Wheat amylase-trypsin inhibitors (ATIs) are a family of plant defense proteins with an important role in human health for their involvement in allergies, celiac disease and non-celiac wheat sensitivity. Information about the differences in ATI activities among wheat genotypes and the influence of the growing environment is scarce. Therefore, ten selected wheat accessions with different ploidy level and year of release, previously characterized for their ATI gene sequences, were grown during three consecutive crop years at two growing areas and used for in vitro ATI activities. The contributions of the genotype and the crop year were significant for both activities. The hexaploid wheat genotypes showed the highest inhibitory activities. Einkorn had a peculiar behavior showing the lowest alpha-amylase inhibitory activity, but the highest trypsin inhibitory activity. It was not possible to observe any trend in ATI activities as a function of the release year of the wheat samples. The two inhibitory activities were differently affected by the growing conditions and were negatively correlated with the protein content. This information can be important in understanding the extent of variation of ATI inhibitory properties in relation to the wheat genotype and the growing environment and the impact of ATIs, if any, on human health and nutrition. Wheat amylase-trypsin inhibitors (ATIs) are a family of water-soluble proteins with low molecular weight and account for about 2–4% of total wheat protein . Four grATIs are accumulated at high levels in the endosperm of the grain kernel where they give an important contribution to plant defense against pests and pathogens . They caRegarding the impact on human health, ATIs are involved in bakers’ eczema, asthma and food allergy,  and, recIn recent years, there has been an increasing interest from consumers and farmers in landraces and old wheat varieties which are wheat species that remained unchanged over the last hundred years, in contrast to modern species released after 1950, i.e., after the phase commonly referred to as the “Green Revolution” . SeveralThere are few and, in some cases, contrasting data in the literature about the differences in ATIs existing between different wheat genotypes and, in particular, between ancient and modern wheat varieties. Data concerning the inflammatory potential of ATIs (determined as pro-inflammatory cytokine release in TLR4-responsive mouse and human cell lines) from different wheat varieties showed that modern hexaploid wheat contained higher ATI inflammatory activity than some ancient variants like diploid (einkorn) and tetraploid wheat  or older hexaploid variants like spelt . RegardiIn the present study, ten selected wheat accessions with different ploidy level and year of release, previously characterized for their ATI gene sequences , were grTriticum species were grown under organic farming management for three consecutive crop years at two locations in the USA and in Italy. At the American location, it was not possible to harvest Peliss wheat in 2016 and 2017 and emmer harvested in 2016 was not included in the analyses because it was destroyed while in storage.Ten selected samples belonging to different The meteorological data (temperature and rain) recorded at both sites are shown in All the wheat genotypes were tested for their protein contents and levels of mycotoxins (DON), and results are shown in In general, samples grown in the USA showed higher protein contents than those cultivated in Italy. This agrees with the fact that, compared to Italy, in Montana (USA) the average wheat yields are typically lower and the protein contents are higher . All theThe results of the alpha-amylase inhibitory activity showed that there was high variability among the ten wheat genotypes analyzed .T. aestivum, triticale and rye were significantly affected by the variety, genus and year of harvest as sources of changes [The general linear model (GLM) highlighted that the contribution of the fixed (genotype) and the random (year) factors, as well as their interactions, to the variability of the alpha-amylase inhibitory activity was significant in both growing areas. Similarly, another study showed that inhibitory activities against human saliva alpha-amylase in two cultivars of  changes . HoweverIn this study, the mean values for the alpha-amylase inhibitory activity ranged between 1.12% and 66.63%, with the lowest activity observed in einkorn samples in both growing areas. In general, except for emmer, the hexaploid wheat genotypes showed higher inhibitory activities than tetraploid and diploid wheat genotypes. Interestingly, these results are supported by the ATI gene sequences obtained previously from the same samples used in this study . In factConsidering the results of the alpha-amylase inhibitory activities, it was not possible to highlight differences between ancient and modern wheat genotypes. The same conclusions were reported by Gelinas et al.  who alsoAs mentioned above, the lowest activity was observed in einkorn samples in both growing areas with values much lower than the other wheat samples or even close to zero. These results agree with previous studies based on immunological, mass spectrometry and colorimetric tests ,19,20,21The mean values for each genotype in the three crop years are shown in In the USA, 2016–2017 and 2017–2018 crop years showed higher alpha-amylase inhibitory activities than 2015–2016. All the wheat genotypes showed a similar trend among the three crop years with the exceptions of einkorn and turanicum samples which significantly increased and decreased, respectively, their activities in 2017–2018. As it is shown in In Italy, the highest alpha inhibitory activities were in 2016–2017 and the lowest in 2018–2019. Overall, there were not samples showing a different trend compared to the average. The precipitation was most abundant in the last months before the harvest in 2019 (period April–July). In particular, the amount of precipitation in May was double compared to the averages for that area , and as In both growing areas, higher precipitation resulted in higher DON content in wheat genotypes, but corresponded to lower alpha-amylase inhibitory activities, differently from what could be expected for a class of protein involved in plant defense. However, it should be taken into consideration that it is known in the literature that each type of ATIs shows different specificities against different enzymes , and in Regarding the mean high temperatures for the same period analyzed for precipitation, the lowest values were registered in 2016 in the USA and in 2019 in Italy  which coThe two cultivation areas affected the wheat genotypes differently. Einkorn, Peliss, Alzada and Turkey Red all showed significantly higher alpha-amylase inhibitory activities in USA .p < 0.01) and in Italy . This agrees with Prandi et al. [There were negative correlations between the protein content and the alpha-amylase inhibitory activities both in the USA  showed that the contribution of the fixed (genotype) and the random (year) factors to the variability of the trypsin inhibitory activity were significant, but not their interactions. Previously Piasecka et al.  showed tThe mean values of the trypsin inhibitory activity ranged between 18.61% and 100%, with the lowest activity observed in the Peliss sample and the highest activity observed in the einkorn sample in both growing areas and in all three crop years.After einkorn, the hexaploid wheat genotypes Turkey Red, Judee and Marquis showed the highest inhibitory activities in both growing areas. Moreover, as for the alpha-amylase inhibitory activity, it was not possible to observe any trend as a function of the release year of wheat samples. These results are in line with the ATI gene sequence analysis results on the same wheat samples  and withThe hard red winter common wheat genotypes (Turkey Red and Judee) showed higher activities than the hard red spring common wheat genotypes (Marquis and Vida) in the USA where they were cultivated on a winter and on a spring cycle, respectively. However, in Italy where all the genotypes were cultivated on a winter cycle, Marquis showed a similar inhibitory activity as Turkey Red and Judee, but Vida seemed not to be influenced by the different growing cycle adopted in Italy and showed similar inhibitory activities as in the USA. Interestingly, the same behavior of Marquis and Vida samples were observed in this study for the alpha-amylase inhibitory activity. So, besides the genotype, it seems that also the crop cycle can influence the expression of these inhibitors. However, further studies on more samples are needed to confirm this hypothesis.T. aestivum.Data regarding trypsin inhibitory activity of wheat are very scarce in the literature. A few studies compared trypsin inhibitory activity in wheat with other cereals and legumes and showed that it is much lower in wheat than in rye, triticale and soybean ,30. RegaThe absence of correlation between the release year of wheat samples and the trypsin inhibitor activity and the high activities observed in einkorn samples are in line with what is observed in a recent paper where the authors analyzed the ATI concentrations and trypsin inhibitor activities in a set of different Triticum species . They alRegarding the interaction of the trypsin inhibitory activity with the crop year, as shown in Considering the same period analyzed for alpha-amylase inhibitory activities, the highest precipitation corresponded to the lowest trypsin inhibitory activity in Italy (2018–2019), but to the highest trypsin inhibitory activity in the USA (2015–2016). Similarly, the lowest values for the mean high temperatures corresponded to the lowest trypsin inhibitory activity in Italy (2018–2019), but to the highest trypsin inhibitory activity in the USA (2015–2016) . So, theGenotypes responded differently to the growing areas; in particular emmer, Judee, Turkey Red and Alzada showed significantly higher trypsin activities in the USA, even three times more considering emmer .p < 0.01) and in Italy .As for the alpha-amylase inhibitory activities, there were negative correlations between the protein content and the trypsin inhibitory activities both in the USA  have been reported in The wheat genotype had the most effect on the inhibitory activities of both locations, representing 55–65% of the variability, which was higher for trypsin inhibitory activity especially in Italy where it exerted a predominant effect (91.5%). Genotype × Year interaction was small but significant for alpha-amylase inhibitor activity and non-significant for trypsin inhibitory activity. This indicates a higher stability of trypsin inhibitors which seem to be less influenced by the climatic conditions compared to alpha-amylase inhibitors, especially in Italy. A possible explanation could be provided by the type of soil in the two test areas. In fact, the soil in Italy was clay–loam which is known to be able to retain moisture and so to keep more constant the moisture level in the soil unlike the soil in the USA which was sandy–loam and so prone to moisture loss. A more constant level of moisture in Italy could have stimulated less enzyme activity, especially of trypsin inhibitors which were less affected by the environment than alpha-amylase inhibitors in both Italy and the USA. As a result, the genotype had a significantly higher contribution on the variability of trypsin inhibitory activity in Italy.Besides genetic factors, the results shown in A previous study also highlighted a negative correlation between the monthly precipitation (particularly in January and in March) and the alpha-inhibitory activities in two cultivars of T. aestivum against the hog pancreas alpha-amylase, but not against human alpha-amylase and insects α-amylase . Other sThe inhibitory activities were measured in this study against mammalian enzymes. It is known from the literature that each type of ATI has a different specificity and that it can be differentially activated by different types of stress. Further studies are needed to understand possible mechanisms able to modulate the enzymatic activity in response to different types of biotic stress.ATIs have also a relevant place in human nutrition. Besides their role in allergies and in the etiology of celiac disease and non-celiac wheat sensitivity , ATIs caInhibition of alpha-amylase activity leads to accumulation of undigested and unabsorbed starchy constituents which are fermented in the distal gut by the microbial flora and can cause mild to moderate symptoms, including flatulence and bloating, in sensitive individuals to carbohydrate malabsorption . These sHigh trypsin inhibitory activities can be negative from a nutritional point of view because these can limit the absorption of protein during food assumption which decreases its nutritional usefulness . It was According to the results of this study, the genotypes which showed in general the lowest inhibitory activities against both alpha-amylase and trypsin and could be better tolerated by people suffering from wheat sensitivities were turanicum and Alzada, while the highest activities were shown by the hexaploid Judee, Turkey Red and Marquis. In line with these results, consumption of T. turgidum ssp. turanicum wheat-based products has been previously shown to improve symptoms in people suffering from irritable bowel syndrome . MoreoveThe effect of ATIs on the human organism highly depends on how these proteins are processed by the digestive enzymes. ATIs are members of the prolamin superfamily, and these proteins are characterized by having high resistance to digestion and denaturation . HoweverRecent studies showed that activity and pro-inflammatory properties of ATIs can be lost also as a consequence of food processing, such as bread making, sourdough fermentation and baking ,46 as weTriticum species, i.e., T. monococcum L. subsp. monococcum , T. turgidum L. subsp. dicoccum, T. turgidum L. subsp. turanicum, T. turgidum L. subsp. durum , T. aestivum L. subsp. aestivum, T. aestivum L. subsp. spelta . The selection of these genotypes was made to include different wheat species with a different genome composition  and with a different year of release, when available. A full description of these wheat accessions was reported in Simonetti et al. (2022) and in The present study included ten selected accessions belonging to different The ten selected wheat samples were grown under organic farming management during three consecutive crop years at two locations: at the Quinn Farm & Ranch located near Big Sandy, Montana, USA  and at the experimental farm Podere Santa Croce located at Argelato, Bologna, Italy . The field trial soil in the USA was sandy–loam while in Italy was clay–loam. The soil parameters prior to sowing are shown in The three crop years were 2015–2016; 2016–2017; 2017–2018 in the USA and 2016–2017; 2017–2018; 2018–2019 in Italy. In the USA, the wheat samples einkorn, emmer, turanicum, Peliss, Alzada, Marquis and Vida were cultivated on a spring cycle (planted in May and harvested in August), while spelt, Turkey Red and Judee were cultivated on a winter cycle (planted in September and harvested in early August). In Italy, all ten wheat samples were planted in November and harvested in July. The meteorological data (temperature and rain) were recorded at both sites from the National Center of Environmental Information  for the Grain samples from different genotypes were tested for their protein contents by employing the Foss Infratec 1229 NIT spectrophotometer .The grain samples were milled with the domestic stone mill (100% flour extraction) , and the flour obtained was used for the assessment of the mycotoxins content and for the enzymatic tests.® Deoxynivalenol ELISA test kit  and following the manufacturer’s instructions. The color produced was read at 450 nm with the Labsystems Multiskan MS Microplate Reader .The content of deoxynivalenol (DON) was determined with an enzyme-linked immunosorbent assay using the AgraQuantThe whole wheat flour samples previously obtained from the ten wheat genotypes cultivated for three consecutive years in two growing areas were extracted in duplicate and, two replicates from each sample extract were tested independently in the following enzymatic assays.In duplicate, the flour samples were extracted using cold water. Briefly, 100 mg of each of the flour samples was homogenized with 500 μL of distilled water using an automatic shaker at 160 rpm for 30 min. The samples were then centrifuged at 7000 rpm for 5 min, and 400 μL of supernatant was collected. The remaining pellet was again homogenized with 400 μL of distilled water at 160 rpm for 5 min, and after a centrifugation at 7000 rpm for 5 min, 350 μL of supernatant was collected and added to the first supernatant. Then, the supernatant was heated at 70 °C for 20 min to inactivate endogenous enzymes, diluted 1:100 in distilled water and used the same day for alpha-amylase inhibitory activity assessment.p-nitrophenol) which is proportional to the amount of the substrate (ethilidene-pNP-G7) hydrolyzed by the amylase. The absorbance was measured at 405 nm with the Labsystems Multiskan MS Microplate Reader. The inhibitory activity of the extracts was calculated as a percentage with respect to the control according to the following formula:initial and Tfinal and calculated using the standards provided by the kit; Tinitial is the first absorbance measure taken 3 min after the addition of the substrate; Tfinal is the absorbance measured when the most active sample was close to but not exceeding the end of the linear range of the standard curve.In duplicate, 1.7 μL of each sample extract was transferred to a 96 well flat-bottom clear plate and incubated for 10 min at 25 °C with 48.3 μL of amylase assay buffer  containing 20 mU of alpha-amylase from human saliva (Sigma–Aldrich Canada Co.). The control was the amylase assay buffer containing 20 mU of alpha-amylase from human saliva without the addition of the sample extract. The amylase activity was tested with the Amylase Activity Assay Kit (Sigma–Aldrich Canada Co.) following the manufacturer’s instructions. This test is based on the production of a colorimetric product at 405 nm  which is proportional to the amount of the substrate hydrolyzed by the trypsin. The absorbance was measured at 405 nm as described for the alpha-amylase inhibitory activity assay  containing 8 U of trypsin from bovine pancreas (Sigma–Aldrich Canada Co.). The control was the trypsin assay buffer containing 8 U of trypsin from bovine pancreas without the addition of the sample extract. The trypsin activity was tested with the Trypsin Activity Colorimetric Assay Kit (Sigma–Aldrich Canada Co.) following the manufacturer’s instructions. This test is based on the production of a colorimetric product at 405 nm .The general linear model (GLM) was used to assess the variance significance for the fixed (genotype) and the random (year) factors, as well as their interactions, for all measured variables. Tukey’s (HSD) test was used to determine differences between means at The tested in vitro inhibitory activities showed high variability among the ten wheat genotypes analyzed, and the contributions of the genotype and the crop year were significant for both the alpha-amylase and the trypsin inhibitory activities in the two growing areas. In general, the hexaploid wheat genotypes showed the highest ATI inhibitory activities, and it was not possible to highlight any trend in ATI activities as function of release year of wheat samples. Einkorn had a peculiar behavior showing the lowest alpha-amylase inhibitory activity, but the highest trypsin inhibitory activity. The tetraploid turanicum and Alzada wheat samples showed an interesting profile, with the lowest inhibitory activities against both alpha-amylase and trypsin The two inhibitory activities were differently affected by the growing conditions and were negatively correlated with the protein content.Although the number of samples tested was limited, the information collected may be important to understand the extent of the variation of ATI inhibitory properties in relation to the wheat genotype and the growing environment and its impact, if any, on human health and nutrition.


Several studies have shown an association between sarcopenia and clinical outcomes in patients with Crohn's disease (CD). However, studies have shown different results, and the association between prognosis and wasting conditions in pediatric patients with CD is uncertain. In this study, we evaluated the clinical significance of wasting in pediatric CD patients.We retrospectively analyzed data on wasting syndrome in patients diagnosed with CD at the Pediatric Department of Gachon University Gil Medical Center between January 1995 and January 2018.P < .001 and P < .001, respectively). Additionally, wasting was significantly associated with low levels of hemoglobin (P < .001), high levels of inflammatory markers, including C-reactive protein (P = .005) and erythrocyte sedimentation rate (P = .04), and a smaller surface area of the gluteus maximus muscle (P < .001). Interestingly, since the site of CD involvement and other markers for nutrition did not correlate with wasting syndrome, wasting appears to be a marker for the severity of pediatric CD. Lastly, the wasting group tended to have a greater use of biologic therapy after first-line therapy failed to improve wasting syndrome.Of 105 patients diagnosed with CD, 39.0% were classified into the wasting group (weight-for-age z-score ≤−1) and 61.0% into the nonwasting group (weight-for-age z-score >−1). Height-for-age and body mass index-for-age z-scores at the time of diagnosis were significantly associated with wasting (Wasting syndrome, including sarcopenia, can serve as a marker for the severity of pediatric CD. Weight loss associated with sarcopenia remains a cornerstone for the diagnosis of CD. Children with CD have features of nutritional cachexia with normal fat stores but depleted lean mass. They present with a lower concentration of plasma micronutrients, anemia, and alterations in trace elements. However, changes in nutrition are not sufficient to explain sarcopenia because antagonizing the sarcopenic TNF-α antibody with infliximab can improve sarcopenia. Patients of different sexes with CD differ in body composition, and improvement of lean body mass can be achieved with physical activity and anti-inflammatory medicines.Crohn's disease (CD) is a chronic inflammatory bowel disease that usually develops in adolescence and early adulthood.,10 Several possible explanations for sarcopenia in CD have been suggested. First, the inflammatory process, in particular, high levels of sarcopenic inflammatory mediators, such as TNF-α and IL-6, are believed to be related to increased muscle-specific protein degradation and decreased muscle synthesis.–13 Second, patients with CD seem to be unable to maintain a positive postprandial protein balance and could be deficient in vitamin D. and growth hormone. Lastly, there may be alterations in the intestinal microbiota.–20 The etiology of CD is believed to be multifactorial, including changes in the intestinal microbiota. Dysbiosis in CD comprises a decrease in Bacteroides and Firmicutes bacteria and an increase in Gammaproteobacteria and Actinobacteria. As a result of this increase, bacteria cross the mucosal barrier, attach to the intestinal epithelial cells, and replicate within the macrophages, stimulating the secretion of TNF-α. The significance of the interplay between gut microbiota and other organs has recently emerged.Although the exact mechanism of CD-related sarcopenia is currently unknown, repeated intestinal inflammation, disuse, or debility, and glucocorticoid therapy may play a role.Min+ mice, and prominent manifestations of pediatric CD, we hypothesized that wasting syndrome could be a marker of severe systemic disease in pediatric CD. Therefore, we evaluated the significance of wasting syndrome in pediatric patients with CD.Based on studies of wasting syndrome, including sarcopenia in cachexia models that were associated with gut barrier dysfunction, intestinal polyposis, inflammatory responses in APC/22.1Patients aged <18 years who were diagnosed with CD at Gachon University Gil Medical Center  between July 1995 and January 2018 were eligible for the study. Patients whose diagnoses were confirmed by primary colonoscopy, biopsy, or other investigations were included. Patients for whom CD could not be confirmed with colonoscopy and those who had already been diagnosed and treated for CD at another hospital were excluded. The study complied with the principles of the Declaration of Helsinki. The study protocol was reviewed and approved by the Institutional Review Board of the Gachon University College of Medicine (GCIRB 2018-202). Data collected from medical records included age at diagnosis, sex, height, weight, laboratory findings, medication use, colonoscopy results, and abdominopelvic computed tomography (CT) results. BMI was calculated as weight divided by height in meters squared (kg/m2). The use of mesalamine, azathioprine, infliximab, and adalimumab were documented. Our center only prescribed biologics as a step-up strategy when primary therapy failed. We investigated the use of medication based on wasting syndrome and whether a top-down strategy was required.CD diagnosis was based on conventional clinical, radiologic, endoscopic, and histopathological criteria.2.2Sex-specific height-for-age z-score (HAZ), weight-for-age z-score (WAZ), and BMI-for-age z-score (BMIZ) were calculated using the LMS method. Patients were divided into the following 2 groups based on their WAZ: patients with a WAZ ≤−1 were classified into the wasting group and those with a WAZ >−1 were classified into the nonwasting group. The standard deviation scores (z-scores) were used to evaluate and compare anthropometric measurements among children of various ages and sexes. The z-score, which is a standardized value based on data from a normal pediatric population, is calculated using values appropriate for the child's age and sex. The HAZ, WAZ, and BMIZ for each patient were calculated to 2 decimal places after accounting for age and sex.2.3Venous blood samples were obtained at the time of the diagnosis. Complete blood cell count, serum albumin (g/dL), C-reactive protein (CRP) (mg/dL), erythrocyte sedimentation rate (ESR) (mm/h), calprotectin (μg/mg), and micronutrients such as 25-OH vitamin D were measured using standard techniques. The association between wasting syndrome and laboratory findings at diagnosis was also investigated.2.4gluteus maximus muscle surface area (cm2) was measured on a single image at the thickest section of the left ischiofemoral space  as well as the extent of organ involvement were documented. Images were obtained from digital storage using image-viewing software. CT image analysis was performed using the INFINITY PACS software. The left ace Fig. . This su2.5Ver. 18.2.1 . Student t test or the Mann–Whitney U test was used to test the associations between continuous variables, and the Chi-Squared test or Fisher exact test was used to test the associations between categorical variables. Statistical significance was set at P < .05. Univariate regression was used to evaluate the association between each variable and wasting. Multivariate regression was used to evaluate the association between wasting and selected variables while controlling for confounders.Analyses were performed using MedCalc 3P < .001) (Table P < .001) (Table P < .05) (Table P < .05)  Table . Initial1) Table ; however5) Table . These d5) Table . We wondgluteus maximus    Fig. . Since t1) Table .P < .05). However, in the multivariable analysis, only lean mass deficit and CRP were significant predictors of wasting syndrome (P < .05) (Table P < .01)  Table . Since w1) Table .4In this study, we found that sarcopenia was associated with low height-for-age, low BMI-for-age, high levels of inflammatory markers such as CRP and ESR, and low levels of hemoglobin, hematocrit, and albumin in 105 children diagnosed with primary CD. Since there was no association between wasting syndrome and disease location or nutritional derangement, malabsorption was not the main cause of wasting. Fewer patients with wasting responded to primary therapy and received biological therapy. We concluded that in addition to current markers for assessing CD severity, clinical evaluation of sarcopenia should be mandatory for pediatric CD. reported an association between decreased lean mass and postoperative complications. In children, Thayu et al did not find an association between body composition and disease activity, but they did show a correlation between body composition and inflammatory markers. Another study by Burnham et al showed that lean mass deficit was positively correlated with disease activity. Conclusively, altered body composition might reflect “inflammatory activities” in inflammatory bowel diseases (IBD). However, sarcopenia is a manifestation of altered body composition, and our study suggests that sarcopenia is associated with increased disease activity in pediatric CD, independent of nutritional derangement. A major difference between our study and previous studies is that patients were divided into 2 groups based on weight-for-age z-scores. For adults, wasting, especially lean mass deficit, is well-recognized for its clinical significance in many diseases.,24–27 These data suggest that wasting syndrome, deficit, and lean mass can indicate poor prognosis in pediatric CD, and more efforts to restore weight should be considered for patients with wasting syndrome.Some studies have shown an association between altered body composition and clinical outcomes in patients with CD. In adults, Zhang et al,29 Since malnutrition and growth retardation are important issues in pediatric CD, Song et al investigated the prevalence of nutritional and growth status in Korean children with CD and found subnormal serum levels of hemoglobin, albumin, iron, ferritin, calcium, magnesium, folate, vitamin B12, and zinc. However, in our study  with TNF-α antagonists, such as infliximab or adalimumab, not only improved cachexia but also improved the prognosis of cancer.,36 However, to measure lean mass, only CT imaging data were used in our study. In addition to radiologic data, bioelectrical impedance, isotope dilution, total body potassium, and dual-energy X-ray absorptiometry can be used to measure body composition. Some researchers argue that a high resolution CT is required. We included pediatric patients who required evaluation of the gastrointestinal conditions in whom sarcopenia was evaluated. Therefore, assessment of sarcopenia can be performed with other types of evaluation in pediatric patients. Another limitation of our study is the small sample size; the study was performed at a single medical center study, and it was a retrospective analysis. The small sample size can be explained by the fact that we only included cases diagnosed with primary CD and excluded referral cases. It is worth mentioning that our institute is one of the biggest medical centers in Korea. Lastly, because the study was retrospective, we were not able to obtain additional data for disease severity, such as the PCDAI score from the early participants. For the prospective study, additional data on disease severity should be included.A potential limitation of this study is the lack of consideration of the Tanner stage. In the case of pediatric CD, the Tanner stage has profound effects on height, weight, and body composition such as lean mass, especially in adolescents. Magnetic resonance imaging has recently been developed to offer 3-dimensional volume calculation to allow for calculation of muscle volume.In conclusion, we analyzed cases according to z-scores  reflecting objective measurements. The characteristics of wasting syndrome can be summarized as having low HAZ and BMIZ, high levels of inflammatory markers, a deficit in lean mass, and low response rates to primary treatment other than biologicals. Therefore, wasting syndrome, including sarcopenia, can be a marker of the severity of pediatric CD.Conceptualization: Hann Tchah, Jung-Ho Kim, Ki-Baik Hahm, Kwang-An Kwon, Su-Jin Jeong.Data curation: Dong-Hwa Yang, Hann Tchah, Ki-Baik Hahm.Formal analysis: Dong-Hwa Yang, Wook Jin.Investigation: Dong-Hwa Yang, Wook Jin.Supervision: Hann Tchah, Ki-Baik Hahm.Writing – original draft: Wook Jin.Writing – review & editing: Wook Jin.


Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo-electron microscopy (cryo-EM). The amorphous atomic structure of the ice that stabilizes and protects biological samples in cryo-EM grids also imprints some additional noise in cryo-EM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables, also usable in SerialEM and Leginon, using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the “ice channel” technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin sample, achieving a 1.88 Ångstrom resolution in subsequent refinement of the atomic map. MeasureIce is an ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. Sub-3 Å resolutions—which allow the fitting of amino acid side chains in the three-dimensional density map3—are now common and the current resolution record is just above 1.2 Å4. The history of the development of cryo-EM has principally been one of surmounting the inherent challenges of signal-to-noise. Biological macromolecules are susceptible to electron beam radiolysis, the breaking of chemical bonds by the high-energy electron beam of the transmission electron microscopy (TEM) instrument5. Vitrification, plunge-freezing of grids so that the water freezes into amorphous (vitreous) ice, provides some protection by reducing the diffusion of radiolysis products, and so permits stability of at least the low-resolution features of a protein up to an electron dose of around 40–70 e/Å6. Even with this dose level cryo-EM images are dominated by Poisson counting or “shot” noise, the quantum-statistical fluctuations in the numbers of electrons arriving at any given camera pixel during acquisition, overwhelming the atomic resolution structure that would be otherwise evident in single images. In the single particle analysis (SPA) workflow, sophisticated algorithms7 combine tens or even hundreds of thousands of images into a single three-dimensional reconstruction such that the available signal then becomes sufficient to finally reach the resolutions approaching what the TEM is inherently capable of.Single particle cryogenic transmission electron microscopy (cryo-EM) is a characterization technique for biological macromolecules that has seen rapid growth in the past decade8. The disordered atomic structure of the vitreous ice that proteins are necessarily encapsulated within in cryo-EM is itself an additional source of noise9 and thicker ice means noisier images. Therefore, a key step in the cryo-EM single particle analysis (SPA) workflow is the inspection of samples on an economical screening TEM to ensure that sufficient densities of particles can be found in thin ice regions of the grid before these samples advance to data collection on expensive high-end acquisition instruments and consume much needed computational resources. This is hampered by the fact that most methods for quantitative ice thickness measurement require either time-consuming post-experiment analysis or pre-experimental calibration so are difficult to easily incorporate in screening workflows. Commonly used methods are summarized in Fig. 10. The longer acquisition and data processing time means it is unsuited to on-the-fly analysis. The ice channel method, Fig. 13. In the energy filter approach, Fig. 13. The ALS approach, Fig. 14. We focus on the ALS method in this work since, once calibrated, ice thickness is measured directly from single micrographs meaning the method is fast, only requires the most basic of screening microscopes without an energy filter or tomography software and minimal user training. In addition, the ALS method is best used at low magnifications where the beam is typically spread so wide that the intensity is much <1 e/Å2. Large regions of the grid can be surveyed with minimal damage to the specimen, making this an appropriate technique for selecting holes for acquisition15, unlike tomography (which requires higher magnification and dose) and the ice channel method which is necessarily destructive to the region of interest.Since signal to noise is inherently limited in cryo-EM yet critical to successfully solving structures via the technique, much research effort is applied to reducing additional sources of noise. The most striking example is the development of direct electron detectors which feature an improved detector quantum efficiency (DQE), a measure of the additional noise the detection system adds to the measured micrograph14I0 is the incident intensity of the electron beam (that measured in the absence of any sample in the path of the beam), Teff is the effective mean-free-path for elastic and inelastic scattering of an electron to an angle large enough that the objective aperture will remove it from the image and is often referred to as the ALS coefficient14. This effective mean-free-path, Teff, is dependent on a complex interplay between elastic and inelastic scattering, which we will review in the methods section of this paper. For a well-aligned microscope it is a function of the accelerating voltage, which determines the degree of elastic and inelastic scattering for a given ice thickness, and aperture size, which determines the scattering angle beyond which scattered electrons will be removed from the image. Note that the relevant value of the aperture size is in units of inverse length or angle  measured using a diffraction standard such as gold nanoparticles, not the aperture physical size in micron values typically provided by the instrument manufacturer. In previous work Teff was measured by extensive experimental calibration14. In this work, we demonstrate how Teff can be estimated through first principles simulation and thus the method be applied to a TEM without the need for the time-consuming calibration step. We benchmark this approach using the ice channel and tomography ice thickness measurements and present a software package named MeasureIce that allows for ice thickness measurements using ALS during data collection. Finally, we demonstrate the utility of quantitative ice thickness measurements pre-acquisition via a 1.88 Å reconstruction of equine apoferritin, which is a 0.22 Å improvement on previously reported results17. We refer to our technique of measuring ice thickness using aperture limited scattering informed by simulated look-up tables as the “MeasureIce approach”.For ALS images recorded in-focus (so that defocus-induced phase contrast is minimized) the intensity for a region of amorphous ice is well approximated byr2), indicated on the plots, give the quality of agreement between the MeasureIce simulation calibration curves and experimental datapoints. Good agreement between experiment and theory was found for all experimental accelerating voltages suggesting that the simulations correctly incorporate all of the important and necessary physics for the energy ranges typically used in cryo-EM and can be relied on to produce accurate ice thickness maps.Simulated ice thickness-image intensity curves were generated for three different microscopes operated at different accelerating voltages and are plotted in Fig. By way of an additional benchmark, we compared ice thickness measurements from the MeasureIce approach to estimates from tomography. Since information loss in the z direction resulting from the missing wedge in a standard tilt series acquisition makes it difficult to detect the top and bottom surfaces of the ice in a pure water sample, a 7 mg/ml apoferritin sample was used for this experiment. Apo-ferritin particles were used as an indicator of the extent of the water volume.Four tomograms were acquired using a ThermoFisher Talos Arctica, operated at 200 keV, for different holes in the grid, selected using MeasureIce to give a spectrum of ice thicknesses up to about 100 nm. We then used a custom script to fit a surface function to these points to estimate the profile of the ice layer; this is shown for a representative Quantifoil® hole in Fig. Since de-ionized water was used in the ice channel measurements, using an apo-ferritin sample for this benchmark had the additional benefit of testing whether the presence of a standard cryo-EM protein sample, both protein and buffer, would affect the accuracy of ice thickness measurements. Protein typically has a shorter inelastic mean-free path than the surrounding water so the presence of protein will mean the solution has a slightly shorter inelastic mean-free path on average and MeasureIce might be expected to underestimate thickness. This effect is not observed in our experiments for thicknesses <80 nm though the departure from agreement between the approaches for thicknesses >80 nm might be accounted for by the shorter electron inelastic mean free path of protein-containing solution. This gives confidence in using the MeasureIce approach for standard cryo-EM samples for thicknesses approaching ideal ice thicknesses for SPA acquisition (<50 nm). Caution is advised with samples containing large amounts of additives such as glycerol, or with a concentrated salt buffer.16 and the conductive film ameliorates beam induced charging and thus ice movement18. Since a graphene backing should be only a few monolayers thick (therefore less than a nanometer) we expect this to have negligible effect on ice thickness measurement. Amorphous carbon backings are usually thicker, typically 5–10 nm, so will impact ice thickness measurements. Simulations we present here demonstrate that accounting for a carbon backing in ice thickness measurements is straight-forward and does not even require a specialized calibration for different thicknesses of carbon film. All that is required is that the reference intensity, I0, instead be set to the measured intensity for the carbon film no ice (a dry region of the backed grid). Then a standard I/I0 calibration curve prepared for a setup assuming a non-carbon-backed grid can be used to measure ice thickness. A 10 nm amorphous carbon layer was added to the MeasureIce simulation for a 200 kV microscope using a realistic carbon atomic model generated by Ricolleau et al.19 and an inelastic mean free path for an electron in carbon from Shinotsuka et al.20. As can be seen when the fractional intensity I/I0 is plotted against ice thickness for two different hypothetical objective apertures, 10 mrad and 15 mrad, in Fig. I0 to the intensity measured for just the carbon film, that is for an ice thickness of 0 nm, would then shift any of these calibration curves back to that observed for no carbon film.Adding an amorphous carbon or functionalized graphene backing to a cryo-EM grid is becoming an increasingly common practice, since this sometimes addresses preferred orientation effectsTimfp, is a key parameter in MeasureIce simulations and its accuracy will have consequences for ice thickness measurement. In this section, we will discuss the considerable variation in literature values of inelastic mean free path but show that the possible impact of this variation on the estimated ice thickness is of the order 10%. The mean free paths for inelastic scattering, Timfp21 for 120 kV and 300 kV. For other values electron energy we use the following equation23 as a basis for interpolation between measured values of T21Mw, ρ, and Z, are the molecular weight, density, and atomic number of the sample and U0, m, c, and 26 and those predicted by Eq. (21 (labeled “Eq. 21 the most rigorous to date since mean free path is directly measured from electron energy-loss spectroscopy (EELS) data recorded for an electron probe scanned across water channels of known geometry. Grimm et al.24, whose value for the inelastic mean free path of an electron at 120 kV agrees well with Yesibolati et al.21, conducted a rigorous error analysis with their experiment and this presents an opportunity to investigate the effect of errors in inelastic mean free path. Figure 24 error bars and plotted with dashed lines. Inset in this figure is a plot of the implied thickness error for both the L120C apertures as a function of nominal thickness . It can be seen that this implied ice thickness error is consistently well below 10%, sufficient for estimating ice thickness in cryo-EM.Energy-dependent inelastic mean free path, entclass1pt{minimaentclass1pt{minimaentclass1pt{minimahttps://www.pyqtgraph.org/) graphics and user interface Python library, though stress that the code outputs values of Teff that can be used in existing software, notably Leginon14 and SerialEM15. The source code for the MeasureIce python script is available on GitHub (https://github.com/HamishGBrown/MeasureIce). It can be deployed using a python interpreter installed on the microscope control or support PC or compiled into a stand-alone Windows executable using the py_installer  package.Once ice thickness image intensity look-up tables are simulated for a microscope’s given aperture-accelerating voltage combination this information can be used to infer ice thickness on the fly. We have developed a Python software tool to facilitate thickness measurements using these simulated look-up tables using the pyqtgraph  suggest that deviating from parallel illumination induces minimal error.https://www.hdfgroup.org/HDF5/), for example for different microscopes or for different accelerating voltages on the same microscope, from the drop-down menu at the bottom left, Fig. https://github.com/ercius/openNCEM, package developed by Peter Ercius at Lawrence Berkeley National Lab) or mrc [using the Python mrcfile module27] files by clicking the “Load raw image” button, Fig. I/I0), the image intensity recorded for regions of the image with no material in the path of the beam, which we will refer to as the “vacuum intensity”. MeasureIce needs the user to manually input I0 and this is measured from a region of the image without ice or TEM grid overage. The vacuum intensity I0 can be selected by moving with the mouse pointer the red line, labeled I0, which is atop the histogram immediately to the right of the raw image, Fig. I0 are highlighted in red in the interface, Fig. I0 has been set, the user generates an ice thickness map by pressing the “Generate Ice thickness map” button, Fig. 28 plot in png or pdf format by clicking the “save ice thickness map” button, Fig. A screenshot of the graphical user interface is shown in Fig. The ultimate goal of MeasureIce is to ensure that cryo-EM practitioners can more rapidly and quantitatively achieve minimum possible ice thicknesses for a given sample, which in turn reduces the effective signal to noise of micrographs resulting in higher resolution reconstructions, potentially from fewer total particles. To this end MeasureIce was evaluated on a standard cryo-EM single particle workflow with Equine Apoferritin (Sigma) vitrified on UltrAuFoil® gold grids.7 and Cryosparc29 and resulted in a 1.88 Å map. This exceeds the current record resolution 2.1 Å map of the same protein17. The reconstructed map is shown overlaid with the fitted molecular model 6RJH from ref. 1 in Fig. 30.Compared to carbon Quantifoils® where ice thickness gradients are visible even on the carbon substrate and give some qualitative indication of ice thickness across a grid square, gold grids can be challenging for qualitatively evaluating ice thickness for inexperienced users. This is because the gold foil scatters the electron beam strongly and Bragg scattering by the polycrystalline foil dominates TEM images outside the grid holes, rendering the subtle ice thickness gradients invisible. Using MeasureIce on the microscope support PC we were able to identify the relative intensity that corresponded to ice holes having <15 nm thickness and select these holes for acquisition using the “Ice filter” setting on the EPU automated acquisition software of our Titan Krios—which selects holes for acquisition based on the mean image intensity inside the hole. Data processing was performed with RelionThe aperture limited scattering (ALS) approach provides a fast and convenient way to measure ice thickness on the fly, though till now the time-consuming nature of calibration means that the method is not as widespread in TEM as might ideally be the case. In this paper we have presented an approach to calculate look-up tables to convert image intensities in the ALS approach to ice-thickness values that allows the time-consuming process of experimental calibration to be skipped. This approach has been packaged for user convenience in software which we have named MeasureIce meaning that barriers to including quantitative ice thickness measurements are substantially reduced. Thickness measurements compare favorably with alternative ice thickness measurement techniques, and we have demonstrated the advantages of quantitative ice thickness measurements in general with a 1.88 Å resolution reconstruction of Equine apoferritin. This improvement cannot wholly be attributed to ice thickness measurement but does, however, make the case for including quantitative ice thickness tools like MeasureIce within cryo-EM screening and acquisition workflows, especially considering the convenience of the MeasureIce software tool.3 cube of vitreous ice was generated using the GROMACS molecular dynamics simulation package31 according to the method detailed in Souza Junior et al.32. The multislice algorithm has been previously used to simulate electron elastic scattering in the cryo-EM context34 and here simulations were performed using the open source py_multislice package35. To incorporate multiple inelastic scattering we adapted the approach suggested by Egerton36. For a given thickness θ = tan−1(λk), where k is the diffraction plane coordinate in units of inverse length and λ is the electron wavelength, is parameterized as21 for 120 kV and 300 kV and interpolated for other values. Since α = tan−1(λkα) of the objective aperture, is then given byTeff, the ALS coefficient, to the user so that it can be used in the Leginon14 and SerialEM software15.In simulations, we found that it was important to include both elastic and inelastic scattering and the realistic structure of amorphous ice. A 10 nmβ in the opposite direction. The thickness of the ice layer can be inferred from the size of the ice channel measured in projection,To confirm the validity of the simulation approach we benchmark it using the ice channel approach. Here the sample is tilted to angle 37] and the objective aperture is accurately placed in the back focal plane. Own tests which we discuss in the following section indicate that measuring ice thickness with the beam strongly deviated from this condition should only introduce imperceptible errors. The reference intensity I0 also needs to be known accurately. If there is no hole in the specimen through which I0 can be estimated, then a separate reference image, recorded with the same beam intensity, magnification, and acquisition time should be recorded to measure I0.MeasureIce images must be recorded in focus and with the objective aperture centered with respect to the TEM optic axis. MeasureIce simulations assume that the beam is in a parallel illumination condition [see e.g., ref. To explore the effect of non-parallel illumination, we compared the fractional intensity measured in an image of a carbon quantifoil grid with vitreous ice layer recorded at a parallel illumination condition in our Arctica G2 cryo-TEM (a C2 lens excitation of 48.6% at spot size 5) with TEM images of the same area but with different values of the C2 lens excitation, causing some departure from the parallel condition. The 70 micron (9.2 mrad) objective aperture was inserted. Per pixel values are plotted in Fig. Select the appropriate calibration file from the bottom left-most drop-down menu. These must be generated beforehand using the Generate_measureice_calibration.py utility.2.Load a TEM raw image  using the “Load raw image” button. Users must be cautious that microscope software is not compressing the bit depth of images. For example, saving with the tiff format in ThermoFisher’s TIA software compresses to the 8-bit or 16-bit integer datatype. Bit depth compression will mean intensity measurements are not comparable between acquisitions even if beam current and camera acquisition time is kept constant. To ensure accurate ice thickness measurements, users must be also careful that the image was recorded in focus (with minimum defocus induced phase contrast) and with the objective aperture aligned to be concentric with the microscope optic axis. The use of the “counting” mode for direct electron detectors such as the Gatan K2 and K3 or ThermoFisher Falcon cameras is discouraged since exceeding the recommended dose-per-pixel for accurate electron counting will mean a high rate of electron coincidence loss and that the detector response will not be linear. We have observed that the MeasureIce software noticeably underestimates ice thickness values when this happens. The low magnifications at ice thickness maps that will normally be recorded  mean that the dose efficiency improvements resulting from electron counting are anyway irrelevant since the dose applied to the specimen should be a fraction of an electron/ÅI0, the image intensity recorded for regions of the image with no material in the path of the beam, by moving with the mouse pointer the red line superimposed on the intensity histogram immediately to the right of the raw image. The MeasureIce interface will identify regions of intensity greater than this value with a red mask overlayed on the image. This is shown for the case of a crack in the holey carbon film visible in the TEM image of Fig. I0 needs to be measured from a separate image of an empty part of the grid using identical beam current and acquisition time and manually inputted into the I0 section of the interface.Set the vacuum intensity Select the aperture used to record the image from the drop-down menu in the center of the bottom panel of the interface—MeasureIce needs the aperture sizes in diffraction units of inverse Angstrom or mrad but the apertures can be labeled with the micron values that will actually appear in the microscope user interface.Press the “Measure ice thickness” button. The ice thickness map will be shown in the right-hand panel of the user interface and hovering the mouse above regions of the ice thickness map will cause the ice thickness for the region below the cursor to be displayed in the image title. Since there can be apparent large pixel-to-pixel variation in the ice thickness map, due mainly to Poisson counting noise, there is the option of filtering the image to remove much of this noise using a Gaussian low-pass filter. The amount of filtering is controlled by setting the filter kernel size in the “Map spatial-filter kernel” radio box.Save the ice thickness map for future analysis and presentation by clicking the “Save ice thickness map” button.Equine Apoferritin at 7 mg/ml  was prepared on 1.2/1.3 gold Ultrafoil grids, glow discharged in a PELCO easiGlow system for 2 min with a 15 mA current, for single particle reconstructions and on 1.2/1.3 carbon Quantifoil grids, glow discharged for 30 s with a 15 mA current, for tomography using a Leica EM GP2 automated plunge freezer. A 4 μL volume was applied to the grids which were back-side blotted using the ‘auto-blot’ function of the EM GP2. The sample chamber was set to 22° and 95% humidity.38, with frames aligned via cross-correlation and binning of 4 K images by a factor 4 to enhance the contrast of the apo-ferritin particles. Particles at the extremities (in z) of the reconstructed volume were taken to indicate the upper and lower layers of ice and manually picked.Images were recorded on a Talos Arctica (FEI) fitted with a Gatan K2 camera operated in electron counting mode and energy filtered with a 20 eV slit inserted to improve contrast. Before tilt-series acquisition, low magnification (×5000) images were recorded for later MeasureIce analysis. The tomograms were reconstructed using IMOD2 on the sample with 8 frames per movie. The last frame was deleted to avoid unevenness of the dose distribution resulting in 7 frame movies with a total dose of ~16 e/Å2. Acquisition parameters are compared with those of Naydenova et al.16 in Table Grids were imaged on a Krios G4 cryoTEM (ThermoFisher) equipped with a Falcon 4 direct electron detector. The fractional intensity implied by MeasureIce for ice thickness <15 nm was used to select holes in the ThermoFisher EPU software using the “Ice filter” feature. The samples were imaged at a nominal magnification of 120,000 corresponding to a pixel size of 0.637 Å, calibrated using the 220 lattice reflection of a standard orientated gold specimen, with a defocus of −0.4 μm to −1.0 μm. Exposures of 1.52 s were taken with a fluency of 5.34 e/px/s resulting in a nominal dose of ~20 e-/Å8 and Cryosparc 3.029. Motion correction was performed on gain-corrected movies with RELION 3.1’s own motion correction implementation. Motion corrected movies were imported into Cryosparc with CTF estimation done using patch CTF estimation. A total of 97,513 particles were extracted. Two rounds of 2D classification in Cryosparc resulted in a working dataset of 68,025 particles. Particles were imported back into Relion using Pyem39. Particles were used to create a de novo model then subjected to 3D refinement, CTF refinement, and polishing follow by another round of CTF refinement. Octahedral symmetry was enforced on the 3D model in 3D refinement. The ultimate 3D refinement resulted in a structure at 1.9 Å with a B-factor of −66. Ewald sphere correction was applied to the refinement results and improved the structure to a resolution of 1.88 Å and a B-factor of −54. The resolution was estimated using the gold standard FSC = 0.143, calculated using a relaxed solvent map (deposition EMD-25619).Reconstruction was carried out using a mixed workflow of RELION 3.116 was fitted into the Coulomb potential map using rigid body fitting in Chimera-X40.Pdb structure 6RJHFurther information on research design is available in the Peer Review FileReporting Summary


For the most potent inhibitor of BuChE, Cornish-Bowden plots were used, which demonstrated that it behaves as a mixed inhibitor. Modelling studies of the same inhibitor demonstrated that the benzotriazole and 1-deoxynojirimycin moiety is accommodated in the peripheral anionic site and catalytic anionic site, respectively, of AChE. The binding mode to BuChE was different as the benzotriazole moiety is accommodated in the catalytic anionic site.The synthesis of four heterodimers in which the copper(I)-catalysed azide-alkyne cycloaddition was employed to connect a 1-deoxynojirimycin moiety with a benzotriazole scaffold is reported. The heterodimers were investigated as inhibitors against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The heterodimers displayed preferential inhibition (> 9) of BuChE over AChE in the micromolar concentration range (IC AD is a multifactorial disease in which all the factors involved in the progression of the disease have not been pinpointed; however, the two major pathogenic mechanisms are believed to include accumulation of amyloid-beta (Aβ) plaques between the neurons that causes neuronal loss4) (5),6),7)6) lowers the level of Aβ protein,Iminosugars, such as naturally occurring 1-deoxynojirimycin (1-DNJ) (4)  are carbca. 20 Å deep active gorge.,X-ray analysis has demonstrated that the overall structure of AChE and BuChE is very similar and that both enzymes host a catalytic triad consisting of histidine, glutamate, and serine almost on the bottom of a 4) has been employed as a binding unit in the development of dual binding site ChE inhibitors when it is connected to a second aryl-substituted selenourea (exemplified by 8),9),10)9 indicated that its 1-DNJ binding unit, in its acidic form, and tacrine binding unit are able to interact simultaneously with the Trp residues in PAS and CAS, respectively.10, on the other hand, in complex with AChE and BuChE demonstrated that its 1-DNJ binding unit, in its acidic form, and catechol binding unit bind to the catalytic active site and PAS, respectively.Because iminosugars can be protonated at physiological pH, and as such they were thought to establish cation–π interactions with the tryptophan residue in CAS of AChE in the same way as the quaternary ammonium groups of ACh and of the AChE inhibitors edrophonium and decamethonium.11 in viz. compound 11, exhibited inhibition potency against AChE and copper-promoted aggregation of Aβ protein. In addition, 11 displays chelation properties with biometal ions  and is therefore potentially able to tackle metal homeostasis imbalance, which is thought to be involved in the progression of AD. Dimer 11 displayed mixed AChE inhibition, which was consistent with docking studies in which the coumarin and benzotriazole moiety binds to PAS and CAS, respectively. When the benzotriazole ring was connected to a naphthoquinone and anthraquinone system interesting multitarget agents for AD were obtained as they inhibit both ChEs and monoamine oxidase (MAO) B.Bedi and co-workers reported a series of coumarin-benzotriazole heterodimers . The reactions were carried out under an argon atmosphere, unless otherwise specified. Microwave reactions were performed in a CEM Discover-SP, max power 300 W. TLC analyses were performed on Merck silica gel 60 F254 plates using UV light for detection. Silica gel NORMASIL 60® 40–63 µm pore size was used for flash column chromatography. NMR spectra were recorded on a Bruker Avance NMR spectrometer; 1H NMR spectra were recorded at 400.13 MHz, 13 C NMR spectra were recorded at 100.61 MHz, in CDCl3 or CD3OD. Chemical shifts are reported in ppm relative to an internal standard of residual chloroform  and residual methanol . High-resolution mass spectra (HRMS) were recorded on a Qexactive spectrometer in positive electrospray ionisation (ESI) mode.DMF, CH17  in dry acetonitrile (15 ml) were added the corresponding alkyl dibromide (12 equiv.) and potassium carbonate (1.3 equiv.). The resulting mixture was heated under microwave irradiation at 100 °C for 1 h. The reaction mixture was then cooled to room temperature and the solvent was evaporated under reduced pressure, followed by addition of 10 ml of water and extraction with EtOAc (2 × 20 ml). The combined organic layers were dried (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography .To a solution of iminosugar 18a, 18 b, 18c, or 18d  in DMF (2 ml) was added NaN3 (4 equiv.). The reaction mixture was heated overnight at 45 °C under Ar-atmosphere. The mixture was then cooled to room temperature and the solvent was evaporated under reduced pressure; 10 ml of water was then added, and the aqueous phase was extracted with CH2Cl2 (2 × 20 ml). The combined organic layers were dried (MgSO4), filtered and concentrated under reduced pressure without the need for further purification .To a solution of the alkyl bromide 20 , azide 19a, 19b, 19c, or 19d , and copper (II) sulphate pentahydrate (0.4 equiv.) in DMF (4 ml) in a foil covered round bottom flask was added sodium ascorbate (0.8 equiv.). The mixture was kept stirring at room temperature under Ar-atmosphere overnight. The solvent was then removed under reduced pressure, and the concentrate was suspended in water (10 ml) and EtOAc (20 ml). The layers were separated, and the organic extract was dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography .To a mixture of alkyne 21a, 21b, 21c, or 21d  in anhydrous CH2Cl2 (10 ml) under an Ar-atmosphere at −78 °C was slowly added BCl3 . After addition, the mixture was kept stirring at −78 °C for 2 h and then at 0 °C overnight. The volatiles were then removed under reduced pressure and the concentrate underwent purification by silica gel column chromatography .A solution of compound 12a–12d against AChE and BuChE was carried out following minor modifications of the Ellman assay,TM VarioskanTM LUX microplate reader and Greiner F-bottom 96-well plates.The inhibitory activity of Structures for all proteins  were retrieved from the Protein DataBank.12a–12d commenced from commercially available methyl α-D-glucopyranoside (13)  per-O-benzylation, (2) acidic cleavage of glycosidic bond, (3) reduction of pyranose 15, (4) double Swern oxidation of diol 16, and (5) double reductive amination to obtain 17.N-alkylation of 17via a nucleophilic substitution reaction is challenging when carried out under conventional heating,O-benzyl group.N-alkylation challenge of 17 is to use microwave irradiation. Thus, bromoalkyl derivatives 18a–18d of 1-DNJ were obtain in 44–77% when 17 was treated with an excess of dibromoalkanes in acetonitrile in the presence of K2CO3 under microwave irradiation. Treatment of 18a–18d with sodium azide provided the azide cycloaddition partners 19a–19d in excellent yield with no need for purification before they underwent copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) with N-propargyl benzotriazole 2021a–21d in 71–84% yield. BCl3 promoted de-O-benzylation of 21a–21d provided the target compounds 12a–12d.The synthesis of 1-DNJ-benzotriazole heterodimers ide (13) 1, which 12a–12d were tested against eeAChE (AChE from Electrophorus electricus) and eqBuChE (BuChE from equine serum) by using minor modification of the Ellman assay.50) of the ChE enzymes are tabulated in 2) as a positive reference and parent 1-DNJ (4) to assess the impact of the benzotriazole binding group on the inhibitory properties of 12a–12d.The inhibition potencies of 1-DNJ-benzotriazole heterodimers 12a, 12c, and 12d exhibited very poor inhibition potency against AChE. Heterodimer 12b was an outlier from this triplet of heterodimers as it displayed inhibition in the micromolar concentration range, which shows that the arming of 1-DNJ with a benzotriazole group can strengthen its AChE inhibition potency when the number of CH2-groups in the linker is optimised.As shown in 12a–12d, became inhibitors in the micromolar concentration range (IC50 = 7–50 μM) with a clear preference for BuChE over AChE (BuChE selectivity ≥ 9.7). From 12b is a 1.5-fold more potent BuChE inhibitor than 1-DNJ. Heterodimers 12a, 12c, and 12d, on the other hand, are 1.7 up to 5 times less potent BuChE inhibitors than 1-DNJ.When testing for activity towards BuChE that includes an active site gorge with a larger volume compared to AChE and different combination of residues, which affect the inhibitor selectivity of such enzymes,50-values provide useful information about inhibition potency they do not provide any information about the type of inhibition and affinity for inhibitors to their enzymes.vs. [I] and [S]/V vs. [I]) were created for the inhibition of BuChE by 12b  is smaller than the uncompetitive inhibition constant (αKi = 12 ± 1 μM), it is reasonable to expect that 12b possesses a greater affinity for the active site than for PAS.Even though ICE by 12b . Since a12b in recombinant human acetylcholinesterase (rhAChE) is shown in 12b binds in its acidic form, is that no cation–π interactions are observed between the positively charged nitrogen atom of 12b and any of the aromatic residues in the active gorge. However, because increased polarity of a X-H bond increases the strength of a X − H···π interaction,12b further polarise the C–H bonds of the neighbouring endocyclic CH2-group and thereby strengthen its CH–π interaction with Tyr341. In addition, the CH2-group between the benzotriazole and triazole moiety was observed to participate in a CH–π interaction with Trp286 in PAS.The preferred binding pose of 12b to the active gorge of human BuChE (hBuChE) is presented in 12b to AChE and BuChE is very different as the benzotriazole moiety of 12b is accommodated in the CAS of BuChE in which it participates in π–π interactions with the Trp82 moiety.The most stable binding pose of 12a–12d are stronger inhibitors for BuChE than for AChE. The inhibition of BuChE by 12a–12d is susceptible to the number of CH2-groups between the 1-DNJ and benzotriazole binding groups as 12b is a more potent BuChE inhibitor than 1-DNJ whereas 12a, 12c, and 12d are less potent than 1-DNJ. Modelling studies suggest that the 1-DNJ binding group is placed in the PAS of BuChE and the benzotriazole binding group in the CAS. This suggested dual binding site inhibition is supported by Cornish-Bowden plots, which demonstrate that 12b exhibit mixed type of inhibition against BuChE. The hydroxyl groups of heterodimers 12a–12d open the gate to prepare derivatives, which include additional pharmacophores and thereby target more of the factors that are involved in the progression of AD. Indeed, such studies are currently in progress in our laboratories.Heterodimers Click here for additional data file.


Hirundo rustica) mainly breed in temperate regions and parts of the tropics. These two regions have remarkably different environments, especially pertaining to their weather conditions. It is interesting to see how the breeding performances of Barn Swallows differ between the two regions and how the weather conditions affect the reproductive success of the Barn Swallow. We monitored the breeding activities of two Barn Swallow populations simultaneously in both tropical and temperate regions in China from 2019 to 2021 and investigated the effects of weather conditions on the reproductive success of the Barn Swallow in the study sites. We found that the reproductive success of the Barn Swallow at the tropical site was lower than that at the temperate site, i.e., swallows at the tropical site laid fewer eggs and had fewer nestlings/successful breeding attempts than swallows at the temperate site. Temperature, rather than rainfall, influenced the breeding performances of Barn Swallows at both sites. In particular, nestling survival decreased with increasing temperatures at the tropical site, but not at the temperate site. We suggest that researchers pay more attention to the reproduction and population changes of the Barn Swallow in the tropics in the context of climate warming.Barn Swallows (Hirundo rustica) simultaneously at a single tropical site and a single temperate site in China, compared the breeding performances of the two populations, and investigated the effects of weather conditions on reproductive success separately. The clutch and brood sizes of the Barn Swallow at the topical site were significantly smaller than those at the temperate site. Furthermore, the breeding success of the Barn Swallow at the tropical site was significantly lower than that at the temperate site. The mean daytime temperature had a negative effect on the clutch size and brood size at both sites; it had a negative effect on nestling survival at the tropical site, but not the temperate site. This study will help us understand the adaptation strategies of widely distributed bird species in different environments, and how climate change will affect birds in different climate zones.Temperate–tropical comparisons of avian life history traits are helpful to understand the different selective pressures placed on birds by different climate zones. Although there have been many comparative studies targeting multiple species in different regions, there are few comparative studies on the reproductive successes of the same species between tropical and temperate regions. In this study, we monitored the breeding activities of the Barn Swallow ( Troglodytes aedon) found that tropical wrens had a lower field metabolic rate during reproduction and produced fewer chicks per brood [Variations in breeding performances are common phenomena in birds living in different regions. Tropical birds are generally thought to have higher predation pressures, smaller clutch sizes, and lower reproductive success rates than temperate birds . Many lier brood . Althouger brood .Lanius collaris), thereby reducing nestling growth rates and muscle development [Differences in the life history characteristics of birds in different regions may be due to factors such as climatic conditions , predatielopment . Moreoveelopment . Howeverelopment . While selopment ,18.Hirundo rustica) is an aerial insectivore that breeds widely in a variety of habitats and climate zones across Eurasia and North America [The Barn Swallow . One America . HoweverTemperate–tropical comparisons are fundamental to understanding the different selective pressures that birds face in different climate zones and have been applied most frequently to study life history evolution ,31. Here2, with 6103 km2 of arable land, mainly planted with rice, bananas, and sugarcane [2; the main terrain consists of flood plains and tidal flats [This study was conducted in Zhanjiang city, Guangdong Province, China, from March to July 2020 and 2021, and in Panjin City, Liaoning Province, China, from May to August 2019 and 2021 . Zhanjiaugarcane . Zhanjiaugarcane . Panjin al flats . The stup = 0.16). In Zhanjiang, due to the high mortality rate of nestlings, we extended the monitoring period and defined the fate of nests with live 16-day-old nestlings as successful. We recorded the causes of breeding failure and classified them into the following categories: (1) nest damage—refers to the suspension of breeding activities due to the damage or fall of nests caused by bad weather or human destruction; (2) hatching failure—refers to the nest beyond the incubation period without chicks; (3) egg disappearance—refers to the disappearance of eggs observed in the nest during the incubation period; (4) nestling disappearance—refers to nestlings that disappeared within 15 days of age; (5) nestling death—refers to the phenomenon of nestling death observed during the brooding period; (6) interspecies competition—refers to the phenomenon where Barn Swallow nests were occupied by other bird species and the reproduction was forced to stop.During the study period, all Barn Swallow nests found at the study sites were numbered and inspected every 3 to 5 days to record the laying dates of the first eggs, clutch sizes, hatching dates, brood sizes at hatching , brood sizes of 11/16-day-old nestlings, nest fate, and the cause of breeding failure. GPS data of the nests were recorded. It was recorded as a breeding attempt if newly laid eggs were found in the nest. In Panjin, we defined the fate of nests that still had live nestlings 11 days after hatching as successful breeding attempts because nestlings rarely died afterward before fledging . The number of Zhanjiang meteorological stations is 59,658, about 15–40 km away from our study sites in Zhanjiang; the number of Panjin meteorological stations is 54,337, about 60 km away from our study site in Panjin. The meteorological data consisted of average temperatures, precipitation, etc., for no longer than every three hours, from which daily rainfall and average temperatures were extracted. For each stage, we calculated the mean daytime temperature and daytime precipitation totals.The breeding process of the Barn Swallow could be divided into the following stages: the egg-laying period (from the first egg to the penultimate egg ), incubaStatistical analyses were performed in R software  and RStFor each nest, we calculated hatching success and nestling survival. The hatching success was calculated by dividing the brood size at hatching by the clutch size. The nestling survival was calculated by dividing the brood size of 11-day-old nestlings by the brood size at hatching.p < 0.01), Welch’s t-tests were used to compare the clutch size and the brood size of 11-day-old nestlings between tropical and temperate populations. Wilcoxon rank-sum tests were used to compare the hatching success and nestling survival because the data were not normally distributed.A chi-square test was used to compare the breeding success of the Barn Swallow populations at the tropical and temperate sites. Since the variances of data were not equal between the two studied populations  were established to analyze the effects of weather conditions on the breeding performances of the Barn Swallow. GLMMs were established for the two study sites , respectively. The response variables were clutch size, hatching success, the brood size of 11-day-old nestlings, and nestling survival. The fixed factors were the mean daytime temperature and precipitation of the corresponding reproductive stage , and the random factor was the year . For the brood and clutch sizes, a normal distribution was selected to describe the error structures of the response variables, while a binomial distribution with a logit link function was used for hatching success and nestling survival. Apart from constructing GLMMs separately for each study site, we also constructed GLMMs with the dataset including both sites and with the site, the interaction between the site, and weather conditions as fixed factors for each response variable.p < 0.001).At the tropical site (Zhanjiang), the breeding season of the Barn Swallow started from the end of February to the end of July, lasting about five months in total. At the temperate site (Panjin), the breeding season of the Barn Swallow started from early May to mid-August and lasted about three and a half months . The repp = 0.345). However, the nestling survival of the Barn Swallow at the tropical site was significantly lower than at the temperate site , and the egg disappearance showed no difference between the two Barn Swallow populations at the tropical and temperate sites .The most important reason for reproductive failure at the temperate site was nest damage caused by rainfall (54.35%), followed by nestling disappearance (15.22%) and egg disappearance (13.04%). At the tropical site, the most important reason for reproductive failure was nestling disappearance (60.58%), followed by egg disappearance (12.50%), hatching failure (12.50%), and nestling death (11.54%). The nestling disappearance of Zhanjiang was significantly higher than that at the temperate site (n = 304). At the temperate site (Panjin), the mean daytime temperature during the breeding season (from May to July 2019 and 2021) was 19.08 ± 7.72 °C . The mean daytime temperature at the tropical site was significantly higher than at the temperate site . In terms of precipitation, the distribution of precipitation at the tropical site from March to July was not regular, while precipitation increased after May at the temperate site  in tropical Yunnan, China, was 31.76% [Tyrannus savana) in tropical south-eastern Brazil showed a breeding success of 25% [H. r. gutturalis [H. r. rustica) [H. r. erythrogaster) [Many studies have found that tropical birds generally have relatively lower breeding success than temperate species. For instance, the breeding success of the White-rumped Munia  ,46,47,48ogaster) ,49, whilSpilopelia senegalensis) have shown that the daily nest survival rate increased with the increasing nest age, possibly due to the increased intensity of parental defensive behavior with reproduction progress [For the daily survival rate, the daily egg survival rate was the same in the tropical and temperate sites in this study, indicating that the daily survival in both sites was similar during the egg stage, which was also supported by the similar hatching success. The daily nest survival rate appeared to be higher than egg daily survival at the temperate site. Studies on the nest survival of the Laughing Dove (progress . The difprogress .Overall, the reasons for reproductive failure in this study are similar to those in other Barn Swallow studies, including nest damage, hatching failure, and egg or nestling disappearance ,27,52,53Studies have shown that weather conditions can directly ,56 or inContrary to our expectations, precipitation does not affect the nestling survival of the Barn Swallow at either temperate or tropical sites. It was reported that the average mass of insects brought to swallow nestlings was positively related to the temperature and negatively related to rainfall . AnotherIn this study, we focused on the reproductive performances of Barn Swallows in different climate zones and the influence of weather factors on reproductive performances. However, many other factors can also affect the reproductive performances of Barn Swallows, such as habitat , parasitWe provided the first report on the breeding success of the Barn Swallow in the tropics. By comparing the reproductive performances of tropical and temperate Barn Swallows, we found that the reproductive success of Barn Swallows at the tropical site was lower than that at the temperate site. Temperature, rather than precipitation, had a strong effect on the breeding performance of the Barn Swallow. The nestling survival decreased with increasing temperatures at the tropical site, but not at the temperate site. Further identifying the effects of weather conditions on the reproduction of the Barn Swallow, especially in the tropics, requires collecting data from more populations. We suggest that more attention be given to the reproductive success and population changes of the Barn Swallow in the tropics in the context of global warming.


For patients with early American Joint Committee on Cancer (AJCC)‐stage melanoma the combined loss of the autophagy regulatory protein AMBRA1 and the terminal differentiation marker loricrin in the peritumoral epidermis is associated with a significantly increased risk of metastasis.The aim of the present study was to evaluate the potential contribution of melanoma paracrine transforming growth factor (TGF)‐β signalling to the loss of AMBRA1 in the epidermis overlying the primary tumour and disruption of epidermal integrity.Immunohistochemistry was used to analyse AMBRA1 and TGF‐β2 in a cohort of 109 AJCC all‐stage melanomas, and TGF‐β2 and claudin‐1 in a cohort of 30 or 42 AJCC stage I melanomas, respectively, with known AMBRA1 and loricrin (AMLo) expression. Evidence of pre‐ulceration was analysed in a cohort of 42 melanomas, with TGF‐β2 signalling evaluated in primary keratinocytes.P < 0·05), ulceration (P < 0·001), AMLo high‐risk status (P < 0·05) and metastasis (P < 0·01). TGF‐β2 treatment of keratinocytes resulted in downregulation of AMBRA1, loricrin and claudin‐1, while knockdown of AMBRA1 was associated with decreased expression of claudin‐1 and increased proliferation of keratinocytes (P < 0·05). Importantly, we show loss of AMBRA1 in the peritumoral epidermis was associated with decreased claudin‐1 expression (P < 0·05), parakeratosis (P < 0·01) and cleft formation in the dermoepidermal junction (P < 0·05).Increased tumoral TGF‐β2 was significantly associated with loss of peritumoral AMBRA1  in the tumour microenvironment has recently been identified as a prognostic biomarker associated with significantly increased risk of metastasis in early‐stage melanoma.Whatdoes this study add?Here we demonstrate paracrine transforming growth factor (TGF)‐β2 signalling in high‐risk AJCC early‐stage melanomas causes a loss of peritumoral AMBRA1 leading to the disruption of epidermal integrity and facilitating tumour ulceration and/or metastasis.Whatis the translational message?The discovery of the underlying mechanisms leading to loss of epidermal AMBRA1 overlying primary melanomas suggests TGF‐β blockade as an attractive adjuvant treatment strategy for patients with AJCC early‐stage I/II tumours stratified as high risk by loss of epidermal AMLo.Linked Comment:Br J Dermatol 2022; 186:606–607.G. Paragh. Plain language summary available online Cutaneous melanoma is an aggressive form of skin cancer with an increasing worldwide incidence, particularly in the younger population.AMBRA1‐mutant mice show embryonic lethality due to hyperproliferation of neuronal precursors,AMBRA1 haploinsufficiency is associated with increased cellular proliferation and spontaneous tumour formation in an animal model.V600E/PtenBraf‐deleted mouse model of melanoma.AMBRA1 is a scaffold protein with key roles in autophagy, cell survival and proliferation. AMBRA1 promotes autophagy through initiation of autophagosome formation,in vitro and in the epidermis, whereas AMBRA1 knockdown results in deregulated keratinocyte differentiation and a concomitant increase in cell proliferation.AMBRA1 levels increase in parallel with keratinocyte differentiation in vitro,Melanoma cells can influence the tumour microenvironment through secretion of growth factors, including transforming growth factors α and β .The aim of this study was to determine whether paracrine TGF‐β signalling results in the loss of AMBRA1 and loricrin (AMLo) levels in the epidermis overlying early‐stage melanomas. We show that TGF‐β2 is able to drive the downregulation of AMBRA1 in keratinocytes, and loss of peritumoral AMBRA1 is associated with a loss of epidermal integrity.The study included a retrospective cohort of AJCC all‐stage melanomas  defined by the 8th edition of the AJCC staging criteria, with a minimum of 10 years of clinical follow‐up  prepared on adhesion slides derived from primary melanomas or from epidermal skin equivalents were subjected to immunohistochemical (IHC) analysis for AMBRA1, TGF‐β2, TGF‐β3, claudin‐1 or AMLo expression, as described previously,–1 glucose  and 10% fetal bovine serum, and grown on cell culture dishes coated with 2% gelatin . Primary keratinocytes isolated from surplus human foreskin following informed consent (REC: 19/NE/004_Lovat) were cultured as previously described (Methods –1) medium for up to 6 days. Recombinant TGF‐β2 , ALK5 inhibitor , and rapamycin  were added in DMSO, and chloroquine  added in water, with an equal volume of vehicle used in all experiments as the control. TGF‐β2 secretion was measured using the TGF‐β2 Quantikine ELISA Kit  according to the manufacturer’s instructions.Human umbilical vein endothelial cells (HUVEC)  were cultured in Dulbecco’s Modified Eagle Medium supplemented with 4·5 g L Methods .30 Ker–1) using Lipofectamine RNAiMAX , according to the manufacturer’s instructions. After 6 h, medium was replaced with EpiLife  medium for 18 h prior to induction of differentiation, incorporation into skin equivalents or drug treatment.Small interfering RNA (siRNA) for AMBRA1 (Dharmacon (Horizon Discovery), Lafayette, CO, USA; ON‐TARGETplus Human AMBRA1 siRNA SMARTpool), ATG7  or nontargeting control  were transfected into cells in OPTIMEM containing siRNA , coated with human type I collagen , and cultured in low‐calcium EpiLife for 48 h. Equivalents were then grown at the air/liquid interface in high‐calcium EpiLife supplemented with vitamin C (50 μg mL–1) for 7 days, replacing medium every 48 h. Skin equivalents were fixed in 4% (w/v) paraformaldehyde, embedded in paraffin and processed for immunohistochemistry or haematoxylin and eosin staining. Cell proliferation was assessed by counting nuclei in 500‐μm fields of view.Epidermal equivalents were generated by seeding 1 × 10Total protein was obtained from cell pellets in extraction or a modified RIPA extraction buffer Methods , and sepTM RNA Cell Miniprep System  and reverse transcribed using an AMV Reverse Transcriptase kit  or High Capacity Reverse Transcription Kit , according to the manufacturer’s instructions. Real‐time polymerase chain reaction (PCR) was performed using TaqMan or QuantiTect gene expression assays, or primer pair sequences were designed by using IDT PrimerQuest Tool software (Table Ct method (2−ΔΔCt) was used for relative quantification of gene expression.Total RNA was isolated from cell pellets using Trizol reagent [Invitrogen (ThermoFisher Scientific)] or the ReliaPrept‐test, with Welch correction if standard deviations were not equal (F test), or by one‐way or two‐way anova with appropriate post hoc correction for multiple comparisons, or the Kruksal–Wallis test for non‐normally distributed data. Fisher’s exact test was used to compare the distribution of precursor features of melanoma ulceration .Data were analysed by Student’s in vivo, levels of TGF‐β2 and β3 increase in parallel with tumour progression.P < 0·05; Figure P < 0·05; Figure P < 0·01; Figure While all three TGF‐β isoforms are overexpressed in melanoma compared with melanocytes P < 0·05; Figure P < 0·001; Figure We next tested whether TGF‐β2 signalling could regulate expression of AMBRA1 in keratinocytes. TGF‐β2 treatment reversed the increase in AMBRA1 and loricrin and decrease in cytokeratin (CK) 14 expression observed during calcium‐induced keratinocyte differentiation Figure . To inveP < 0·05; Figure We have previously shown that knockdown of AMBRA1 results in decreased expression of loricrin in keratinocytes.P < 0·01; Figure The highest expression of tumoral TGF‐β2 was in ulcerated melanoma tumours, defined as the absence of an intact epidermis overlying most of the primary tumour.P < 0·05; Figure P < 0·01, Figure To examine epidermal integrity in both the epidermis adjacent to the melanoma and the epidermis overlying the tumour, IHC for AMBRA1 and claudin‐1 were performed on a subcohort of nonulcerated early‐stage melanomas. Loss of AMBRA1 in the epidermis overlying nonulcerated stage I melanomas was associated with decreased claudin‐1 levels (The identification of prognostic biomarkers able to stratify patients with early‐stage melanoma at high risk of metastasis will enable early therapeutic intervention. We have previously identified loss of AMLo (combined loss of AMBRA1 and loricrin in the epidermis overlying the tumour) as a novel independent prognostic biomarker in early‐stage melanoma and identified a functional role for AMBRA1 in epidermal homeostasis.TGF‐β has a complex role in cancer progression, with a tumour‐suppressive role in early‐stage tumours whereas in advanced cancers, TGF‐β signalling promotes angiogenesis, invasion and metastasis.in vitro studies and animal models suggests that TGF‐β1 signalling negatively regulates keratinocyte proliferation and is required for normal epidermal differentiation;Evidence from The highest expression of tumoral TGF‐β2 was observed in ulcerated melanoma tumours known to be associated with a worse prognosis.In summary, these data suggest a paracrine mechanism whereby melanoma secretion of TGF‐β2 causes peritumoral loss of AMBRA1 and reduced epidermal integrity facilitating erosion of the epidermis and tumour ulceration. Targeting TGF‐β2 signalling may therefore represent a novel adjuvant treatment strategy for high‐risk early‐stage tumours with loss of epidermal AMBRA1.Ioana Cosgarea: Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Validation ; Visualization ; Writing‐review & editing . Ashleigh McConnell: Formal analysis ; Investigation ; Methodology ; Supervision ; Validation ; Visualization ; Writing‐review & editing . Tom Ewen: Formal analysis ; Investigation ; Methodology ; Software ; Validation ; Visualization ; Writing‐review & editing . Diana Tang: Formal analysis ; Funding acquisition (supporting); Investigation ; Methodology ; Supervision ; Validation ; Visualization ; Writing‐review & editing (supporting). David Hill: Conceptualization (supporting); Formal analysis ; Investigation ; Methodology ; Validation ; Visualization ; Writing‐review & editing (supporting). Maria Eleni Anagnostou: Formal analysis ; Investigation ; Methodology ; Validation ; Visualization ; Writing‐review & editing (supporting). Martina Elias: Formal analysis ; Investigation ; Methodology ; Validation ; Visualization ; Writing‐review & editing (supporting). Robert Alexander Ellis: Conceptualization ; Formal analysis ; Funding acquisition ; Investigation ; Methodology (supporting); Resources (supporting); Supervision (supporting); Validation (supporting); Visualization (supporting); Writing‐review & editing (supporting). Aisling Murray: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Validation (supporting); Visualization (supporting); Writing‐review & editing (supporting). Lindsay Spender: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Validation (supporting); Visualization (supporting); Writing‐review & editing (supporting). Paola Giglio: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Validation (supporting); Visualization (supporting); Writing‐review & editing (supporting). Mara Gagliardi: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Validation (supporting); Visualization (supporting); Writing‐review & editing (supporting). Alison Greenwood: Formal analysis (supporting); Investigation (supporting); Methodology ; Validation (supporting); Visualization (supporting); Writing‐review & editing (supporting). Mauro Piacentini: Conceptualization (supporting); Funding acquisition ; Resources (supporting); Supervision (supporting); Writing‐review & editing (supporting). Gareth Inman: Funding acquisition ; Resources ; Supervision ; Writing‐review & editing . GianMaria Fimia: Conceptualization (supporting); Funding acquisition ; Supervision ; Writing‐review & editing . Marco Corazzari: Conceptualization (lead); Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Supervision ; Validation ; Visualization (lead); Writing‐review & editing . Jane Armstrong: Conceptualization (lead); Formal analysis ; Funding acquisition ; Investigation (lead); Methodology ; Project administration (supporting); Supervision ; Validation (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing . Penny Emma Lovat: Conceptualization (lead); Formal analysis ; Funding acquisition (lead); Investigation ; Methodology ; Project administration (lead); Resources (lead); Supervision ; Validation (supporting); Visualization ; Writing‐original draft ; Writing‐review & editing .Methods S1 Semiquantitative immunohistochemistry; Cell culture; Western blotting.Table S1 Patient demographic data (AJCC Stage 8th edition), Newcastle upon Tyne Hospitals NHS Foundation Trust (n = 109).Table S2 Nonulcerated AJCC Stage I and II (8th edition) patient demographic data, South Tees NHS Foundation Trust (n = 72).Table S3 Antibodies used for Western blotting.Table S4 PCR primer sequences or product number.Click here for additional data file.Figure S1 Peritumoural AMBRA1 loss does not correlate with melanoma secretion of TGF‐β3.Click here for additional data file.Figure S2 AMBRA1 functions as an autophagy regulatory protein in keratinocytes.Click here for additional data file.Figure S3 TGF‐β2 does not decrease autophagy gene expression in differentiated keratinocytes.Click here for additional data file.Figure S4 AMBRA1 loss correlates with decreased claudin‐1 in the peritumoral epidermis of stage II melanomas.Click here for additional data file.Video S1 Author video.Click here for additional data file.