior in small spaces.


Previous studies on the impacts of racism on adolescent development have largely overlooked Indigenous youth. We conducted a scoping review of the empirical literature on racism against Indigenous adolescents to determine the nature and scope of this research and to establish associations with developmental outcomes. Our literature search resulted in 32 studies with samples from the United States, Canada, Australia and New Zealand. Studies were limited to self‐reported experiences of racism and thus primarily focused on perceived discrimination. Quantitative studies found small to moderate effects of perceived discrimination on adolescent psychopathology and academic outcomes. Qualitative studies provided insight into structural forms of racism. We offer recommendations for future investigations into the impacts of overt and covert racism on Indigenous adolescents. Overt racism is largely condemned in settler‐colonial nations; it has clear intent to disempower, exclude or harm and is thus easy to identify and sanction. Covert racism is harder to track and often manifests as ambiguity and paradox. For example, covert racism is found in both intolerance s Chong, ; in negaStudies from settler‐colonial nations indicate that Indigenous adolescents experience poorer mental health outcomes compared to non‐Indigenous adolescents  and adolescent development. We took a broad definition of development so that studies were included if they examined salient tasks of adolescence  or risks specifically associated with adolescence . Consistent with previous reviews  for study inclusion were adolescents aged 12–25 years  included Native American participants (USA), followed closely by Aboriginal and Torres Strait Islander (AUS) youth (n = 10). Five studies were exclusive with First Nations Canadian (CAN) youth and an additional five included both the United States and Canadian populations. Only one study included Māori (NZ) youth. Seven studies included participants under the age of 12  included youth aged 12–18 years.The majority of studies , First Nations (CAN), Aboriginal and Torres Strait Islander (AUS), and Māori (NZ), without specifying tribes/Nations.Native American and First Nations Canadian youth were largely recruited through research partnerships with local Reservations . Johnston‐Goodstar and VeLure Roholt (The majority (75%) of studies employed quantitative survey methodology. Surveys were often administered verbally to maximize comprehension. Eight studies included both youth and caregiver reports  reported at least one aspect of culturally grounded methodology  of studies measured racism via perceived discrimination. The most popular measures were the Perceived Discrimination Measure , which includes three subscales; global discrimination; authority discrimination; and school, and the Schedule of Racist Events  adapted for Indigenous groups. The frequency of perceived discrimination varied across the studies but always fell within the low to mid ranges (see Table The vast majority (n = 3), Canada (n = 2), and the United States (n = 3). Only the Australian studies focused exclusively on school contexts, and all found negative associations between racism and academic outcomes, including achievement, engagement, and academic selfsabotage, with small to moderate effect sizes . These studies also associated racism with negative impacts in the academic domain. For example, Johnston‐Goodstar and VeLure Roholt . Eight of these were quantitative and included samples from Australia , Substance use and Conduct Disorder were the dominant externalizing behaviors measured. Dickerson et al.  found thFour studies  examined relations between racism and bullying  and North America (n = 2). Schinke et al.  Indigenous‐developed models of health and well‐being will be essential for informing culturally specific and culturally relevant outcomes. For example, while Dickerson et al.  developmental theory risks overlooking the significant areas which contribute to both risk and thriving among Indigenous youth. As developmental science progresses toward antiracist research practice  on Indigenous adolescents' academic outcomes and internalizing and externalizing symptoms. Greater breadth and depth of focus on outcomes of racism is needed with both local and international studies to enable cross‐cultural commentary on the frequency and severity of the impact of racism. Likewise, only by considering racism against Indigenous adolescents as embedded within settler‐colonialism and as distinct from other racial and ethnic populations will developmental science be able to fully unveil and unpack this deeply complex, nuanced, and the insidious system of oppression.Table S1. Search Strings used in Literature Search.Click here for additional data file.Table S2. Summary of Studies Reviewed.Click here for additional data file.