CpG islands (CGIs) are vertebrate genomic landmarks that encompass the promoters of most genes and often lack DNA methylation. Querying their apparent importance, the number of CGIs is reported to vary widely in different species and many do not co-localise with annotated promoters. We set out to quantify the number of CGIs in mouse and human genomes using CXXC Affinity Purification plus deep sequencing (CAP-seq). We also asked whether CGIs not associated with annotated transcripts share properties with those at known promoters. We found that, contrary to previous estimates, CGI abundance in humans and mice is very similar and many are at conserved locations relative to genes. In each species CpG density correlates positively with the degree of H3K4 trimethylation, supporting the hypothesis that these two properties are mechanistically interdependent. Approximately half of mammalian CGIs  are “orphans” that are not associated with annotated promoters. Many orphan CGIs show evidence of transcriptional initiation and dynamic expression during development. Unlike CGIs at known promoters, orphan CGIs are frequently subject to DNA methylation during development, and this is accompanied by loss of their active promoter features. In colorectal tumors, however, orphan CGIs are not preferentially methylated, suggesting that cancer does not recapitulate a developmental program. Human and mouse genomes have similar numbers of CGIs, over half of which are remote from known promoters. Orphan CGIs nevertheless have the characteristics of functional promoters, though they are much more likely than promoter CGIs to become methylated during development and hence lose these properties. The data indicate that orphan CGIs correspond to previously undetected promoters whose transcriptional activity may play a functional role during development. In the decade since the sequence of the human genome was announced, efforts have been made to annotate all genes with their regulatory sequences. CpG islands are short regions containing the sequence CG at high density that map to regions controlling the expression of most human genes (known as promoters). Using a biochemical method, we have identified and mapped all CpG islands in the human and mouse genomes and find that over half are remote from known gene promoters—so-called “orphans.” Mice, which were thought to possess far fewer CpG islands than humans, turn out to have a very similar number. Surprisingly, orphan CpG islands in both species often mark hitherto unknown promoters. The activity of these novel promoters is particularly dynamic during normal development, as they are often silenced by DNA methylation. In colorectal cancers, however, aberrant DNA methylation affects all CpG islands equally. In the decade since the human genome sequence was published CGIs encompass the transcription start site (TSS) of approximately 60% of human protein coding genes. Extensive genome-wide mapping of histone modifications by chromatin immunoprecipitation (ChIP) has established that trimethylation of lysine 4 of histone H3 (H3K4me3) is a signature mark coinciding with most promoter CGIs, even when the associated gene is not expressed The notion that CGIs facilitate promoter function fits well with their presence at TSSs, but is challenged by two observations that appear to weaken the link with genes. Firstly, genomic analysis has indicated that the number of CGIs in humans and mice is very different, with mice apparently possessing little more than half the number present in humans −16; Welch Two Sample t-test; CGIs are characteristic of most human and mouse gene promoters, but biochemical and computational studies have suggested that the total number in the mouse genome is over one third less than for human bona fide biological differences  mapped to annotated gene promoters, the remainder being evenly distributed between intra- and intergenic regions . This protocol has the advantage that fluctuations in base composition that can erroneously register as CGIs using algorithmic methods are not recovered by CAP unless they are free of CpG methylation. As the vast majority (∼99%) of genomic DNA is both CpG-deficient and heavily methylated (70–80%), false-positive identification of CGIs is minimal. The absence of significant background signal in CAP-seq profiles verifies that CGIs represent a discrete fraction of the mammalian genome that is conserved during evolution. Computational prediction methods suggest that the human genome contains almost twice as many CGIs as mouse α-globin gene is CGI-associated, whereas this is replaced in mouse by a methylated CpG-deficient promoter Retinoblastoma (RB1) gene. This CGI, which is absent in mouse, is an imprinted promoter that gives rise to a transcript regulating RB1 transcription in cis Although broadly similar, the number of CGIs in humans exceeds that in mouse by ∼2,500. Specific examples contributing to this difference are known; for example the human More than half of all CGIs in human and mouse  can be classed as orphans that are remote from annotated promoters, being either embedded within coding regions or between transcription units. Trimethylation of H3K4, a signature chromatin modification associated with sites of transcriptional initiation Xist, Tsix, Air and HOTAIR, are transcribed from CGIs embedded within or between the coding regions of other genes There is evidence that CGIs are intrinsically poor substrates for nucleosome assembly and therefore have a reduced requirement for chromatin remodelling to induce transcription Pax6Orphan CGIs resemble annotated promoter CGIs in their sequence properties, promoter-like chromatin state and general lack of DNA methylation. Their enhanced propensity to become methylated during development, however, is distinctive. To visualise CGI methylation, we applied the reciprocal technologies of CXXC and MBD affinity purification to DNA from human and mouse tissues. Using this positive-negative screen, we established that both human and mouse methylated about 11% of all CGIs in the somatic cell types that were tested. Strikingly, the great majority of CGI methylation events occurring in these normal tissues involve orphan CGIs. Annotated promoter CGIs, by contrast, are infrequently methylated during development. Among orphans, we found that intragenic CGIs were almost twice as likely as intergenic CGIs to become methylated. An intriguing possibility is that many intragenic orphans represent alternative promoters which are utilised in a spatially or temporally restricted fashion, as described for HOXA locus where a conserved domain of orphan CGIs showed blood-specific methylation in both species. This aligns with previous suggestions that developmental genes, typified by HOX, are enriched for methylated CGIs HOTAIRConservation of methylation patterns at orphan CGIs in humans and mice, which diverged from a common ancestor about 75 million years ago, suggests a functional role for these putative promoters. The phenomenon is illustrated by the In cancer, aberrant silencing of tumour suppressor genes often coincides with the abnormal acquisition of CGI methylation Colorectal tumour-specific CGI methylation affects annotated promoter and orphan CGIs equally. In contrast, normal colon showed a typical somatic distribution of methylated CGIs as seen in blood and cerebellum, whereby orphan CGIs were preferentially methylated . InteresOur results establish that CGIs represent a distinctive fraction of the mammalian genome that is conserved between humans and mice. The relationship between CpG density and degrees of H3K4me3 supports a role for CpG in signalling a “promoter-friendly” chromatin conformation. Although about half of CGIs in both species are not previously annotated, our data suggests that these nevertheless represent thousands of functional promoters, often as novel genes for non-coding RNAs. In terms of transcription and DNA methylation, expression of orphan CGI promoters is more highly developmentally regulated than CGIs at annotated protein-coding genes. This observation raises the possibility that the resulting transcripts play functionally important roles during development.Work involving the use of human post-mortem brain samples was approved by the Lothian Research Ethical Committee (Ref. 2003/8/37). All donors consented to the use of this material for DNA extraction and all samples were anonymized prior to DNA extraction. DNA from normal and neoplastic colon tissues were prepared under ethics 08/S1101/41 through the Edinburgh Experimental Cancer Medicine Centre. All mouse work was carried out in accordance with Home Office regulations. No liscenced procedures were required for this work.Human whole semen , whole male blood  and whole female blood (n = 5) was collected from healthy donors. Human semen was centrifuged at 5000 g for 5 min then washed three times in PBS to yield pure sperm. All donors consented to the use of this material for DNA extraction and all samples were anonymized prior to DNA extraction. Approximately 500 mg of human cerebellum was provided for three males  and three females  by the MRC Sudden Death Brain Bank, Edinburgh.57Bl/6J mice. Testis (n = 4) and cerebella  were dissected from a subset of mice used for blood extraction. Testes were dounce homogenised on ice in 310 mM sucrose, 3 mM MgCl2, 10 mM potassium phospahate (pH 6) and 0.05% v/v Triton X-100 and cells were then centrifuged at 800 g for 20 min at 4°C. Cells were resuspended in water and subjected to 3×10 bursts of sonication on ice at setting 2 with a duty cycle of 20% using a Branson digital sonifer. Sonicated cells were then pelleted twice through a 2 ml 1.5 M sucrose cushion at 1000 g for 30 min at 4°C. Pelleted cells were visually inspected to ensure the presence of pure sperm heads.Mouse blood was extracted from male  and female  wild type CAll human and mouse tissues were pooled prior to DNA extraction. DNA was extracted from 10 mls of whole blood using the Genomic-tip 500/G kit according to manufacturers instructions . Purified sperm cells were incubated in 6 M guanidinium hydrochloride, 30 mM sodium citrate, 0.5% w/v sarkosyl, 0.2 mg/ml proteinase K, 0.2 mg/ml RNase A and 0.3 M β-mercaptoethanol and incubated at 55°C for 4 hours. DNA was isopropanol precipitated as previously described Human Shef 4 ES cells Bisulfite sequencing was performed as previously described MAP was performed using two sequential rounds of chromatography as previously described ChIP was performed on human and mouse ES cells as previously described http://maq.sourceforge.net/). Reads with a mapping score greater or equal to 30 where retained.MAP and CAP input DNA was ligated to solexa sequencing adaptors prior to purification as previously described for MAP http://main.g2.bx.psu.edu/) Mapped solexa sequence in the form of. WIG files was processed and analysed using a set of novel tools based on R and perl scripts interfaced with the Galaxy server  were scaled to a constant approximating the “average” read number for that purification in order to account for variable sequence depth. In the case of MAP samples, background was removed prior to normalisation as background reads could skew the normalisation procedure. Parameters applied for normalisation are outlined in Peaks of enrichment were identified using H, L and G parameters as outlined in Regions of CAP-seq enrichment in sperm, blood and cerebellum were combined to give a comprehensive set of CGIs for both human and mouse. For mouse CGIs, CAP-seq enriched regions identified at both high and low stringency (see above) were combined to account for the relative CpG deficiency of mouse CGIs  and each of the somatic tissue samples. This gave a ratio for each window. If both windows being compared contained less than 4 reads this ratio was set to 1 in order to remove bias due to small fluctuations at low read depth.Differentially methylated CGIs were defined as those containing 9 out of 10 contiguous windows with a log2 ratio of >2. CGIs were then scored as −1 (less methylated than sperm), 0 (same methylation as sperm) and 1 (more methylated than sperm). These parameters were verified using CGIs on the X chromosome which are methylated specifically in females.All analytical results are summarised in Dataset S1Summary of Human CGI data. Summary of all analysed data with respect to Human CGIs.(8.56 MB XLS)Click here for additional data file.Dataset S2Summary of Mouse CGI data. Summary of all analysed data with respect to Mouse CGIs.(6.47 MB XLS)Click here for additional data file.Figure S1Preliminary characterisation of Human and Mouse CAP-seq results. (A) Sperm CAP-seq read density profiles (blue) for human and mouse sperm generated by washing DNA bound to the CXXC column with 600 mM NaCl prior to elution. CpG density  at 4 human and mouse syntenic chromosomal locations is shown below the read profiles. Genes (Refseq) are annotated below the CAP-seq profiles with those mapped to the positive and negative strand displayed above and below the chromosome (grey line) respectively. The CpG density of 5 CpGs per 100 bp (dashed black line) is indicated for reference. Mouse regions assessed by bisulfite sequencing are indicated . (B) Bisulfite sequencing of four putative mouse CGI island promoters. Open circles represent unmethylated CpG sites. Each column represents a single PCR amplicon and horizontal lines represent single sequenced DNA clones. Vertical strokes represent the relative CpG position within each amplicon. (C) Histogram depicting the CpG observed/expected (o/e) values for all human (pink) and mouse (black) CGIs identified by CAP with washing at 600 mM NaCl. Statistical significance (**) was determined using a Welch Two Sample t-Test and CpG o/e values of 0.21  and 0.6  are indicated. (D) Sperm CAP-seq read density profiles (blue) for mouse sperm generated by washing with the optimised NaCl concentration (560 mM) in comparison with CpG density .(0.19 MB PDF)Click here for additional data file.Figure S2Pairwise analysis of mouse CAP-seq data. Scatter plots of CAP-seq data representing the mean sequence read depth for every contiguous 1 kb window in the mouse genome. Each pairwise comparison was assessed by calculating a Pearson correlation coefficient, which is presented above each plot. Tissue and replicate status for pairwise comparisons are noted above and to the left of the plots.(1.26 MB PDF)Click here for additional data file.Figure S3Proportional relationship between CpG density and H3K4me3 at Human CGIs. Box plots of H3K4me3 reads per base (averaged across 500 bp with a 100 bp slide) spanning 5 kb of all human CGIs at different CpG densities (CpGs per 100 bp). CpG density categories applied are ≤5, 5–6, 6–7, 7–8, 8–9 and >9 CpGs per 100 bp, arranged in ascending order from top to bottom. Box plots represent the distribution of the central 50% of the data (filled box) and the median (black bisecting line). The numbers of islands in each category (n) is noted in parenthesis.(0.04 MB PDF)Click here for additional data file.Figure S4Characterisation of MAP enrichment. Histograms representing the CpG density of MAP-enriched genomic loci in human (hMAP) and mouse (mMAP). The vertical dashed red line represents the lower tenth percentile of the data indicating that the majority of characterised MAP enriched DNA fragments have a CpG density of at least 1 and 1.3 CpGs per 100 bp in human and mouse respectively.(0.05 MB PDF)Click here for additional data file.Figure S5Global scatter plots reveal a reciprocal relationship between CAP- and MAP-seq data for human sperm, blood, and cerebellum. Scatter plots display pairwise comparisons of CAP- and MAP-seq data for every contiguous 1 kb window in the human genome using normalised data for human sperm, blood and cerebellum. Plots are represented as for (0.12 MB PDF)Click here for additional data file.Figure S6Pairwise comparisons of MAP-seq data reveal consistent tumour-specific methylation. Scatter plots displaying pairwise comparisons of MAP-seq data for every colon (C) and colorectal tumour (T) sample screened by MAP-seq. Data represents the mean sequence depth for every 1 kb window in the human genome. Data is presented as for (4.94 MB PDF)Click here for additional data file.Figure S7PDX1. MAP-seq profiles (red) for five colon mucosa  and five matched colorectal tumour  biopsy samples for human chr13: 27,325,000–27,402,000. CGIs (blue bars) and sites of hES H3K27 trimethylation Click here for additional data file.Table S1Sample information and data analysis parameters. Summary of the sequence and analysis statistics for each biological sample presented.(0.04 MB XLS)Click here for additional data file.


As part of our programme for developing predictive tests for normal tissue response to radiotherapy, we have investigated the efficacy of the cytokinesis-block micronucleus (MN) assay as a means of detecting interindividual differences in cellular radiosensitivity. A study was made of nine fibroblast strains established from vaginal biopsies of pretreatment cervical cancer patients and an ataxia telangiectasia (A-T) cell strain. Cells were irradiated in plateau phase, replated and treated with cytochalasin B 24 h later. MN formation was examined 72 h after irradiation as the number of MN in 100 binucleate cells. The method yielded low spontaneous MN yields (<7 per 100 cells), and mean induced MN frequencies after 3.5 Gy varied between cell strains from 18 to 144 per 100 cells. However, in repeat experiments, considerable intrastrain variability was observed (CV = 32%), with up to twofold differences in MN yields, although this was less than interstrain variability (CV = 62%). An analysis was made of the relationship between MN results and previously obtained clonogenic survival data. There was a significant correlation between MN yields and clonogenic survival. However, when the A-T strain was excluded from the analysis, the correlation lost significance, mainly because of one slow-growing strain which was the most sensitive to cell killing but had almost the lowest MN frequency. With current methodology, the MN assay on human fibroblasts does not appear to have a role in predictive testing of normal tissue radiosensitivity.


Distinguishing management effects from the inherent variability in a system is a key consideration in assessing reserve efficacy. Here, we demonstrate how seascape heterogeneity, defined as the spatial configuration and composition of coral reef habitats, can mask our ability to discern reserve effects. We then test the application of a landscape approach, utilizing advances in benthic habitat mapping and GIS techniques, to quantify this heterogeneity and alleviate the confounding influence during reserve assessment. Seascape metrics were quantified at multiple spatial scales using a combination of spatial image analysis and in situ surveys at 87 patch reef sites in Glover's Reef Lagoon, Belize, within and outside a marine reserve enforced since 1998. Patch reef sites were then clustered into classes sharing similar seascape attributes using metrics that correlated significantly to observed variations in both fish and coral communities. When the efficacy of the marine reserve was assessed without including landscape attributes, no reserve effects were detected in the diversity and abundance of fish and coral communities, despite 10 years of management protection. However, grouping sites based on landscape attributes revealed significant reserve effects between site classes. Fish had higher total biomass (1.5×) and commercially important biomass (1.75×) inside the reserve and coral cover was 1.8 times greater inside the reserve, though direction and degree of response varied by seascape class. Our findings show that the application of a landscape classification approach vastly improves our ability to evaluate the efficacy of marine reserves by controlling for confounding effects of seascape heterogeneity and suggests that landscape heterogeneity should be considered in future reserve design. No-take marine reserves have been increasingly promoted as a management tool to conserve biodiversity and prevent over-exploitation of marine communities Given the paucity of baseline data, Control-Impact (CI) comparisons are the most commonly used marine reserve assessment methodology, in which control sites outside of the reserve are compared to impact sites within In both terrestrial and aquatic systems, the response of organisms to heterogeneity in a landscape varies across spatial scales In comparison to the numerous terrestrial-based landscape studies, a landscape ecology approach in marine systems is still in its infancy 2  is located 30 km offshore of Belize, Central America, and comprises an area of 560 km2 . The atoPatch reefs served as the focal habitat for this analysis. Several features of the patch-reef array at Glover's Atoll make this reserve an ideal model system to test the applicability of landscape ecology approach to marine reserve evaluation. First, patch-reef complexes are pervasive, often containing hundreds of individual patches enabling ample replication within the reef system. Second, the discrete boundaries of patch reefs, often surrounded by sand or seagrass, enables spatial metrics of patch composition and configuration to be readily quantified through remote sensing and spatial analyses. Third, due to the geographic isolation and deep waters (>400 m) surrounding the atoll We assessed the fish and benthic communities at 87 submerged patch reefs in 2008–09 using a spatially explicit stratified random sampling design in which the entire lagoon area was divided into 23, equally sized blocks. A random point generator in ArcGIS was used to select a minimum of 3 patch reef sites within each block. A total of 56 non-reserve sites and 31 reserve sites were sampled in three field efforts: May 2008, February 2009 and April 2009. To investigate possible temporal changes over the 10-month sampling period, fish and coral surveys were repeated at 15 randomly selected patches from the total 87. No significant differences in coral cover, coral diversity, fish abundance or fish diversity were detected in this subset from 2008 sampling to 2009, and we therefore pooled the two years of data. Fish abundances were determined using the stationary point-survey method 2 were taken every 2 m from 0.5 m above the substrate along transects running the long and short reef axes. Depending on the total patch size, 25–100 images were generated per patch. Images were analyzed to species for scleractinian corals (>2 cm min. diameter), to functional group for benthic biota or to substrate class for non-biotic substrates. Using point-intercept methods, 100 random points were scored per image, on 20 randomly selected images per patch using CPCe v3.5 software The benthic composition of each patch reef was determined through the use of digital photography. Photographs of the benthos encompassing a reef area of approximately 0.25 mwww.spatialecology.com/htools) on polygons drawn around the patch boundaries using multi-band, high-resolution (4×4 m ground resolution) IKONOS imagery. An adjusted patch area was also calculated using the percentage of sand and seagrass to account for differences in hard substrate on each patch. Patch volumes were obtained from bathymetric maps generated in ENVI v4.7 based on depth and GPS data collected throughout the atoll at 183 points  was used. In CCA, regression analysis is used to find the best possible relationship between multiple environmental variables and multivariate community response data, assuming key environmental variables have been measured and the community response is unimodal in relation to these variables. Multicollinearity between seascape metrics was explored through correlation matrices. When evident (r>0.2), a principle component analysis was conducted on the co-linear metrics and the first principal component was used in subsequent analyses as an independent explanatory variable Separate hierarchical clustering analyses were preformed for coral and fish to classify patches together into ‘seascape groups’ sharing similar attributes of the significant seascape metrics identified for fish and for corals in the CCA analyses. Reserve effects were then evaluated using a modified Control-Impact design, in which reserve effects were only tested among patch reefs sharing the same seascape grouping for fish and corals, respectively. Comparisons of the fish assemblage  and coral assemblage  between management zones were conducted using one-way analysis of variance (ANOVA). We then repeated our analyses for each response variable using a traditional Control-Impact methodology with all 87 patch reef sites. Reserve effects were then compared between the two Control-Impact assessments.th root-transformed abundance data in order to reduce the contribution of common species Following detection of reserve effects, additional analyses were conducted to determine which organisms were influenced by reserve protection. Community similarity within coral and fish communities with respect to reserve protection and patch type were calculated in multidimensional space using a two-way crossed analysis of similarity (ANOSIM). Community similarity matrices were calculated using a Bray-Curtis index on 4Three seascape-level metrics of spatial configuration were identified in CCA analyses as explaining the greatest amount of variation in the fish community: distance from channel, patch reef area within a 500 m buffer, and nearest neighbor distance . Using tCCA analysis was used to identify 3 seascape level metrics that explained the greatest amount of variation in the coral community: distance from channel, ‘patch size’, and structural complexity of the patch . ‘Patch 1,43 = 8.05, P = 0.007). A similar significant increase of 50% was seen in total fish biomass from outside the reserve to inside . There was no difference in fish species richness inside versus outside reserve for either site grouping approach . Similarly, coral:macroalgal ratio increased 80% for Type II clustered sites within the reserve versus outside .As with the fish community, no significant differences between reserve and nonreserve sites were detected in coral community parameters when all patch reef sites were pooled . However1,56 = 9.037, P = 0.004; coral:macroalgal: one-way ANOVA, F1,56 = 5.362, P = 0.024). Similarly, Fish Type II patches showed positive responses, while Type I patches showed no differences between reserve and nonreserve sites  results revealed that coral communities were statistically indistinguishable between both Type I and Type II patches and across the reserve boundary (P>0.05). ANOSIM of the fish community revealed significant differences by patch type and reserve protection, but only Patch Type II reefs showed separation of community composition across the reserve boundary . Non-commercial fish species showed no significant response to reserve protection within either Type I or Type II patch reefs , suggesting that the positive reserve effect detected among Type II patch reef was driven by commercially important fish species sensitive to seascape heterogeneity and reserve management. Further investigation of the differences in commercial fish species composition on Type II patches showed no significant difference across the reserve boundary based on fish diet or trophic level . Species-specific responses within Type II patches revealed significantly greater biomass within the reserve for 3 species; two snappers (Lutjanus griseus and L. synagris) and the hogfish  factors of nearest neighbor and reef area within a 500 m buffer were significant factors explaining composition of fishes. Interestingly, benthic complexity of the patch reefs (i.e. rugosity) at the fine or medium grain scale was not found to be an important predictor for fish or coral assemblage parameters. This suggests that when patterns of community composition are assessed and constrained to a single, topographically complex habitat type, landscape level parameters may be better predictors of marine assemblage structure.For the large number of marine reserves lacking baseline data, augmenting the traditional Control-Impact reserve assessment with the seascape approach can improve reserve evaluation by controlling for influential aspects of seascape variability that affect target populations. While applied here to shallow water patch reef environments, this approach is repeatable in other marine systems given the increased access to high-resolution benthic habitat maps and GIS technology We stress the need to control for spatial heterogeneity in the evaluation of marine reserves, but application of these landscape ecology principles may improve criteria for reserve placement and design The establishment of marine reserves as a conservation tool has increased rapidly over the past decade. Yet the absence of baseline data, even within relatively well-replicated studies, makes it challenging to separate management effects from natural variability in populations driven by seascape differences. A weak assessment design that fails to capture reserve effects when they are present can generate false conclusions about reserve efficacy, seriously crippling management efforts to expand the use of marine reserves as a conservation tool. The burden of proof rests on managers and scientists to clarify how marine reserves can function as viable strategies for conservation and population replenishment. Therefore, we need a better understanding of the effects of reserves, which can be positive, negative or mixed. The use of a robust assessment methodology should be implemented to ensure that, when present, positive or negative effects can be properly ascertained. We suggest that the seascape approach applied in this study is one such method, and will serve as a powerful tool to improve our ability to distinguish management effects from natural system variation in future assessments of reserve efficacy.Table S1Commercially important fish species observed during sampling for Glover's Atoll.(0.04 MB DOC)Click here for additional data file.Table S2ANOVA results of reserve effects on fish and coral response variables using varying site classification scenarios.(0.05 MB DOC)Click here for additional data file.


We examined the hypothesis that elevation in urinary albumin creatinine ratio (ACR) in adolescents with type 1 diabetes is associated with abnormal retinal vascular geometry (RVG) phenotypes.10 ACR into tertiles: upper tertile ACR was defined as ‘high-risk’ for future albuminuria and the lower two tertiles were deemed ‘low-risk’. RVG analysis, using a semi-automated computer program, determined retinal vascular calibres (standard and extended zones) and tortuosity. RVG measures were analysed continuously and categorically (in quintiles: Q1–Q5) for associations with log10 ACR and ACR risk groups.A cross-sectional study at baseline of the relationship between ACR within the normoalbuminuric range and RVG in 963 adolescents aged 14.4 ± 1.6 years with type 1 diabetes (median duration 6.5 years) screened for participation in AdDIT. A validated algorithm was used to categorise log10 ACR was associated with narrower vessel calibres and greater tortuosity. The high-risk group was more likely to have extended zone vessel calibres in the lowest quintile  and tortuosity in the highest quintile . The effects of retinal vascular calibres and tortuosity were additive such that the participants with the narrowest and most tortuous vessels were more likely to be in the high-risk group . These effects were independent of duration, blood pressure, BMI and blood glucose control.Greater logHigher ACR in adolescents is associated with narrower and more tortuous retinal vessels. Therefore, RVG phenotypes may serve to identify populations at high risk of diabetes complications during adolescence and well before onset of clinical diabetes complications. Despite significant advances in the management of type 1 diabetes, premature cardiovascular mortality has not been significantly ameliorated, with most of the increase in cardiovascular risk explained by diabetic nephropathy –4. FurthThe retina offers a unique opportunity to visualise and study the vasculature  in vivo. Previous retinal vascular geometry (RVG) studies considered the proximal branches of central retinal vessels only . We recehttp://isrctn.com registration number 91419926) is the first interventional study in a group of normoalbuminuric adolescents with type 1 diabetes determined to be at high risk of developing kidney disease based on their urinary albumin creatinine ratio (ACR)  vs low-risk [the two lower tertiles]). Thresholds were explored and quintiles deemed the optimal category for analysis. RVG measures were analysed as both continuous and categorical variables (quintiles). Mean differences in retinal vascular measures between high-risk and low-risk groups were determined using independent samples t tests for normally distributed variables or Mann–Whitney U tests for non-parametric variables. A χ2 test was used to compare the likelihood of being in the high-risk ACR group depending on retinal vascular measure quintiles.Descriptive data are summarised as mean ± SD for parametric data and median (interquartile range [IQR]) for non-parametric data. Urinary ACR was log10 ACR, log10 eGFR and retinal vascular measures as continuous variables were examined using linear regression models. Binary logistic regression was used to explore threshold effects of retinal vascular measures, arriving at quintiles as the optimal grouping as predictors of high-risk and low-risk ACR groups. For vessel calibres, Q2–Q5 were combined into a single category for analysis. For tortuosity Q1–Q4, which were not statistically significantly different, were combined into a single category for analysis. Univariable regression analyses were carried out and multivariable regression models built using variables that were significant in univariable analysis, including HbA1c, BMI, SBP, lipids and duration. To test our proposed hypotheses that both arteriolar and venular components of the microvascular system contribute to the risk of microvascular dysfunction, we created two compound binary variables: (1) exMWaQ1 & CTvQ5 (the lowest exMWa quintiles and highest CTv quintile) and (2) exMWaQ1 & exMWvQ1 (the lowest quintiles of exMWa and exMWv). All statistical analyses were conducted using IBM SPSS version 22, Armonk, NY, USA.Associations between log1c or lipid profiles between the two groups. Participants in the high-risk ACR group had narrower arteriolar and venular calibres in both standard  and extended zones   Fig.  as well 10 ACR was inversely associated with BMI SDS  and diabetes duration  but not with HbA1c , SBP SDS  or cholesterol .In univariable analysis, log10 ACR was inversely associated with the calibres of the six largest arterioles (CRAE) in extended zones (exMWa and exMWv) but not the calibres of the six largest venules (CRVE). The compound variables  showed an additive effect on log10 ACR  and greater arteriolar and venular tortuosity (CTa and CTv). There was a similar trend for venular calibres (exMWv) but this did not reach statistical significance. The compound variable exMWaQ1 & CTvQ5 remained significant with an additive effect on log10 ACR. The associations remained significant after adjusting for BMI SDS and diabetes duration. The compound variable exMWaQ1 & exMWvQ1 showed a similar trend but did not reach statistical significance , younger age , lower BMI SDS  and higher SBP SDS , while its association with HbA1c  was not significant.In univariable analysis, high-risk ACR was associated with shorter diabetes duration  and the highest quintile for vessel tortuosity . The compound variables  showed an additive effect for high-risk ACR  in the standard zone have previously been associated with vascular dysfunction and adverse cardiovascular events [2+-activated K+ (BK) channels that are important in vasodilatation [Our study adds significant new knowledge to this field. First, we provide detailed and extensive analysis over a wider area of the retina (extended zone), measuring not only vessel calibres but also vessel tortuosity. Second, we report that extended zone calibres, both arteriolar and venular  rather than CRAE and CRVE, were more robust predictors of logr events –21; herelatation . Such rilatation .10 ACR. Notably, the highest quintile of venular tortuosity was associated with greatest probability of being in the high-risk ACR group. We previously demonstrated that greater tortuosity was associated with increased risk of retinopathy [We also demonstrate that greater tortuosity was associated with higher loginopathy  and renainopathy , 26. We Third, we provide evidence supporting a unifying haemodynamic model integrating afferent (arteriolar) and efferent (venular) components of the microcirculation whereby both components contribute to risk in an additive manner. We report an adverse retinal phenotype comprising narrower vessel calibres and greater tortuousity that was associated with a significantly greater likelihood of a person being in the high-risk ACR group than when either of these measures were used alone.We observed that individuals in the high-risk ACR group were younger, had shorter diabetes duration and higher blood pressure but no difference in glycaemic control compared with the low-risk group. Thus, our data suggest the high-risk ACR group, screened through ACR, may indeed be a population predisposed to earlier onset of complications and likely to benefit from earlier intervention. Current evidence supports both genetic and metabolic mechanisms that protect against and predispose to diabetes complications , 10, 27.The strengths of our study include the multinational collaboration and large sample size, standardised retinal photography images and single-centre grading of retinal images masked to clinical and biochemical participant data. However, these are the baseline data and therefore cross-sectional in nature. The longitudinal follow-up from AdDIT will provide invaluable insights into the mechanisms of diabetes complications and potential benefits of early ‘pre-complications’ intervention in paediatric cohorts. In addition to their role as potential biomarkers for risk stratification of future diabetes complications, RVG measures may provide an opportunity to quantify the effect of intervention on the microvasculature in vivo. Furthermore, these RVG quantification techniques may be applied to other previously conducted interventional study cohorts such as RASS and the DCCT. Long-term follow-up of such cohorts may determine the potential to shift the current screening paradigm from one of detection and treatment of diabetes complications to one of identification of persons at risk and prevention of microvascular complications.In conclusion, we demonstrate that urinary ACR is associated with RVG variables prior to the onset of clinical complications in adolescents with type 1 diabetes. Higher ACR and greater likelihood of being in the high-risk ACR group were associated with narrower and more tortuous retinal vessels, particularly in the extended zones. RVG may assist in identifying individuals at high risk of complications early in the disease process and may provide useful biomarkers by which to quantify response to diabetes therapy.


The influenza virus RNA-dependent RNA polymerase (RdRP) cleaves the 5′ end of nascent capped host RNAs and uses the capped RNA fragment to prime viral transcription in a mechanism called ‘cap snatching’. Cap snatching requires an intimate association between influenza RdRP and cellular RNA polymerase II (Pol II), which is the source of nascent capped host RNAs targeted by influenza virus. Recent structural studies have revealed how influenza RdRP binds to Pol II and how this binding promotes the initiation of viral transcription by influenza RdRP. In this review we focus on these recent insights into the mechanism of cap snatching by influenza virus and the impact of cap snatching on host gene expression during influenza virus infection. Influenza virus RdRP associates with cellular Pol II. This provides influenza RdRP with a source of nascent capped host RNAs which it targets in a process called ‘cap snatching’ to generate primers for viral transcription.Influenza RdRP makes direct interactions with the Pol II C terminal domain (CTD). Molecular details of these interactions have recently been determined, and there is evidence that Pol II CTD binding promotes viral transcription by modulating the conformation of influenza RdRP.Influenza RdRP cap snatches small nuclear/nucleolar RNAs most frequently. When cap snatching generates suboptimal capped RNA fragments, influenza RdRP can use a prime-and-realign mechanism to promote transcription initiation.Influenza virus infection reduces Pol II abundance in gene bodies, suggesting that cap snatching contributes to host shut-off. Influenza viruses are negative-strand RNA viruses of the family Orthomyxoviridae. Influenza A and B viruses are responsible for annual epidemics which cause up to half a million deaths worldwide each year. Influenza C viruses are less prevalent and they usually cause mild infections, while influenza D viruses are not known to infect humans Influenza viruses have a genome consisting of eight viral RNA (vRNA) segments, each encoding one or two major viral proteins. Each genome segment is packaged with viral nucleoprotein (NP) and the RdRP in viral ribonucleoprotein complexes (vRNPs). Influenza virions must contain a full complement of eight vRNPs to be productively infectious. Virions bind to sialic acid on the cell surface, are endocytosed, and fuse with the endosomal membrane. vRNPs are released into the cytoplasm and are trafficked to the nucleus where they begin synthesising viral mRNAs to produce viral proteins 7G) cap and 3′ polyA tail Influenza RdRP is a heterotrimer consisting of a core polymerase subunit with a canonical right-hand fold (PB1), and two auxiliary subunits, PB2 and PA 7G capping of mRNAs, some negative-sense single-stranded RNA (ssRNA) viruses, such as Ebola, measles, and rabies viruses, synthesize an m7G cap de novo using their own viral mRNA capping machinery 7G capped 5′ end of nascent host RNAs 7G on nascent host RNAs using a cap-binding domain on the RdRP PB2 subunit For 5′ m7G capped and 3′ polyadenylated, and nascent Pol II transcripts with a completed m7G cap are the target of cap snatching by influenza RdRP. Understanding of influenza RdRP interactions with Pol II at a molecular level has improved greatly in recent years, thanks to biochemical studies and multiple crystal structures of the complete influenza RdRP heterotrimer Cap snatching by the influenza RdRP requires an intimate association with the host transcriptional machinery RPB1 subunit (see Cellular Pol II is a complex composed of 12 core subunits. The unit see  has a flCDK7 (a component of TFIIH) phosphorylates Ser5 (Ser5P) on the Pol II CTD. The Ser5P CTD modification is recognised by cellular machinery required early in Pol II transcription, such as mRNA capping enzymes P-TEFb complex releases paused Pol II so it can continue transcription CDK9, a component of the P-TEFb complex, phosphorylates Ser2 on the Pol II CTD (Ser2P). The Ser2P CTD modification occurs on elongating Pol II, and Ser5 on the CTD is gradually dephosphorylated throughout elongation Early in Pol II transcription, The Ser5P CTD modification on Pol II is most prevalent close to transcription start sites, but is still present at lower abundance in gene bodies HTat-SF1 and SPT5RNA exosome complex, involved in RNA quality control in the nucleus, which may enhance the targeting of influenza RdRP to gene promoters for subsequent cap snatching The specificity of influenza RdRP for Ser5P CTD indicates that cap snatching most likely occurs early in Pol II transcription. In agreement with this, chromatin immunoprecipitation (ChIP) data show that influenza RdRP is primarily associated with regions close to gene promoters Improved understanding of influenza RdRP structure has led to elucidation of the molecular mechanisms of cap snatching. Recent structural studies have revealed how the RdRPs of different influenza types bind to the Pol II CTD, and how this interaction promotes cap snatching and viral transcription initiation at a molecular level.The interaction between influenza RdRP and Pol II CTD was shown to be direct by using Pol II CTD mimic peptides Critical amino acid residues identified in the CTD binding sites are conserved across most FluA strains. Interestingly, amino acid sequence alignments predict that only Site 1 is conserved in influenza B (FluB) RdRP Amino acid sequence alignment of the FluA RdRP CTD binding sites with influenza C (FluC) RdRP indicates that neither Site 1 nor Site 2 is strongly conserved in the FluC RdRP Recently, FluC RdRP has also been cocrystallised with a Pol II CTD peptide et al. suggest that the presence of multiple binding sites may be a mechanism to increase avidity of the Pol II CTD and influenza RdRP interaction These studies show that the mode of influenza RdRP binding Pol II CTD has diverged between influenza virus types. Despite this, the RdRPs of influenza A, B, and C appear to have two Pol II CTD binding sites. Serna Martin in vitro. These data led to the suggestion that binding to the Pol II CTD may influence the conformation of influenza RdRP in order to improve the efficiency of cap snatching and viral transcription initiation Recent evidence shows that influenza RdRP binding to Ser5P Pol II CTD promotes cap snatching and transcription initiation activity 7G cap-binding domain, and the N terminal PA endonuclease undergo major rearrangements between the two conformations. In the ‘transcriptionally inactive’ conformation, the PB2 cap-binding domain is occluded by an adjacent linker region of the PB2 subunit et al. propose that Ser5P Pol II CTD binding, in combination with vRNA promoter binding, promotes a ‘transcription preinitiation’ RdRP conformation to facilitate cap snatching when close to a source of nascent capped host RNAs  and small nuclear RNAs (snRNAs) Just as structural techniques have given insight into the molecular mechanisms of cap snatching, several recent RNA sequencing-based studies have built a picture of which nascent Pol II transcripts are targeted by influenza virus There is some debate over whether specific host transcripts are targeted, or if cap snatching is opportunistic and influenza RdRP simply cap snatches the most abundant nascent capped RNAs. Studies have aimed to examine the cap snatching repertoire by sequencing the 5′ end of viral mRNAs and mapping the 10–13 nt originating from capped RNA fragments back to the donor host transcripts. These studies show that sequences originating from host snoRNAs and snRNAs are very abundant in viral mRNAs Interestingly, multiple studies have observed heterogeneity in cap snatching between different influenza virus genome segments. Specifically, these studies show that different genome segments use different lengths of capped RNA fragments et al. observed that up to 30% of influenza mRNAs contain short insertions at the 5′ end, often GCG or GCA trinucleotides depending on the vRNA template in vitro data have provided mechanistic insight into this process, and suggest that the function of prime and realign is to enable transcription initiation using suboptimal capped RNA primers – for example, capped RNAs which are shorter than normal or which base pair weakly with the vRNA template Using the same sequencing approach, Koppstein et al. have shown that influenza virus infection causes a dramatic loss of Pol II occupancy on host genes on a genome-wide basis Influenza virus uses a variety of methods to suppress host antiviral responses, including inhibition of Pol II transcription Recent studies have provided insights into molecular details of the cap snatching process and how this links to transcription initiation by influenza RdRP. Biochemical and structural studies have revealed that influenza RdRP in a vRNP is targeted to Pol II using Ser5P CTD binding sites, which vary between influenza types. While these interactions are clearly important, it is unlikely that Pol II CTD binding alone accounts for all the interplay between influenza RdRP and Pol II in cap snatching (see Outstanding Questions). Pol II Ser5P CTD binding to influenza RdRP promotes a transcriptionally competent conformation which enhances nascent capped RNA cleavage and transcription initiation 7G cap-binding activities Outstanding QuestionsWhat is the structure of the influenza vRNP–Pol II complex? Do host proteins aid this interaction during cap snatching?When, in viral transcription, do influenza vRNPs dissociate from the Pol II CTD?Is heterogeneity in cap snatching between viral genome segments important for influenza virus?How does cap snatching lead to Pol II termination? How important is cap snatching for host shut-off by influenza virus?Nascent Pol II-transcribed RNAs appear to be targeted based on abundance, and heterogeneity in cap snatching between different viral genome segments has been observed


Coronary microvascular dysfunction and/or vasospasm are potential causes of ischaemia in patients with no obstructive coronary artery disease (INOCA). We tested the hypothesis that these patients also have functional abnormalities in peripheral small arteries.P = 0.0047]. Endothelium-independent relaxation [sodium nitroprusside (SNP)] was similar between all groups. The maximum contractile response to endothelin-1 (ET-1) was greater in MVA (median 121%) vs. controls . Response to the thromboxane agonist, U46619, was also greater in MVA (143%) vs. controls . Patients with VSA had similar abnormal patterns of peripheral vascular reactivity including reduced maximum relaxation to ACh  and increased response to constrictor agonists including ET-1 . In all groups, resistance arteries were ≈50-fold more sensitive to the constrictor effects of ET-1 compared with U46619.Patients were prospectively enrolled and categorised as having microvascular angina (MVA), vasospastic angina (VSA) or normal control based on invasive coronary artery function tests incorporating probes of endothelial and endothelial-independent function (acetylcholine and adenosine). Gluteal biopsies of subcutaneous fat were performed in 81 subjects . Resistance arteries were dissected enabling study using wire myography. Maximum relaxation to ACh  was reduced in MVA vs. controls [median 77.6 vs. 98.7%; 95% confidence interval (CI) of difference 2.3–38%; Systemic microvascular abnormalities are common in patients with MVA and VSA. These mechanisms may involve ET-1 and were characterized by endothelial dysfunction and enhanced vasoconstriction.ClinicalTrials.gov registration is NCT03193294. The 2013 European Society of Cardiology (ESC) guidelines on the management of stable coronary artery disease (CAD) were notable as the first international practice guidelines to address the diagnosis and management of functional coronary disorders.Coronary microvascular dysfunction and/or vasospasm are potential causes of ischaemia in patients with no obstructive coronary artery disease (INOCA).,Figure ). We hypothesised that small vessel damage in the heart may be a systemic phenomenon and that patients have generalized endothelial dysfunction leading to abnormal vascular reactivity assessed using wire myography. To test this hypothesis, we designed a case–control study to investigate peripheral small artery changes in two distinct groups of INOCA—those with microvascular angina (MVA) and those with vasospastic angina (VSA). Our aim was to compare peripheral endothelial function and vascular reactivity in these two groups with matched control subjects .Importantly, microvascular disease may be a multi-system entity with links between coronary microvascular dysfunction and small vessel disease of the kidney,We screened elective adult referrals to two large cardiac catheterization laboratories with clinical suspicion of significant coronary artery disease based on the presence of angina. Patients were prospectively recruited from the larger British Heart Foundation Coronary Microvascular Angina (CorMicA) study which is a randomized controlled trial evaluating the effect of diagnostic strategy and linked therapy on symptoms and quality of life in INOCA.Table 1). The CorMicA study design paper describes the coronary artery function testing procedure.We defined MVA according to the COVADIS diagnostic criteria: stable angina,,−1·min−1) was administered via a large peripheral vein to induce steady-state maximal hyperaemia. Increased IMR (≥25) was representative of microvascular dysfunction.In brief, we measured coronary flow reserve (CFR) and the index of microcirculatory resistance (IMR) using thermodilution as previously described.−6, 10−5, and 10−4 mol/L, respectively) at 1 mL/min for 2 min via a mechanical infusion pump.−4 mol/L over 20 s—reduced to 50 μg for the right coronary artery).The target vessel was the left anterior descending coronary artery. If technical factors, e.g. vessel tortuosity, precluded assessment of this artery then the left circumflex or right coronary artery was selected. We assessed endothelium-dependent coronary vasomotor function using intra-coronary infusions of ACh via the guiding catheter at concentrations of 0.182, 1.82, and 18.2 µg/mL  within 4 weeks of coronary angiography. Vasoactive medications were withheld for at least 24 hours prior to the surgical biopsy. Small resistance arterioles  were studied in a Mulvany-Halpern 4-channel wire myograph  with isometric tension recordings made as previously described.,max) for vasoconstrictors was expressed as a percentage of the mean response of the contraction to 62.5mM potassium chloride solution. For relaxation data, the maximum response (Emax) to ACh was expressed as percentage relaxation after preconstruction with thromboxane analogue . Sensitivity of the arterioles to each compound was expressed as the pEC50 (constrictors) or pIC50 (inhibitors) derived from the CCRC. Prism 7.0 The pEC50 value represents the minus log concentration required to produce 50% of the maximum response. Similarly, pIC50 is the -log of concentration required to inhibit 50% of the maximum response. Higher numbers indicate more potency (less concentration required to achieve the median response).After a standard normalization and start-up protocol involving repeated washes with high potassium chloride solution (62.5 mM), the arterioles were pre-constricted with thromboxane-A2 analogue . Previous work on human resistance arteries support its application in myography due to its consistent vasoconstriction with a steady plateau from which to assess arteriolar relaxation. Blood vessels with no responses were discarded. We averaged the relaxation response to each incremental concentration of acetycholine  in multiple arterioles in each subject. This served to increase accuracy by levelling out the regional heterogeneity of endothelial function in vivo.Emax) between two groups: MVA and control subjects. Sample size calculations were based on this measure. Using an allocation ratio of 5:1 (MVA:controls), we calculated that a sample size of 52 patients and 10 controls would have 80% power to detect between group difference in ACh Emax of 10% at the 5% significance level. A minimum sample size of 62 subjects was estimated using G*Power 3.1 . This calculation used the Mann–Whitney U test reflecting small sample size and likelihood of non-parametric distribution.The study population analysed comprised all of the participants who underwent a biopsy and no patients were excluded even if no viable vessels were dissected. The primary endpoint of this study was the difference in maximum relaxation induced by ACh . We applied the Shapiro–Wilk normality test to establish whether the data followed a parametric distribution. Best-fit cumulative concentration curves (CCRCs) were compared with the extra sum-of-squares F test. We performed two-tailed analysis and considered a P-value <0.05 to be significant. Statistical analyses were performed with Prism 7.0  and SPSS 24.0 .Results are reported as mean (± standard deviation) for parametric data and median (25th and 75th percentile) for data that were not normally distributed. The Fisher’s exact test or the χTable 2). Non-obstructive epicardial CAD severity, assessed using Gensini scoreP = 0.15). All 59 MVA patients had demonstrable evidence of coronary microvascular dysfunction including 50 (85%) with abnormal CFR and/or IMR, 23 (39%) with evidence of microvascular spasm to ACh and 14 (24%) with abnormalities to both adenosine and ACh. Coronary flow reserve was reduced in MVA (2.4 ± 1.1) vs. VSA (3.6 ± 2.2) and control subjects . Coronary microvascular resistance was greater in MVA (mean IMR 28.2 ± 16.3) compared with VSA (15.8 ± 1.6) and control subjects controls .From December 2017—June 2018, 81 subjects gave informed consent and underwent clinically-indicated invasive coronary angiography coupled with guidewire-based measurements of coronary artery function and vasoreactivity testing. A gluteal skin fat biopsy was then obtained within 4 weeks of the angiogram. The mean age of the MVA patients was 63 years which was similar to the VSA and control subjects. Overall, the characteristics of the patients including demographics, risk factors, and treatment were similar between the groups . Additional information on vessels including length and number of viable vessels is demonstrated in Table 4. In total, 81 biopsies were performed . Three surgical biopsies had no functional small resistance arteries . The normalized internal diameters of resistance arteries did not differ between MVA, VSA, and control subjects . The maximum relaxation to ACh did not differ between patients with MVA and VSA . Maximum relaxation to the endothelium-independent dilator, SNP, was similar in small resistance arteries from MVA patients compared with controls .Acetylcholine and SNP evoked concentration-dependent relaxations in preconstructed resistance arteries from all groups. For the primary study endpoint, the maximum relaxation to ACh was significantly lower in patients with MVA compared with controls [median 77.6% vs. 98.7%; 95% confidence interval (CI) of difference in medians 2–38%, Mann-Whitney U = 106, pIC50 ET-1 9.6 vs. 7.9; 95% CI of median difference in pIC50 1.4–1.9; Mann-Whitney U = 247, P < 0.001). Importantly, the maximum constrictor response to ET-1 was greater in MVA compared with the control group . The maximum response to U46619 was also greater in MVA . The VSA group had similar patterns of increased vasoconstriction to both ET-1  and U46619 . These were not significantly different from the MVA subjects . The key findings of the study are summarized in the Take home figure.All arteries responded in a concentration-dependent manner to both endothelin-1 (ET-1) and U46619. In all subjects, blood vessels were ≈50-fold more sensitive to the constrictor effects of ET-1 compared with the thromboxane agonist U46619  cases of minor wound dehiscence post procedure without clinically significant wound infection. Wound healing occurred by secondary intention in all cases without additional treatment.We have shown altered function of peripheral small arteries in patients with INOCA compared with control subjects. Specifically, both MVA and VSA patients had peripheral microvascular abnormalities characterized by reduced maximum relaxation to ACh  and increased responses to vasoconstrictor stimuli. Vasoconstriction in response to ET-1 and the thromboxane agonist U46619 was greater in MVA and VSA patients. Overall, our results stimulate the provocative concept that patients with MVA or VSA are at risk of developing systemic small vessel disease. Our study helps explain the associations between coronary microvascular dysfunction and small vessel disease in other organs, such as the brain and kidney.To our knowledge, this is the first study to prospectively investigate patients with INOCA and objective invasive evidence of coronary microvascular function using wire myography to assess peripheral vascular function. These assessments were temporally approximated within a 4 week period. Furthermore, a control group of subjects with normal coronary microvascular function who underwent the same interventional diagnostic procedures were also included. This protocol included reference invasive coronary testing based on ESCin vitro research of small resistance arteries and is well established in our laboratory. This method enables vascular mechanisms to be specifically investigated, which otherwise would not be possible in vivo. Our findings extend prior studies that identified abnormalities in peripheral vascular function in patients with both MVAEmax between MVA and VSA, so we cannot draw definitive conclusions about comparative endothelial function between the MVA and VSA groups. It would be expected that MVA patients would have reduced endothelial function compared with VSA patients although confirmation of this would require a much larger study. Notwithstanding the distinct differences in the pathogenesis of these disorders, endothelial dysfunction is implicated in the aetiology and progression of both these INOCA endotypes.,,Wire myography is a reference technique for in vitro were from a different vascular bed from those implicated in the pathogenesis of coronary microvascular dysfunction. The cross-sectional study design limits understanding of the natural history and causality, and concomitant cardiovascular treatment is a potential confounder. As such, our findings are associative but the structural and functional changes that occur in human subcutaneous small arteries in response to vascular risk factors, such as diabetes mellitus and hypertension, have prognostic relevanceOur work provides new insights into the presence and mechanisms of systemic arteriolar dysfunction. The small arteries studied ,in vitro vasorelaxation in response to the NO donor, SNP, consistent with normal responses to exogenous NO. The SNP-mediated effects are independent of NO derived from eNOS activation and accordingly have been considered as endothelial-independent vasorelaxation.We did not measure nitric oxide (NO) and it is possible that bioavailable NO was not deficient, rather the mechanisms of vascular dysfunction occurred in the vascular smooth muscle cells (VSMCs). It is known that high doses of ACh provoke constriction via direct action on vascular smooth muscle muscarinic receptors in the coronary circulation. However, this effect does not occur in human small peripheral arterioles.A receptor down-regulation in response to elevated ET-1 levels. Differences between in vitro and in vivo studies of human endothelial function are well recognized.Our results contrast with a study of forearm blood flow (FBF) that reported preserved ACh mediated relaxation in patients with Syndrome X .Our research findings stimulate the provocative concept that MVA and VSA associate with a generalized susceptibility to vasoconstrictor stimuli. We found enhanced contractile responses to both ET-1 and U46619 in small resistance arteries from patients. This finding may be due to endothelial dysfunction contributing towards an imbalance of bioavailable constrictor factors  which predominate over endothelium derived relaxing factors (nitric oxide (NO), prostacyclin ( PGI2) and endothelium derived relaxing factor (EDRF)). We recognize the potential for concomitant epicardial vasospasm in patients with coronary microvascular dysfunction and the potential for exaggerated microvascular constriction in patients with VSA.,Abnormalities vascular at VSMC level with pathological constrictor response to ACh in the smooth muscle is another alternative mechanism . Vascular smooth muscle cells contain several sources of reactive oxygen species that mediate many pathophysiological processes such as growth, migration, apoptosis, and secretion of inflammatory cytokines.As with all case–control studies, the selection of a true ‘reference control’ group is a potential limitation. Within the constraints of any matching criteria, we approached consecutive patients with negative testing of coronary function with similar exposure to risk factors and confounders that were representative of the population at risk. Nevertheless, assignment of participants to the control group in this way may have introduced unmeasured confounding bias.et al.et al.The diagnosis of MVA in our population followed established guidelines, however, the underlying pathophysiology is likely to differ between individual patients reflecting ‘real world’ practice. We recognise other even more in depth endotypes, such as the ′coronary slow flow phenomenon′ proposed by Tambe in vitro myography does not fully replicate physiological conditions in vivo. Considering the primary outcome of the maximum vasorelaxation response to ACh, we averaged the response of arterioles in order to increase accuracy and average within subject variation of microvascular function. Indeed, the same approach is established for other physiological measurements, including guidewire-based coronary thermodilution for measurement of coronary microvascular resistance.,Table 3). We have described one of the largest series of myography experiments in a single study, and the largest to date in patients with INOCA. Nonetheless, the sample size is a potential limitation of our study inherent to the technically demanding procedure of gluteal fat biopsy and wire myograph protocol.Despite the normalization process that serves to standardize each experiment, Recent trials have increased physician awareness of stable coronary syndromes beyond fixed luminal obstruction; half of patients may have ongoing angina despite technically successful percutaneous coronary intervention.2 analogue U46619. The increased constriction to ET-1 observed in the present study raises the question about whether interventions which reduce the effects of this peptide may, therefore, be useful in the treatment of such patients. An appreciable component of the coronary smooth muscle contractile response to ET-1 signalling mechanism is calcium antagonist-resistant.Endothelin-1 is a potent endogenous vasoconstrictor peptide. In this study, ET-1 was ≈50 times more potent than the thromboxane AIn conclusion, we have identified peripheral endothelial dysfunction and enhanced vasoconstriction in MSA and VSA. These processes may involve ET-1. Small vessel damage in the heart may be part of a widespread phenomenon with INOCA patients at risk of other small vessel diseases. Overall, further research is required but this study reminds us to think of angina as a cardiac symptom of a systemic disease where treatment of the entire cardiovascular system (including basic lifestyle measures) should be encouraged.


The asthma gene PCDH 1, encoding protocadherin-1, is a cellular adhesion molecule which plays an important role in epithelial barrier formation and repair. PCDH 1 is a novel susceptible gene not only in childhood asthma but also in eczema and other atopic phenotypes. In this article, we reviewed relevant articles from PubMed, Google Scholar, Science Direct and included all available significant pieces of information about the PCDH 1 association with asthma and other atopic or non-atopic phenotypes. It is very interesting that cigarette smoking can induce changes in PCDH 1 expression but how the changes in PCDH 1 induce asthma is still not clear. PCDH 1 gene polymorphism also sometimes plays role in asthma and bronchial hyperresponsiveness (BHR) pathogenesis as well as in allergic dermatitis. Childhood asthma is one of the most common causes of emergency department visits, hospitalizations and absence in schools. As a disease with increasing incidence in children, asthma became an area to focus on recent clinical research. Asthma is a multifactorial disease including environmental and genetic factors. Bronchial hyperresponsiveness (BHR) develops from chronic pulmonary inflammation leading to recurrent episodes of wheezing, chest tightness, cough at night or in the early morning. The variable airflow in the lungs is reversible spontaneously or with treatment. Different respiratory phenotypic subtypes in children are associated with different risk factors. Some related genes had been discovered in recent years which play a crucial role in the pathophysiology of childhood asthma . ProtocaMost commonly children with asthma also suffer from other atopic diseases like atopic dermatitis which indicate the common environmental and genetic backgrounds shared by asthma and atopic disease. Genetic variation in epithelial cell barrier and repair function is the very significant factor to develop asthma and atopic dermatitis . PCDH 1 Protocadherin expression in the bodyProtocadherins are the largest cadherin subfamily of the cell adhesion molecules. The classification of protocadherin is shown in Figure Delta cadherins have a different structure than classic cadherins due to the presence of seven (Delta 1) and six (Delta 2) extracellular cadherin repeats and variable intracellular signaling domains. Clustered protocadherins express only in the nervous system while non-clustered delta 1 protocadherins express widely in different types of tissues. PCDH 7 is responsible for non-small cell lung cancer, PCDH 9 in autism spectrum disorder and PCDH 11 in Alzheimer’s disease. The recent study identified PCDH 1 as the susceptible gene for asthma. Delta 1 subfamily possesses three conserved motifs  in the intracellular cytoplasmic tail. CM 3, which plays role in lung morphogenesis, is exclusively present in only delta 1 protocadherin subfamily. PCDH 1 shows homotypic adhesion function in cells, but weaker than classic protocadherins. A study proved that PCDH 1 has higher expression in terminally differentiated bronchial cells than the undifferentiated cells which signify the potential role of PCDH 1 in the pathogenesis of asthma. Abnormal regulation of PCDH 1 expression leads to slower or incomplete differentiation of the epithelial layer, eventually causing the dysregulated response to injury and weaker epithelial barrier function . Kozu etPCDH 1 mRNA is expressed in brain, skin, lung, nose and also throughout all epithelial and endothelial lineages during embryonic mouse development. In a skin keratinocyte wounding model, PCDH 1 gene expression is upregulated, indicating the crucial role of PCDH 1 in epithelial barrier function , 5. BarrAetiologies of childhood asthmaAsthma is a genetically heterogeneous disease. Genome-wide association study (GWAS) recently identified a strong association between childhood asthma (wheezy phenotype) and a specific locus on chromosome 17q21 (which encodes ORMDL3 and GSDMB). Chromosome 17q21 is closely associated with the development of recurrent wheeze, asthma exacerbation, and bronchial hyperresponsiveness but no relation with eczema, rhinitis or allergic sensitization. As there was no evidence of atopy in the association of 17q21 and the respiratory symptoms, it can be accepted that 17q21 is an important risk factor for childhood non-atopic asthma. FCER1A is the gene which shows high affinity for the IgE receptors, may influence the serum IgE levels. But polymorphism in FcER1A gene changes serum IgE level in both asthmatic and non-asthmatic patients. Polymorphism in PCDH 1 gene and PDCD 4 gene is also the responsible factor for severe childhood and non-atopic asthma. Several other associated loci with asthma risk are DENND1B, IL1RL1, IL18R1, HLA DQ, IL33, IL2RB, and SMAD3. An SNP rs4950928, in the promoter region of CHI3L 1 expression in the airway epithelium, is also associated with childhood asthma .Another common risk factor for childhood asthma is maternal asthma and child’s sex. Maternal asthma and male child indicate the higher risk of developing childhood asthma while female gender and paternal asthma is significantly associated with adult-onset asthma. Maternal microchimerism  is one of the possible factors which provide protection against childhood asthma. Presence of maternal cells in the child is called maternal microchimerism. Chimeric cells have stem cell-like properties and they can be differentiated to perform immunological as well as multiple other functions .Some environmental factors also contribute to the development of childhood asthma such as air pollution, maternal smoking, diet and microbial exposure in early life like rhinovirus infection which causes decreased methylation in DNA at multiple CpG sites. Interestingly a study found a close connection between adverse childhood experience (ACE) and immunological dysregulation and childhood-onset asthma and other diseases. Even perinatal maternal stress is a causative factor for early-onset asthma in the offspring due to placenta-induced secretion of the corticotropin-releasing hormone, activating the fetal hypothalamic-pituitary-adrenocortical axis and eventually leading to dysregulation of glucocorticoid desensitization. This pathophysiology is also responsible for other atopic diseases like eczema, food allergies .A study showed that PCDH20 gene, which codes protocadherin-20 , has higher methylation rate in the sputum cells of asthmatic patients. To determine the epigenetic state of asthma, both environmental and genetic factors play a crucial role. It is also believed that intrauterine and early life exposure to a farm animal reduces the risk of allergic disorders and asthma in children in their later life. Gestational exposure to cigarette smoking causes differential methylation of cytochrome P450 aryl-hydrocarbon-hydroxylase (CYP1A1 ) gene and aryl hydrocarbon receptor repressor gene (AHRR). Early life DNA methylation causes developing cell memory to metabolize xenobiotics differently which make children vulnerable to have an increased risk of asthma in later life .Pathophysiology of asthmaThe common triggers of asthma are allergens or some foreign pathogens. Most initially, there is constriction of airway smooth muscles and inflammation in the airways . The airway epithelium is the first line defense line in-between the external environment and the submucosa. The conductive portion of airway epithelium is pseudostratified with ciliated columnar epithelial cells, goblet cells, intermediate columnar epithelial cells, populations cells, basal cells and serous cells Figure  10, 11,-16.Several structural and functional abnormalities are seen in asthmatic airway epithelium-like enormous resident stem cells and basal cells, hyperplasia of goblet cells and excessive mucin production along with the reduced number of ciliated cells which indicate dysregulation of cell differentiation. Changes in epithelium increase the risk of oxidative stress, release of cytokines and extracellular matrix, synchronization of mitosis and deficiency of innate immunity which subsequently cause the abnormalities in epithelial barrier function and repair process , 14-16.PCDH 1 gene in asthma and eczemaGenetic variants in the encoding gene of PCDH 1 have been identified to be associated with asthma, eczema, and wheeze in the early childhood. Passive smoking in early childhood is responsible for gene-environment reaction causing the change in PCDH 1 gene to develop asthma. Genetic association studies showed that PCDH 1 gene is associated with BHR, eczema, nonatopic childhood asthma, and transient early wheeze. PCDH 1 dysfunction induces the defect in epithelial barrier function which is significant in asthma and eczema .In a Dutch study, there was an observed association between PCDH 1 polymorphism IVS3-116 and eczema BHR and asthma . Strong Though childhood asthma is a common disease in a pediatric population, very few studies are available to provide in-depth information on genetic association with it. The role of PCDH 1 is explained by several studies. But still, more human studies with large sample size should be done to know about the accurate role of PCDH 1 in asthma and other childhood atopic phenotypes. Detection of PCDH 1 role in the pathophysiology of childhood asthma will not only help to reveal the accurate prevention strategy of childhood asthma but it will also help to do further research for an invention of molecular-targeted therapy. There is a big question how cigarette smoke can induce changes in PCDH 1 and whether other environmental pollution like engine smoke, dust, pollen exposure also can induce a change in PCDH 1 expression in the airway epithelium.


Hyperhidrosis is estimated to affect ~ 4.8% of the US population, and most patients experience a negative psychological impact. Here, we describe development and psychometric evaluation of a patient-reported outcome (PRO) measure to assess severity of axillary hyperhidrosis in clinical trials that meets current U.S. regulatory standards to support product approvals.Three rounds of hybrid concept-elicitation/cognitive-debriefing qualitative interviews were conducted in adults with clinician-diagnosed primary axillary hyperhidrosis, followed by similar interviews in children/adolescents. The draft measure included diary items for presence, severity, impact and bothersomeness (basis of the Axillary Sweating Daily Diary [ASDD]), exploratory weekly impact items, and a single-item Patient Global Impression of Change (PGIC). Phase 2 (adults only) and phase 3 (adults and children ≥9 years) clinical trial data were utilized to evaluate measurement properties of the resulting draft measure: floor/ceiling effects, nonresponse bias, test-retest reliability, construct validity, and responsiveness were assessed. The primary concept of interest was axillary sweating severity (ASDD Item 2); however, additional supportive concepts were explored to allow for development of a comprehensive hyperhidrosis measure.N = 21 adult and N = 8 children/adolescents), resulting in the ASDD  and child-specific ASDD-C (2 items ≥9y to <16y), as well as 6 Weekly Impact items and the PGIC (patients ≥16y). No floor/ceiling effects or response biases were identified. Consistency between hypothesized and observed correlation patterns between ASDD/ASDD-C items and other efficacy measures supported construct validity. Intraclass correlation coefficients supported test-retest reliability . Large effect sizes (− 2.2 to − 2.4) demonstrated that the ASDD/ASDD-C Item 2 could detect changes in hyperhidrosis severity, supporting the measure’s responsiveness. Patients perceiving a moderate improvement in symptoms on the PGIC experienced an average 3.8-point improvement on ASDD axillary sweating severity (Item 2); thus, a 4-point responder threshold was defined as a clinically meaningful change.Twenty-nine patient interviews were conducted (Qualitative and quantitative evidence support the reliability and validity of the ASDD/ASDD-C and its use in the clinical evaluation of axillary hyperhidrosis treatments. Further evaluation of this measure in future research studies is warranted to demonstrate consistent performance across different axillary hyperhidrosis populations and in different study contexts. Hyperhidrosis (HH) is a condition in which sweat production exceeds that which is physiologically necessary to maintain thermal homeostasis . This buA number of tools have been utilized to measure patient-reported severity, impact, and bother associated with hyperhidrosis . GenericAlthough the instruments described above have been used to better characterize patients with hyperhidrosis, it is unclear whether any were developed to support product approvals in accordance with most recent U.S. regulatory standards for PRO measures  in a clinical trial) . This prHere, we describe the development, features, and validation of the Axillary Sweating Daily Diary (ASDD) and Axillary Sweating Daily Diary-Children (ASDD-C), with a focus on the psychometric evaluation of axillary sweating severity item (Item 2).N = 21) in 3 different geographic locations between January 2014 and February 2014. Interview results were qualitatively analyzed in three sets, where each set of interviews built upon the findings of previous interviews to confirm the adequacy of modifications and allow testing of any new items. Consistent with the inclusion criteria for the initial dose-finding trials, all participants were required to exhibit hyperhidrosis symptomology for at least 6 months, as well as report an HDSS score of 3 or 4, indicating “barely tolerable” or “intolerable” sweating and sweating “frequently” or “always” interfering with daily activities. Preliminary concepts were selected for inclusion in a draft item pool based on clinical expertise, review of the literature, and review of other questionnaires . Given that the primary concept of measurement, sweating severity, was both straightforward and clearly important within the context of hyperhidrosis, draft items addressing this concept were developed prior to patient engagement and were comprised of daily diary items covering concepts pertaining to presence, severity, impact and bother of axillary hyperhidrosis (ultimately referred to as the ASDD). Additional supportive concepts were selected for inclusion in a preliminary item pool to further explore the impact of axillary hyperhidrosis on various areas of patients’ lives (ultimately referred to as the Weekly Impact Items). A Patient Global Impression of Change (PGIC) item was also included to assess patient-perceived changes in underarm sweating and facilitate the interpretation of change scores on the diary items. Multiple questions were drafted for each concept so variable wording and response scales could be tested during patient interviews. All interviews were conducted by two experienced qualitative researchers according to a semi-structured interview guide created collaboratively by the developers. Each interview began with open-ended concept elicitation. Participants were asked to describe their experiences with axillary sweating, including variations in severity, the extent and situations in which they were bothered by their sweating, and how their lives were impacted by hyperhidrosis. Following the concept elicitation phase, the same patients were then asked to provide feedback on the draft items via paper forms during a cognitive debriefing phase to support the salience and importance of the concepts, as well as to optimize item wording and response options. Specifically, participants were asked to respond to each item while thinking aloud. Interviews were reviewed individually and compared to other interviews in order to summarize and identify participant patterns in item interpretation and response. An item tracking matrix was created, which included any revisions made to an item and a rationale for the item modification. Additional follow-up questions were also posed by the interviewers to further elucidate comprehension and response processes. If concept saturation was not reached following qualitative data analysis based on the findings during each set of interviews, additional interviews were to be conducted with new patients.A brief overview of the instrument development process is detailed in Fig. N = 8) to evaluate whether the new measure would be relevant for children ≥9 years of age. While the sample size was deemed adequate to achieve concept saturation, if new concepts had been generated during these interviews, testing with additional pediatric patients would have been conducted.Following the decision to include individuals as young as 9 years of age in a future clinical trial program, a similar set of qualitative interviews was conducted with children and adolescents < 18 years with self-reported excessive sweating (N = 102) [To further support use of the ASDD/ASDD-C axillary sweating severity item (Item 2) in clinical trials evaluating the efficacy of new treatments for hyperhidrosis, the measurement properties of ASDD Item 2 were evaluated using data from a 4-week phase 2 trial of glycopyrronium tosylate (GT) in patients ≥18 years of age  . Phase 2N = 344; sites in the U.S. and Germany) and ATMOS-2  [Additional psychometric evaluation of ASDD/ASDD-C Item 2 was subsequently conducted based on pooled data from two, 4-week phase 3 trials of GT, ATMOS-1  . Study des only) . Eligibl. These 3 measures  are collectively referred to in Table PRO measures were completed by patients using an electronic tablet device provided to them during the study period as follows: for patients ≥16 years of age, the ASDD was completed daily (before bed) for 4 to 7 days during screening (7 to 10 days prior to randomization) and during treatment; in the phase 3 studies, patients < 16 years completed the ASDD-C items (Items 1 and 2 only) according to the same schedule (phase 3 only). Patients ≥16 years of age additionally completed two other measures developed alongside the ASDD, namely the Weekly Impact items  and used in exploratory analyses. All statistical tests were two-tailed using a type I error rate of 5% . Rather than relying on statistical significance alone, the interpretation of effect size estimates and patterns of results were emphasized. Observed values were used for all calculations for the phase 2 study. For ASDD Item 2 in the phase 3 studies, the weekly mean from Markov Chain Monte Carlo (MCMC) imputation was used; observed values were used for ASDD Items 3 & 4 as well as the PGIC. For binary response data , missing values were considered a “No” response.Potential floor and ceiling effects , were evaluated based on responses during screening (Baseline) using both summary statistics and graphical techniques. Test-retest reliability for Item 2, 3, and 4 was evaluated to assess each item’s stability through the computation of intraclass correlation coefficients (ICCs) between Week 3 and Week 4, using a threshold of ≥0.70 . Week 3 Construct validity was evaluated at Week 4 based on correlations between ASDD/ASDD-C Item 2 gravimetric measurements and established PRO measures (HDSS and DLQI). Week 4 correlations were also explored between ASDD/ASDD-C Item 2 and ASDD Items 3 and 4, the Weekly Impact items, and the PGIC . CorrelaKnown-groups validity  was evaluated at Week 4 based on two-sample t-tests comparing differences in scores on ASDD/ASDD-C Item 2 between subgroups predefined based on HDSS grades (score of 1 vs score of 3 or 4), DLQI score ranges  , 27, andResponsiveness was evaluated through effect size computation between Baseline and Week 4 to evaluate the ability of the ASDD/ASDD-C items to detect change. A responder definition to establish a threshold for a magnitude of within-person change considered to be clinically meaningful was determined based on methods outlined in the FDA PRO guidance , which rThe cohort of adult interviewees was predominantly white (76%) and female (76%) Table . In geneMinor modifications to item wording were incorporated as needed following each round of interviews to ensure content was clear, comprehensive, and easy to understand. Concepts pertaining to underarm odor severity and interference with sleep were not generally endorsed by patients and deleted, as patients indicated that it was the anxiety about having odor that was most impactful and not necessarily the odor itself, and that hyperhidrosis symptoms typically occurred during the day and not while sleeping. The PGIC was also tested, with no revisions made in all three rounds of interviews as patients found the item clear and easy to answer in the context of receiving hyperhidrosis treatments. No new concepts were introduced as part of the concept elicitation portion of the interviews, and concept saturation was reached across the three sets of qualitative interviews. Thus, no additional patient interviews were required for further input.The level of bother was consistent across both adult and pediatric samples. The most commonly reported impacts of hyperhidrosis in both populations included feeling embarrassed/self-conscious, modifying types of clothes worn, concern about underarm odor, changing shirts throughout the day, anxiety about sweating, ruined clothes, and physically uncomfortable due to excessive underarm wetness. Adults reported work-related impairments such as limiting interactions with work colleagues and nonmandatory work activities, consistent with the school-related impairments such as avoiding school-based activities and difficulty studying reported by the pediatric sample.N = 8) reported being bothered or burdened by the following: feeling embarrassed; sweating a lot more than others in similar circumstances; feeling sweat dripping down sides of the body; needing to cover up their sweating with hair or jackets/sweatshirts, even in hot weather; needing to “air their armpits out”; feeling like they have a bad smell; feeling sticky; not liking the wet feeling; and feeling itchy. Of note, adults did not report feeling itchy in conjunction with their excessive sweating. The three 17-year-old participants reported that their excessive underarm sweating began between during 6th or 8th grade. Three other participants  reported that their excessive sweating began around 4th grade and was most noticeable during sports or other physical activities. The remaining participants (aged 10 and 11 years) did not know when their excessive sweating began, stating that it was likely around age 4 to 5 years.Like the adult interviewees, pediatric interviewees (N = 3) found the instructions and recall period on the ASDD clear and easy to understand. In contrast, 4 of the 5 younger participants (aged 10 to 13 years) had difficulty reading and understanding the instructions and/or understanding the 24-h recall period. Based on these findings, the instructions and recall periods used in the ASDD-C were modified for patients < 16 years of age, as reflected in Table All 17-year-olds  and impact (Item 3) items; there was some ceiling effect 21.4%) for the bothersomeness item  were observed between ASDD Item 2 and HDSS grade at Week 4, as well as between the changes in both measures from Baseline. As expected, ASDD Item 2 was moderately to strongly correlated with the DLQI/C-DLQI (r = 0.61 /0.51 at Week 4 and r = 0.63/0.51 between changes in both measures from Baseline). Correlations between ASDD Item 2 and sweat production were weak to moderate at Week 4 (r = 0.17 to 0.33) and were weak for the changes in both measures from Baseline (r = 0.17 to 0.18), which was expected given the differences in reference period for these measures . Similar trends were noted for both the adult and pediatric populations in the phase 3 study. Exploratory analyses to evaluate the relationship between all newly developed items  showed strong relationships across the different measures. ASDD inter-item correlations were all > 0.80. While severity, impact, and bothersomeness in the ASDD may represent different constructs of hyperhidrosis, they are likely to be strongly associated with one another. Furthermore, ASDD Item 2 was strongly correlated with the Weekly Impact summary. In addition, the correlation between the change from Baseline in ASDD Item 2 was strongly correlated with change from Baseline in the Weekly Impact summary and the PGIC at Week 4 , gravimetrically-measured sweat production quartile  at Week 4, and DLQI (p < 0.0001) (Table n = 32). Only 1 pediatric patient reported a very large to extremely large HRQOL impact due their skin condition on the C-DLQI; this patient reported a mean weekly ASDD score of 9.8 compared to 4.4 in the group with minimal HRQOL impact. Although no specific predictions were preplanned for ASDD Items 3 (impact) and 4 (bother), data in Table Statistically significant differences in mean ASDD Item 2 scores and HDSS grade strata (1) Table  support The responsiveness of ASDD/ASDD-C Item 2 (ability to detect change in sweating severity) was demonstrated by large effect sizes (ranging from − 2.2 to − 2.4), as well as by correlations that were within the expected range for the change in Item 2 and the change in the gravimetric measures of sweat production Table .A PGIC rating of “moderately better” within this scale corresponded to a 3.8-point change on the ASDD Item 2 in both phase 2 and phase 3 studies Table . A “modeThe purpose of this research was to develop the ASDD and ASDD-C and demonstrate validity of Item 2, intended to measure severity of axillary sweating among patients with axillary hyperhidrosis in both adults and pediatric patients aged 9 and above. Qualitative and quantitative analyses presented here demonstrate strong relationships with other established outcomes in hyperhidrosis , good reproducibility (reliability), and establishment of a threshold indicating a meaningful magnitude of response via anchor-based methods (responsiveness) for use in evaluating axillary hyperhidrosis treatments in interventional studies. Validation analyses were highly consistent between the phase 2 and phase 3 studies, providing increased confidence in ASDD measurement properties and particularly the responder definition. This research highlights the importance of capturing the patient perspective for this condition, particularly in light of the limitations associated with more objective methods of assessing sweat production.Given the level of unmet need among hyperhidrosis patients, novel assessment tools are needed to support future research and the development of new treatments. Although the HDSS has historically been used in the clinical trial setting to characterize patients with hyperhidrosis, it cannot be used in support of product approvals because patient input was not formally taken into consideration during its development. In addition, the HDSS combines two concepts within the same item (symptom severity and impact), limiting the ability to ascertain which of the two concepts has changed with treatment. The ASDD was developed in accordance with the recommendations outlined in the FDA PRO guidance to support U.S. product approvals . This inSeveral study limitations should be recognized. Foremost, the development of the ASDD/ASDD-C was based on a small, homogeneous patient population, which may not have reflected different levels of disease severity; additionally, the pediatric patients did not have a physician-confirmed diagnosis of hyperhidrosis for the qualitative development. Patients included in the phase 2 and phase 3 trials were also relatively homogeneous , potentially limiting generalizability of the measure. In addition, though the axillary sweating severity item is equivalent between the ASDD and ASDD-C, the cohort of patients ≥9 and < 16 years in the phase 3 trials was small. Despite this limitation, ASDD-C data in the pediatric population demonstrate results broadly consistent with those of adults. The focus of this research was primarily on the ASDD Item 2; while exploratory analyses showed that all the newly developed items demonstrated consistency with one another, dimensionality was not formally assessed to better understand their relationship with one another and whether it would be appropriate to derive summary scores. It also should be noted that initial draft items pertaining to severity were developed before patient engagement, which as a matter of process may be disadvantageous when seeking to identify constructs directly germane to a patient; however, for this hyperhidrosis measure, the concept of sweating severity was the focus of the study (Item 2) and was considered to be both straightforward and unambiguously relevant.Taken together, the rigorous development and validation of ASDD/ASDD-C axillary sweating severity item support its use in the evaluation of axillary hyperhidrosis treatment in clinical trials. Strong qualitative and quantitative evidence also supports the use of the impact and bothersomeness items as key trial endpoints in adult trials. Further evaluation of this measure in future research studies is warranted to demonstrate consistent performance across different axillary hyperhidrosis populations and in different study contexts.Familiarity with the ASDD/ASDD-C along with the Weekly Impact items among clinicians who treat patients with hyperhidrosis may facilitate a better understanding of the impact of this condition on patients’ lives.