Sexual dysfunction is a private set of disorders that may cause stigma for patients when discussing their private problems with doctors. They might also feel reluctant to initiate a face-to-face consultation. Internet searches are gradually becoming the first choice for people with sexual dysfunction to obtain health information. Globally, Wikipedia is the most popular and consulted validated encyclopedia website in the English-speaking world. Baidu Encyclopedia is becoming the dominant source in Chinese-speaking regions; however, the objectivity and readability of the content are yet to be evaluated.Hence, we aimed to evaluate the reliability, readability, and objectivity of male sexual dysfunction content on Wikipedia and Baidu Encyclopedia.The Chinese Baidu Encyclopedia and English Wikipedia were investigated. All possible synonymous and derivative keywords for the most common male sexual dysfunction, erectile dysfunction, premature ejaculation, and their most common complication, chronic prostatitis/chronic pelvic pain syndrome, were screened. Two doctors evaluated the articles on Chinese Baidu Encyclopedia and English Wikipedia. The Journal of the American Medical Association (JAMA) scoring system, DISCERN instrument, and Global Quality Score (GQS) were used to assess the quality of disease-related articles.P=.002) and JAMA scores (P=.001) for Wikipedia were significantly higher than those of Baidu Encyclopedia; there was no statistical difference between the GQS scores (P=.31) for these websites. Specifically, the DISCERN Section 1 score (P<.001) for Wikipedia was significantly higher than that of Baidu Encyclopedia, while the differences between the DISCERN Section 2 and 3 scores  were minor. Furthermore, Wikipedia had a higher proportion of high total DISCERN scores (P<.001) and DISCERN Section 1 scores (P<.001) than Baidu Encyclopedia. Baidu Encyclopedia and Wikipedia both had low DISCERN Section 2 and 3 scores , and most of these scores were low quality.The total DISCERN scores (Wikipedia provides more reliable, higher quality, and more objective information than Baidu Encyclopedia. Yet, there are opportunities for both platforms to vastly improve their content quality. Moreover, both sites had similar poor quality content on treatment options. Joint efforts of physicians, physician associations, medical institutions, and internet platforms are needed to provide reliable, readable, and objective knowledge about diseases. Knowledge regarding health and well-being is cobbled together from health care professionals, family, friends, books, newspapers, magazines, educational pamphlets, radio, television, and pharmaceutical advertisements . HoweverAs the most common male sexual dysfunctions, erectile dysfunction  and premature ejaculation  greatly affect the quality of life of patients ,10. FurtWikipedia, the most popular and consulted encyclopedia website in English, is a web-based encyclopedia that provides valuable web-based health information . PreviouThe contents analyzed in this study are available on Chinese Baidu Encyclopedia and English Wikipedia. The Chinese Baidu Encyclopedia and English Wikipedia were investigated for articles on ICD-10 version 2016 codes. All possible synonymous and derivative keywords for each term were screened. Two doctors evaluated the articles on Chinese Baidu Encyclopedia and English Wikipedia. Any disagreement was reviewed by and arbitrated by a third reviewer who was an expert on sexual dysfunction. All authors have many years of experience in andrology and urology and are competent in the diagnosis and treatment of male sexual dysfunction and urinary disorders. These reviewers have professional knowledge of male sexual dysfunction and urinary disorders and can make professional evaluations.The Journal of the American Medical Association (JAMA) scoring system , DISCERNU test was conducted to test the significance of different ranks by using SPSS, version 22.0 . The Fisher exact test was used to test the difference in the frequency distribution of DISCERN scores. We used Prism 8 for macOS, version 8.4.0  to conduct statistical analyses and create figures. For the statistical analysis, P<.05 was considered significant.All databases were constructed with Excel 2019 . The Shapiro-Wilk test was used to test the normality of the data. Descriptive analyses are reported as means and SDs for normally distributed variables. Medians and IQRs are reported for non-normally distributed variables. To ensure the quality of these scores, the intraclass correlation coefficient (ICC) was used to evaluate interobserver reliability. ICC values range from 0 (untrusted) to 1 (fully trusted), and any concordance values less than 0.75 were discussed by the research team to clarify the discrepancy. For nonparametric tests, the Mann-Whitney We searched for “erectile dysfunction,” “premature ejaculation,” “chronic prostatitis/chronic pelvic pain syndrome,” and similar keywords on English Wikipedia and Chinese Baidu Encyclopedia. The search results are shown in A 2-way mixed/random effects model was used to analyze the consistency of the ratings by the 2 independent reviewers. The ICC results showed good consistency between the 2 reviewers for the GQS scores (ICC=0.87), JAMA scores (ICC=0.91), and DISCERN scores (ICC=0.82).Comprehensively, the scores for Wikipedia were higher than those for Baidu Encyclopedia A. The coED is one of the most common male sexual dysfunctions. By comparing the content scores for ED articles on Baidu Encyclopedia and Wikipedia, Wikipedia appeared to have numerically higher total DISCERN scores, JAMA scores, and GQS scores, but there were no statistically significant differences A. FurtheA comparison of the scores for PE, the other most common sexual dysfunction disorder, showed that Wikipedia had a significantly higher total DISCERN score than Baidu Encyclopedia A. AlthouCP/CPPS, as one of the most common concomitant diseases of sexual dysfunction, seriously affects the quality of life of male patients. By comparing the overall scores for Wikipedia and Baidu encyclopedia on CP/CPPS, we found that the scores of Baidu Encyclopedia were mostly fair quality, while the scores of Wikipedia ranged from fair quality to good quality A. MeanwhAfter comparing the overall quality of the information for different diseases on Baidu encyclopedia and Wikipedia, the overall scores for Wikipedia seemed to be higher than those of Baidu encyclopedia, but some scores only showed numerical differences without statistical significance. Nevertheless, the differences in the distribution of scores that had numerical differences were seemingly obvious. Therefore, we performed further statistical analyses of the score distributions for Wikipedia and Baidu Encyclopedia. As aforementioned, according to the DISCERN standard, a total DISCERN score <50 (near 60%) is fair or poor quality, while a score >50 is good or excellent quality . Based oThe score distributions for each disease are shown in Internet-based information is playing an increasingly important role in the diagnosis and treatment of patients, especially for privacy-sensitive conditions such as sexual dysfunction and related concomitant diseases. Comprehensive and objective information can help patients understand their condition, choose the right time to visit a doctor, and then improve their prognosis. However, incorrect or incomplete information may leave patients vulnerable to misdiagnosis, leading to delays in treatment and considerable health risks . As a coBy June 2021, the number of online medical users in China was 239.33 million, and the utilization rate of the internet was 23.7%, an increase of 11.4% over December 2020 . In an aCP/CPPS is characterized by localized pain or discomfort in the abdomen, pelvis, and genitals, usually with lower urinary tract symptoms, psychosocial disorders, and sexual dysfunction ,12. The By comparing the contents for ED, PE, and CP/CPPS on Baidu Encyclopedia and Wikipedia, we found that the consistency of Wikipedia is better, with almost all content rated as “good quality,” while the scores for Baidu Encyclopedia were mostly “fair quality.” Take PE-related articles in Baidu Encyclopedia as examples. Both reviewers rated “早发性射精” (early-onset ejaculation) as “poor quality.” After analyzing the content on the web page for “early-onset ejaculation,” we found there was no introduction to “examination, diagnosis, and treatment,” and the content in the article was not objective and scientific. Contrary to the lack of effective information, there are more than 25 irrelevant advertising links and only one reference on this web page. The content on the “早泄” (premature ejaculation) page on Baidu Encyclopedia was rated as “good quality,” and the information was more comprehensive and objective than that for “early-onset ejaculation.” Corresponding to the quality grades for “early-onset ejaculation” and “premature ejaculation,” there was a huge difference in page views . The discrepancy may be related to the inconsistent identity of content providers. The irrelevant advertising links or misleading information obtained by users using Baidu Encyclopedia may be related to the fee-based editing service. There are many third-party underground industries that charge fees to write Baidu Encyclopedia entries on their behalf, so as to insert advertisements and achieve the purpose of attracting patients. In order to improve the quality of the health information, Baidu Encyclopedia announced the “rainbow plan” on December 9, 2012, wherein all medical entries could only be edited and revised by certified medical experts . This isIn contrast, Wikipedia has its own content quality evaluation system, such as the “Wiki-Project article quality grading scheme” and the “Wiki-Project priority assessments” ,39. In tIn the era of rapid internet development, more patients have started to try online consultations . This chSome limitations must be addressed in this study. This study only presents the results of medical professionals’ evaluations of health-related science content on the internet, and further research is needed on the specific impact of this information on the audience and readers. Since information on the internet is updated quickly, there may be some bias between the study results and the actual situation, and the data need to be updated in real time to ensure that the findings are true and valid. In addition, the difference in the number of Chinese and English entries indicates the information received by users will be significantly different because of the entries they choose to click. Therefore, our “combined” evaluation cannot fully represent the quality of the information they really receive. Fortunately, with the availability of infodemiology research, academics can combine content analysis and infodemiology search trends to better elucidate the impact of health-related information on the internet on users, society, and the health care industry.Internet medicine, as a new medical model in the new era, provides strong support for users to understand disease information and choose the timing of treatment in a timely manner. Although it is more formally composited, Wikipedia also provides more reliable, higher quality, and more objective information than Baidu Encyclopedia. They also have a similar impact on patients’ choice of treatment options, and the websites are similar in terms of flow and ease of use. To promote the healthy and sustainable development of internet health care, the joint efforts of physicians, physician associations, medical institutions, and internet platforms are needed to provide more reliable, accessible, and comprehensible disease knowledge to the public.


In this paper, we consider the problem of unsupervised domain adaptation for corner detection in plate images. We trained a model with plate images of one country, the source domain, and applied a domain adaptation scheme so that the model is able to work well on the plates of a different country, the target domain. For this study, we created a dataset of 22,096 Korea plate images with corner labels, which are source domain, and 6762 Philippines, which are target domain. To address this problem, we propose a heatmap-based corner-detection model, which outperforms existing scalar-regression methods, and an image classifier for mixed image of source and target images for domain adaptation. The proposed approach achieves better accuracy, which is 19.1% improvement if compared with baseline discriminator-based domain adaptation scheme.Rectification of vehicle plate images helps to improve the accuracy of license-plate recognition (LPR). It is a perspective-transformation process to project images as if taken from the front geometrically. To obtain the projection matrix, we require the ( License-plate recognition (LPR), in general, consists of two steps ,2. In stThe rectification can be easily achieved by perspective transformation . It is tFor example, we are given imageThis work proposes a novel deep model that locates four corners In this work, another challenge is that we do not assume that training and test data are collected from an identical distribution. The training data are Korean plates, and the test data are Philippines plates. Such a distribution discrepancy has been observed to cause a performance drop in various works. The Korean and Philippines plates are different from each other in regard to shapes, sizes, and characters. Hence, a corner-detection model trained with the Korean plates would not work well with the Philippines. Thus, it is desirable to develop methods that can adapt corner-detection models to unseen domains, which are visually different from the training data. We address this cross-domain corner-detection problem by developing algorithms to transfer knowledge from a labeled source domain of Korean plate images to an unlabeled Philippines.Domain adaptation techniques  aim at rThe contribution of this work can be summarized as follows.We proposed a corner-detection model which outputs a likelihood heatmap. It outperforms existing models based on scalar regression. In addition, we created a new dataset consisting of 22,096 KR plate images and 6762 PH plate images.We presented a novel domain adaptation based on an adversarial discriminative method. It is peculiar in the sense that the discriminator is required to distinguish how Korean plates and target Philippines plates are mixed.We conducted experiments on various corner-detection tasks and validated that our method can bring performance gains by 19.1% if compared with baseline discriminator-based domain adaptation method.This paper is organized as follows: Corner Detection: Finding the four corner positions of vehicle plates for the rectification purpose can be considered as a keypoint detection problem. CNN-based methods [ methods ,7 use fe methods ,9 tried Recently, Vision Transformer (ViT)  is propoKeypoint Detection: 2D keypoint detection is actively studied for its wide applicability in computer vision tasks. Most of the works are based on heatmap-output networks. A multi-resolution framework was proposed that generates heatmaps representing per-pixel likelihood for keypoints [eypoints . Hourglaeypoints . The heaeypoints  was utilDomain Adaptation and Keypoint Detection: When domain shift occurs, most deep models experience performance deterioration because of unseen data. One of active works to deal with such degradation is domain adaptation. It has been actively studied in computer vision. Earlier works have mostly focused on image classification. Recent works have widened its application areas, aiming to improve the domain adaptability of deep neural networks, including References [ferences ,18,19,20Some previous works have investigated domain adaptation in keypoint detection. Most works are related with 3D keypoints detection. A weakly supervised method using depth images was proposed in Reference , domain An overview of our network is shown in Corner-Detection Model: The corner-detection model extracts a set of likelihood heatmaps about the corner positions from input images. The feature extractor is the backbone of ResNet18 pretrained on ImageNet, followed by the up-sampling component using deconvolution layers. The head predicts four corner locations, namely i-th training image, with 2D Gaussian blob centered on the ground truth coordinate of Let us denote Domain Classifier: The domain classifier in our network is the domain adaptation component to align the feature representation distributions on image level. The image-level representation refers to the feature map from the feature extractor. To reduce the difference between the domain distributions on the image level, the classifier consists of repetitions of a sequence of a convolutional layer, batch normalization, and ReLU activation, followed by an adaptive average pooling and a fully connected layer.i-th training image, For the classifier training, images with Korean and Philippines plates cut in half and put together side-by-side are used. The classifier predicts the cut-and-paste label for the images. The output dimension of the classifier, The benefits of this image mix-up are twofold: (1) the mix-up is generally harder dataset. Thus, the domain adaptation phase leads to more robust results. The performance comparison results when using source and target images separately proves this claim in the following section, and (2) the feature extractor is guided to eliminate the character-specific features in the feature maps, which helps to maximize the classification loss in the adversarial training phase. As a result, the feature maps are more likely to contain more plate-shape-related information.By denoting the output of the classifier as To align the domain distributions, we should simultaneously optimize the parameters of the classifier to minimize the er (GRL) , whereasThe overall network of Experiment Setup: The dataset consists of two domains: Korean plate images with a total of 22,096 and Philippines with a total of 6762 images. Following the common terminology in domain adaptation, we refer to the Korean images as source domain, denoted by KR, and to the Philippines images as target domain, PH. All of the images have the same dimension of We construct the ground truth heatmaps from the labels, which have the normalized coordinates of the four corner positions. The heatmaps have 2D Gaussian blobs centered at the corner locations. We determine the blob size by standard deviation, −3. We adopt mini-batch SGD with a momentum of 0.9 and a batch size of 64. The learning rate is adjusted by We finetune ResNet18 of the feature extractor pretrained on ImageNet. The up-sampler and the heatmap head are trained from scratch, with an initial learning rate of 1 × 10Four models are involved in the experiments, as listed in Regarding training data, Conv. and MNet. use only the source images and corresponding labels because the target images have no label information that is required for supervised training. For Classic-DA and Proposed-DA training, both the source and target images are used. Moreover, we train Proposed-DA with only source images and only target images, denoted by Source-Only, and Target-Only, respectively. These are used as baselines to assess the effectiveness of the proposed method.As evaluation metric, we measure error distances between predicted corner positions and the ground truth are measured in pixel units. The errors for the four corner positions, Left-Top, Right-Top, Left-Bottom, and Right-Bottom, respectively, are measured separately. We run two experiments, one using source KR as the test dataset, and the other with target PH as the test dataset. Experimental Results: Observing the results of the heatmap based Source-Only, Classic-DA, and Proposed-DA, we find that they show similar errors. Recall that the training of Source-Only is different from Classic-DA and Proposed-DA; its training uses only the source KR dataset, while two models use both KR and PH dataset. This finding verifies that the target images included in training have little influence on the proposed domain adaptive model. The model is able to maintain accuracy about the source domain, even though it is trained to prepare for domain adaptation. Interestingly, Source-Only slightly outperforms the other two methods. It is obvious because the models trained and tested with same domain dataset perform best.The test results for the target PH dataset are summarized in These experiment results for the source and target dataset highlight that the domain adaptation methods generalize better than the Source-Only model. The proposed method of Proposed-DA is in general more robust to domain shift than the Classic-DA. This suggests that the classifier for mixed images was more effective for decreasing the domain gap between the two distributions than the image discriminator. It is worth noting that the proposed method exhibits a drastic performance improvement of 19.1% on average if compared with the results of the Class-DA.Experiments with deeper backbone networks: We also evaluated with deeper backbones such as ResNet34, ResNet50, etc., to assess its effect on the performance. In the current architecture, ResNet18 is adopted as the backbone. As mentioned in For comparison among deeper backbones, we tested with ResNet34, ResNet50, ResNet101, and ResNet152, along with the original backbone of ResNet18. We prepared a set of Proposed-DA models, each with those different backbone networks. The models were trained with the same dataset and parameters until the loss decrease stabilizes. The evaluation used the same test datasets: source KR test dataset and target PH dataset. We used two comparison metrics: the corner errors and the inference time.As shown in We considered the problem of unsupervised domain adaptation for plate corner detection. To conduct our study, we prepared a new dataset consisting of 22,096 KR plate images and 6762 PH plate images, which we can provide under certain agreement. Our main contributions are twofold; we proposed a heatmap-based corner-detection model, which is better than the existing scalar-regression-based methods, and the domain adaptation using mixed image classifier, an effective approach for cross-domain object detection. Our approach is validated on experiments, and our method outperforms the baseline discriminator-based domain adaptation scheme with 19.1% improved accuracy, as well as existing corner-detection methods, thus demonstrating its effective for corner detection in a domain-shift environment.Although our method can achieve compelling results in the target PH dataset, the results are far from generalization. It may fail when testing with other license plates of different countries. Future work may extend the proposed domain adaptation scheme to become more general, and to other application scenarios beyond license plates and will explore the possibility of combining rotation and flip techniques in adaptation steps to improve performance on the current test dataset. We hope that the proposed dataset will motivate research on this topic and our domain adaptation scheme will serve as a strong baseline for future works.


Radar (SAR) satellites systematically acquire imagery that can be used for volcano monitoring, characterising magmatic systems and potentially forecasting eruptions on a global scale. However, exploiting the large dataset is limited by the need for manual inspection, meaning timely dissemination of information is challenging. Here we automatically process ~ 600,000 images of > 1000 volcanoes acquired by the Sentinel-1 satellite in a 5-year period (2015–2020) and use the dataset to demonstrate the applicability and limitations of machine learning for detecting deformation signals. Of the 16 volcanoes flagged most often, 5 experienced eruptions, 6 showed slow deformation, 2 had non-volcanic deformation and 3 had atmospheric artefacts. The detection threshold for the whole dataset is 5.9 cm, equivalent to a rate of 1.2 cm/year over the 5-year study period. We then use the large testing dataset to explore the effects of atmospheric conditions, land cover and signal characteristics on detectability and find that the performance of the machine learning algorithm is primarily limited by the quality of the available data, with poor coherence and slow signals being particularly challenging. The expanding dataset of systematically acquired, processed and flagged images will enable the quantitative analysis of volcanic monitoring signals on an unprecedented scale, but tailored processing will be needed for routine monitoring applications.The online version contains supplementary material available at 10.1007/s00445-022-01608-x. Globally, over 800 million people live within 100 km of a volcano and the number of fatalities is increasing with time  have demonstrated the potential of machine learning algorithms for detecting volcanic deformation in large InSAR datasets . Sentinel-1 data is taken directly from the Centre for Environmental Data Analysis (CEDA) Sentinel Mirror Archive (SMA) and processed using the COMET-LICSAR automated processing system which is based on the GAMMA SAR processing software package and runs on the UK Centre for Environmental Data Analysis infrastructure JASMIN  produces maps (‘interferograms’) of surface displacement in the satellite line of sight (los) by comparing the phase of the backscattered signal in successive SAR images. Here we use the COMET-LICSAR archive of processed Sentinel-1 radar images spanning November 2015–November 2020 , in particular, deep convolution neural networks (CNNs), is becoming increasingly popular for image classification tasks as they have proven to be efficient and are easy to adapt to many applications. However, to be successful, CNN models require a large training set of labelled data, balanced between classes. We apply the convolution neural network (CNN) proposed by Anantrasirichai et al. , which wFor this study, we use the 2-class model of Anantrasirichai et al. , which iFinally, we use a range of tools to inspect the flagged interferograms and categorise the signals according to the processes responsible. This allows us to identify false positives and investigate the range of mechanisms represented and the associated detection thresholds.We class the volcanoes as ‘persistent detections’ if they have either > 25 detections or > 10% of detections per total number of interferograms, and ‘occasional detections’ if 2–25 inteferograms were flagged. Since the LiCSAR system automatically connects each SAR acquisition to at least three successive acquisitions in time, any rapid transient deformation should appear in at least 6 interferograms. Here we use a conservative threshold of two detections to allow for incoherence, processing errors etc. For volcanoes with persistent detections, we use time-series and atmospheric correction approaches to investigate the characteristics of the signal, while volcanoes with occasional detections are checked visually.To form time series for volcanoes with persistent detections, we apply the method used by Albino and Biggs : the intP < 0.9), while volcanoes with large numbers (> 20) or a high percentage (> 5%) of detections have very high maximum probabilities (P > 0.9). In this section, we first describe the 16 volcanoes with persistent detections in detail, and then highlight interesting examples from the 215 volcanoes with occasional detections.The algorithm flagged 3323 wrapped interferograms of 366 volcanoes Fig. . Of thesP > 0.80. Magma recharge between eruptions caused continuous, but not constant, deformation. The total uplift of 6.5 m between 2005 and 2018  within 30 days and was not sensitive to any further changes in deformation rate  were from volcanoes in the Galapagos Islands, with 904 from Sierra Negra alone Fig. , and the.9 Table . Nonetheate Fig. .Table 1MP > 0.5 after just 3.2 and 4.3 months respectively  on 02-Oct-2019  flags. Many of these are false positives and are easy to filter out during expert review but many are true positives caused by transient or slow deformation. Transient deformation is easy to classify because the flagged interferograms cluster in time. For example, the January 2020 eruption of Taal was associated with lateral magma movement causing large magnitude, complex deformation patterns which were flagged 13 times in individual interferograms, of which 11 spanned January 2020 Fig. . The tim0.4 Fig. . Another0.4 Fig.  or defor0.4 Fig. , but the0.4 Fig. .P = 0.5 after 6.5 months , at others the deformation was too slow to be flagged in many individual interferograms. For example, Alcedo  did not experience any significant eruptive or intrusive activity during the observation window, but slow subsidence (< 3.5 los cm/year) within the caldera was sufficient to cause 12 detections in the short-term interferograms  = (1 + ea(x−b)−) following Anantrasirichai et al. (P∼0 (undetectable) and P∼1 (detectable) occurs over a narrow range and corresponds to the minimum value of displacement that the CNN can identify. The detection threshold for the whole dataset is 5.9 cm, equivalent to a rate of 1.2 cm/year over the 5-year study period. However, it is important to note that this estimate is based on time-series generated for volcanoes where deformation has been detected, and the thresholds might be significantly higher elsewhere.We use the time series results to estimate the detection threshold of the CNN. We plot the output probability, ies Fig.  and fit i et al. . The traThere is considerable variability between volcanoes with individual values ranging from 2.1 cm (0.4 cm/year) at Tambora, Indonesia, to 9.9 cm (2.0 cm/year) at Domuyo, Argentina. In general, detection thresholds are < 5 cm for low relief volcanoes such as Reykjanes and Tambora but are > 5 cm for high relief volcanoes such as Laguna del Maule, Etna and Domuyo. These values are consistent with similar tests conducted on synthetic data by Anantrasirichai et al. . VolcanoP > 0.9. In both cases, the time-series are very noisy, and the detection probability rarely exceeds 0.5  and Masaya (0.08–0.11). In both these cases, tailored processing would be required for the signal to be detectable. In summary, we demonstrate that the CNN can detect slow deformation in individual interferograms if (1) the rate is sufficiently fast, (2) there are interferograms with long time spans and (c) those interferograms are sufficiently coherent.In this section, we focus on the Afar region of Ethiopia where Albino and Biggs  and MoorAt Erte Ale, a dyke intrusion caused short-term deformation rates in excess of 200 cm/year triggering 42 flags; Dabbahu, Nabro and Dallol in Ethiopia were all deforming at steady rates of 3–4 cm/year and were flagged 4–10 times and the small, slow subsidence signal at Gade Ale (1.9 cm/year) was only flagged twice. Although Alu-Dalafilla was flagged 17 times, this was because of its proximity to the large signal at Erte Ale, and the slow deformation signal (1.2 − 1.6 cm/year) reported by Albino and Biggs  was beloSatellite systems are ideally suited for global environmental monitoring, and several pilot studies have shown the potential for using machine learning approaches for characterising and detecting volcanic deformation  and six examples with non-eruptive unrest . The deformation on the Reykjanes Peninsula is retrospectively considered a precursor to the 2021 Fagradalsfjall eruption, which occurred after the end of our observation period. Among these are several new observations that have not been previously published: (1) a completely new deformation signal at Tambora, Indonesia; (2) a change in rate of deformation at Wolf, Galapagos, from 3 cm/year prior to the 2015 eruption  long-duration interferograms or timeseries of unwrapped images are needed to detect slow deformation associated with unrest; (2) tailored processing strategies are needed in regions with dense vegetation and/or snow cover, where automated processing systems may produce incoherent interferograms; and (3) atmospheric corrections using global weather models reduce the total number of false positives, but atmospheric artefacts can still cause false positives or mask real deformation. Many of these recommendations are already been implemented in the LiCSAR system to avoid issues related to cumulated unwrapping errors and fading phase bias . The portal displays a range of LiCSAR products, including interferograms and time series with interactive tools designed to allow observatory volcanologists to (1) search quickly through processed imagery for deformation and (2) to make a critical assessment of whether any apparent signals are likely to be noise or true displacements  are included as a supplementary datafile and is also available at Supplementary file1 (PDF 3089 KB)Below is the link to the electronic supplementary material.


Aptamers, the nucleic acid analogs of antibodies, bind to their target molecules with remarkable specificity and sensitivity, making them promising diagnostic and therapeutic tools. The systematic evolution of ligands by exponential enrichment (SELEX) is time-consuming and expensive. However, regardless of those issues, it is the most used in vitro method for selecting aptamers. Therefore, recent studies have used computational approaches to reduce the time and cost associated with the synthesis and selection of aptamers. In an effort to present the potential of computational techniques in aptamer selection, a simple sequence-based method was used to design a 69-nucleotide long aptamer (mod_09) with a relatively stable structure  and investigate its binding properties to the tyrosine kinase domain of the NT-3 growth factor receptor, for the first time, by employing computational modeling and docking tools. Aptamers, the single-stranded nucleic acid analogs of antibodies, hold a great promise in molecular diagnostics, therapeutics, and drug targeting, due to their sensitivity and high selectivity toward target molecules . The smaO-methyl) are one of the most common adjustments to improve the nuclease resistance [Antibodies have long been applied in molecular diagnostics and clinical medicine ,4. Howevsistance . To decrsistance .The systematic evolution of ligands by exponential enrichment (SELEX) approach is used to select aptamers in vitro, which involves the incubation of the target molecules with a pool of oligonucleotides, followed by a myriad of rounds of selection and enrichment to remove the unbound nucleotides and redeem the final aptamer pool ,10,11. ABioinformatic tools are comprised of a broad range of methods that facilitate aptamer design and investigate their binding capabilities to the proposed targets. The molecular modeling involves the prediction of the secondary structure of RNA from its nucleotides sequence (primary system); then, building a 3D model (tertiary structure) based on this secondary structure . The preThe NT-3 growth factor receptor  is an 839-amino acid transmembrane protein that features a 286-residue intracellular tyrosine kinase (TyrKc) domain . DespiteIn this study, a high-affinity aptamer targeting the TyrKc domain of the NT-3 growth factor receptor was designed, for the first time, computationally, using a sequence-based approach. First, the TyrKc domain was docked with known aptamers present on the nucleic acid database (NDB) . Then, tThe aptamers were obtained from the NDB  by searcThe sequence of the NDB aptamer with the highest affinity to the target domain was used to generate multiple sequences of the same length, by introducing random point mutations (substitutions) in the original sequence.The RNAfold and RNAComposer web servers were used to obtain the minimum free energy (MFE) tertiary RNA analogs of the generated sequences, to be used as ligands in the subsequent analyses ,24,25,26The best-bound aptamer was modified to get a more stable structure, and its interaction with the target domain was visualized in more detail to report the amino acids and nucleotides involved in the interaction, using the Protein-Ligand Interaction Profiler (PLIP) web tool .Vibrio vulnificus (PDB: 5uza) had the highest binding affinity to the TyrKc domain , unsuitably, had a high MFE value, indicating that the structure could be unstable. The structure was modified manually by replacing some of the bases and removing others to address this problem. As a result, a much more stable structure (mod_09) was obtained, as the MFE value decreased from −16.4 to −32.2 kcal/mol. Despite its relatively high stability, the mod_09 aptamer formed a lower stable complex with the target domain than its precursor. However, it can be considered an acceptable result since it was better than the original structure obtained from NDB.Despite being simple, this method may contribute to the process of aptamer design in the future to reduce the number of sequences in the initial SELEX pool, by generating more sequences with similar nucleotides distribution or introducing minor mutations, or through coupling with other more sophisticated approaches.In this study, the TyrKc domain of the NT-3 growth factor receptor was used, for the first time, as a target to demonstrate the potential of the bioinformatics methods and computational approaches as promising tools in the area of aptamer design and selection, by employing a complete set of in silico strategies for the development of aptamers, using a simple sequence-based procedure. As a result, a 69-nt aptamer was optimized to have a relatively stable structure and acceptable binding score to the target domain. Since this computer-based aptamer selection method is cost-effective, simple, and does not require sophisticated devices, it can be applied to obtain more stable aptamers or be implemented in other algorithms for aptamer design.


The resultant hydroxy-functionalized COFs featured high BET-specific surface areas up to 1089 m2 g–1, excellent crystallinity, and superior thermal stability up to 60.44% char yield. When used as supercapacitor electrodes, the hydroxy-functionalized COFs exhibited electrochemical redox activity due to the presence of redox-active 2,3-dihydroxynaphthalene and 2,6-dihydroxynaphthalene in their COF skeletons. The hydroxy-functionalized COFs showed specific capacitance of 271 F g−1 at a current density of 0.5 A g−1 with excellent stability after 2000 cycles of 86.5% capacitance retention. Well-known pore features and high surface areas of such COFs, together with their superior supercapacitor performance, make them suitable electrode materials for use in practical applications.Covalent organic frameworks (COFs) have attracted significant interest because of their heteroatom-containing architectures, high porous networks, large surface areas, and capacity to include redox-active units, which can provide good electrochemical efficiency in energy applications. In this research, we synthesized two novel hydroxy-functionalized COFs—TAPT-2,3-NA(OH) Since the 20th century, the massive usage of fossil fuels including coal, gas, and mineral oil has been essential to economic and industrial advancement ,2. RenewII, NiII, FeIII) in their cavities were pyrolyzed  into N-doped porous graphenes with good specific capacitances in KOH as the electrolyte, possibly making them usable as electrodes for supercapacitors  polycondensations with1,3,5-tris-(4-aminophenyl)triazine (TAPT-3NH2). After that, the resultant hydroxyl-functionalized COFs were evaluated as potential supercapacitor electrodes, and their capacitive properties, such as cyclic voltammetry, galvanostatic charge/discharge, and cyclic durability, were investigated.Taking into account all of this above knowledge, we present in this work the utilization of the redox-active and hydroxyl-functionalized monomers—2,3-dihydroxynaphthalene-1,4-dicarbaldehyde  and 2,6-dihydroxynaphthalene-1,5-dicarbaldehyde —to synthesize two hydroxy-functionalized COFs, namely TAPT-2,3-NA(OH)n-butanol and o-dichlorobenzene, acetic acid, DMF, THF, and acetone were obtained from J. T. Baker . Raw chemical materials and solvents were purchased from commercial resources and then used as they were.2,3-Dihydroxynaphthalene, 2,6-dihydroxynaphthalene, 4-bromoaniline, potassium carbonate, and formamidine were received from Acros. Acetic anhydride, HCl, and triflic acid were ordered from Alfa Aesar. Dioxane, chloroform, 2O (45 mL), and heated at 65 °C for 2 h. Then, we added HCl  and kept heating at 65 °C for 18 h. The solid was filtered and washed several times with hexane. Dihydroxynaphthalene dicarbaldehydes  were obtained after purifying by using column chromatography (Formamidine acetate (14.98 mmol) and dioxane (30 mL) were added into a 100-mL two-neck round-bottom flask and heated under reflux. Acetic anhydride (3 mL) was added when the target temperature reached 95 °C and then stirred for 30 min. Dihydroxynaphthalene (1.87 mmol) was added once all formamidine acetate was dissolved and was kept for two days. After cooling for minutes, dioxane was evaporated at 50 °C, was added Htography .2 (0.196 mmol) (n-butanol (3.5 mL) and o-dichlorobenzene (3.5 mL) with acetic acid  in a 25-mL Schlenk storage tube. The tube was then flame-sealed and heated at 120 °C for three days. After the tube had cooled to room temperature, the precipitate was filtered and washed . The solid was vacuum-dried overnight at 120 °C to provide the TAPT-2,3-NA(OH)2 and TAPT-2,6-NA(OH)2 COFs (Three freeze/pump/thaw cycles were used to degas a solution of TAPT-3NH96 mmol)  and 2,3-H)2 COFs .2 and TAPT-2,6-NA(OH)2 COFs–through solvothermal [3 + 2] polycondensations of TAPT-3NH2 (n-butanol/o-dichlorobenzene (1:1) as a co-solvent over 72 h at 115 °C in Schlenk tubes, in the presence of acetic acid  as an acidic catalyst. In the widely-used organic solvents such as dioxane, methanol, tetrahydrofuran, DMF, acetone, or DMSO, the produced COF powders were insoluble, even at high temperatures.To design novel hydroxyl-functionalized COFs with efficient supercapacitor performances, as well as to investigate the impact of hydroxyl group position on their characteristics and applications, we synthesized two TAPT-2,3-NA(OH)APT-3NH2  with 2,3APT-3NH2  and 2,6-APT-3NH2  as new rAPT-3NH2 A,B. Thei2 and TAPT-2,6-NA(OH)2 COFs to gain knowledge into their crystallinities 2 COFs both featured triclinic networks with long-ordered architectures. In 2 COF displayed a strong signal at 2θ = 2.34°, which we attribute to the 100 facet, as well as three signals at 2θ = 4.11°, 4.79°, and 6.24°, which we attribute to the 110, 200, and 210 facets, respectively. We ascribe the wide diffraction signal at 22.38° to the 001 facet, which emerged due to significant π-stacking between the triclinic interlayers of the TAPT-2,3-NA(OH)2 COF  can be calculated using the Bragg equation; for the TAPT-2,3-NA(OH)2 and TAPT-2,6-NA(OH)2 COFs, the π-interlayer lengths were 3.96 and 3.94 Å, respectively, and the values of d100 were 3.76 and 3.78 nm, respectively 2 COFs 2 COFs 2 COFs both evenly matched those of the relevant AA stacking models. Importantly, the unit cells of the eclipsed AA-stacking simulations revealed the following parameters: for TAPT-2,3-NA(OH)2 COF, a = 43.4 Å, b = 43.2 Å, c = 3.649 Å, α = β = 90°, and γ = 120°  patterns of the TAPT-2,3-NA(OH)linities , which dOH)2 COF . Similarectively . The π-iectively . The theH)2 COFs . The resγ = 120° ; TAPT-2,γ = 120° .13C NMR spectroscopy. The FTIR spectra of TAPT-2,3-NA(OH)2 and TAPT-2,6-NA(OH)2 COFs did not show any of the distinguishable vibration signals for the amino (NH2) group at 13,458–3320 cm–1 for the TAPT-3NH2 monomer or the vibration signals for the aldehydic (CH=O) groups at 1674 and 1641 cm–1, respectively, for the 2,3-NADC and 2,6-NADC, implying complete polycondensations of TAPT-3NH2 with the two hydroxyl-functionalized monomers. New distinguishable vibration signals were observed at 1634 and 1633 cm–1 in the FTIR spectra of TAPT-2,3-NA(OH)2 and TAPT-2,6-NA(OH)2 COFs, respectively, corresponding to their imino (C=N) bonds 2 and TAPT-2,6-NA(OH)2 COFs exhibited distinctive signals at 162.42 and 164.88 ppm for the resonances of the imino-carbon (C=N) nuclei, respectively  nuclei of the TAPT-2,3-NA(OH)2 and TAPT-2,3-NA(OH)2 COFs. In addition, the solid state 13C NMR spectra revealed signals at 176.17 and 176.65 ppm, representing the resonances of triazine units in the TAPT-2,3-NA(OH)2 and TAPT-2,6-NA(OH)2 COFs, respectively.The molecular structures of the hydroxyl-functionalized COFs were confirmed using FTIR and solid state N) bonds . The solectively B. Becaus2 sorption analysis at 77 K to examine the pore structures of the hydroxyl-functionalized COFs. According to the IUPAC technical report on physisorption of gases, N2 sorption isotherms of TAPT-2,3-NA(OH)2 and TAPT-2,6-NA(OH)2 COFs demonstrated type I isotherms, with an unremarkable N2 uptake in the relative pressure range (P/P0) less than 0.1 bar and a steep N2 uptake in the relative pressure range greater than 0.8 bar 2 COFs were microporous. In addition, the adsorption/desorption curves revealed that the Brunauer–Emmett–Teller (BET) surface areas of TAPT-2,3-NA(OH)2 and TAPT-2,6-NA(OH)2 COFs were 429 and 1089 m2 g–1, with pore volumes of 0.17 and 0.22 cm3 g–1, respectively 2 COFs, showing pore diameters of roughly 3.04 and 3.29 nm, respectively 2 COF. The lack of any additional components within XPS detections demonstrates the absence of any perceptible impurities that may have been produced during the synthesis of the hydroxyl-functionalized COFs, as illustrated in 2 COF has three primary kinds of C 1s species on their own surfaces: C–OH at 288.30 eV, C=C at 286.30 eV, and C=N at 284.60 eV . As shown in 84.60 eV . On the ectively . The fitectively  45]..2 COF an2 and TAPT-2,6-NA(OH)2 COFs 2 COFs after solvent exfoliation in ethanol revealed that both the hydroxyl-functionalized COFs were assembled into a significant number of long nanofibers with lengths of up to several micrometers, and such nanofibers were linked by their mesoporous sidewalls 2 COFs revealed average diameters for the nanofibers of 30 ± 50 and 50 ± 70 nm, respectively. As we had documented previously, the degree of planarity of the building monomers can have a significant impact on the morphology of the COFs. We also reported that the COFs constructed from planar building monomers are often built into tubes, rods, or fibers  polycondensations of TAPT-3NH2 with 2,3-NADC and 2,6-NADC, respectively, using n-butanol/o-dichlorobenzene (1:1) as a co-solvent over 72 h at 115 °C in Schlenk tubes, in the presence of acetic acid  as an acidic catalyst. The molecular structures of the hydroxyl-functionalized COFs were verified using FTIR and a solid state 13C NMR spectroscopy. According to the PXRD, BET, TGA, and XPS measurements, hydroxyl-functionalized COFs featured high BET-specific surface areas up to 1089 m2 g−1, excellent crystallinity, and a superior thermal stability up to 60.44% char yield. In addition, we evaluated the applicability of our hydroxyl-functionalized COFs for a supercapacitor application. The resultant hydroxyl-functionalized COFs demonstrated an outstanding electrochemical efficiency (271 F g−1 at a current density of 0.5 A g−1) because of the presence of redox-active 2,3-dihydroxynaphthalene and 2,6-dihydroxynaphthalene in their COF skeletons. Furthermore, we believe that these novel hydroxyl-functionalized COFs might be useful in a variety of applications, including energy storage technologies.In summary, two hydroxy-functionalized COFs, namely, TAPT-2,3-NA(OH)


Astragalus trimestris L. contains the isoflavone formononetin (FMNT). Literature reports show that FMNT can increase insulin sensitivity and potentially target the peroxisome proliferator-activated receptor gamma, PPARγ, as a partial agonist. PPARγ is highly relevant for diabetes control and plays a major role in Type 2 diabetes mellitus development. In this study, we evaluate the biological role of FMNT, and three related isoflavones, genistein, daidzein and biochanin A, using several computational and experimental procedures. Our results reveal the FMNT X-ray crystal structure has strong intermolecular hydrogen bonding and stacking interactions which are useful for antioxidant action. Cyclovoltammetry rotating ring disk electrode (RRDE) measurements show that all four isoflavones behave in a similar manner when scavenging the superoxide radical. DFT calculations conclude that antioxidant activity is based on the familiar superoxide σ-scavenging mode involving hydrogen capture of ring-A H7(hydroxyl) as well as the π–π (polyphenol–superoxide) scavenging activity. These results suggest the possibility of their mimicking superoxide dismutase (SOD) action and help explain the ability of natural polyphenols to assist in lowering superoxide concentrations. The SOD metalloenzymes all dismutate O2•− to H2O2 plus O2 through metal ion redox chemistry whereas these polyphenolic compounds do so through suitable hydrogen bonding and stacking intermolecular interactions. Additionally, docking calculations suggest FMNT can be a partial agonist of the PPARγ domain. Overall, our work confirms the efficacy in combining multidisciplinary approaches to provide insight into the mechanism of action of small molecule polyphenol antioxidants. Our findings promote the further exploration of other natural products, including those known to be effective in traditional Chinese medicine for potential drug design in diabetes research.Isoflavones are plant-derived natural products commonly found in legumes that show a large spectrum of biomedical activities. A common antidiabetic remedy in traditional Chinese medicine,  Isoflavones, including formononetin chromen-4-one), are bioactive phytochemicals that are abundant in legumes such as chickpeas, soy, beans and red clover . These cAstragalus trimestris L.  (Fabaceae), a common antidiabetic herbal remedy in traditional Chinese medicine [FMNT is also found in medicine . Diabetemedicine . Insulinmedicine ,10,11. Cmedicine . In vitrmedicine ,14,15,16To elucidate the biological role of FMNT, we performed several computational and experimental procedures: (1) single crystal X-ray crystallography to obtain FMNT’s molecular structure and information about its intermolecular interactions; (2) determination of FMNT antioxidant activity by measuring its scavenging ability of the superoxide radical; (3) use of computational DFT methods to understand the mechanism of FMNT scavenging of superoxide; (4) characterization of the docking interactions between PPARγ and FMNT using crystal-structure-obtained FMNT atomic coordinates. Upon observing our FMNT encouraging results, an equivalent analysis of superoxide activity was also performed for three related isoflavones: genistein, daidzein and biochanin A. FMNT and biochanin A are converted by 4′-O-demethylation through cytochrome P-450 enzymes to daidzein and genistein, respectively .Single crystal X-ray diffraction data obtained on a suitable crystal of FMNT are reported in The FMNT crystal structure shows intermolecular interactions including a strong hydrogen bond (2.649 Å) between carbonyl O1 and hydroxyl group on C7 A. Other In the Cambridge Structural Database (CSD) , there aScavenging of superoxide was analyzed with DFT methods. In a recent review we described two ways of scavenging this radical by polyphenols: (1) superoxide interaction with an aromatic H(hydroxyl)  and (2) interaction through the π–π approach , see Sch2−, while the remaining FMNT polyphenol comprises the unpaired electron located at ring A, 2−, separated 1.395 Å from O(7). When a proton is van der Waals posed near the more exposed O(superoxide) moiety, DFT minimization shows bond formation, O-H distance = 0.983 Å, while HO2− is separated by 1.540 Å from the polyphenol. However, H7 results as returning to FMNT, O7-H7 = 1.044 Å, 2O2 was formed and became well separated from ring A, 1.636 Å, while the stacked superoxide reagent became trapped within the ring, with a separation between centroids of 3.038 Å, which is shorter than the original van der Waals separation of 3.50 Å, 2O2 was eliminated and upon DFT minimization, 2 molecule (O-O bond length of 1.258 Å) is still bound to ring A, as shown by a centroid separation of 3.015 Å, Scavenging details after the capture of H7 by superoxide FMNT are shown in 2 complex could behave as a catalyst for superoxide scavenging, i.e., beginning a new cycle. Another superoxide radical was σ van der Waals posed (2.60 Å) near H7, and DFT minimization (stopped after 81 cycles) showed initial formation of HO2− ion, separated from FMNT 1.717 Å. More importantly, the π–π superoxide inserted in 2, as shown by its O-O bond distance of 1.289 Å, which is much shorter than 1.373 Å for the superoxide. Thus, 2O2, 2 Next, we explored if this FMNT-η-O2 complex. In addition, a H-bond is formed when superoxide σ approaches H in position 4′ of ring B, In 2, H2O2 forms and is well separated from genistein’s radical, 1.621 Å, which is not shown. 2O2 and posing a proton near O7. Proceeding in a similar way as with FMNT, the additional superoxide posed near H7 induces the π–π molecule of O2 to leave, not shown, while the last superoxide forms a H-bond to H7. Meanwhile the second superoxide relocates above ring C, 3.154 Å, between centroids, 2−. Next, a proton was posed near the most exposed O(HO2−) and DFT minimization resulted in H2O2 formation. Upon H2O2 elimination, a π–π superoxide was posed over ring A and DFT was applied resulting in the superoxide being released as a molecule of O2, 2O2 is eliminated after 2 is released after the final proton is incorporated in Daidzein behavior is similar to that of FMNT and genistein. 2 complex with ring C centroid, 2.844 Å, as is the case with genistein, whereas the attack on ring A occurs as a σ scavenging on H7.2O2 moiety was seen, 1.621 Å from the remaining biochanin neutral radical. Hence, H2O2 was eliminated, the biochanin A radical was minimized and another proton was posed near O7. Upon DFT minimization, O7-H7 formed, and additional superoxide was a van der Waals π–π posed on ring A, 3.50 Å. The subsequent DFT minimized structure is the real catalyst for biochanin A mimicking SOD action, The biochanin A path of scavenging is similar to that of the other three isoflavones. First, H7 is well captured by superoxide, following an H atom transfer (HAT), both through σ or π–π approaches. Next, a proton was added, and upon DFT minimization, a well separated H2-η-biochanin-A complex, 2-η-genistein, it is −147.7 Kcal/mol. As mentioned earlier, when the superoxide resides above ring A, the response of the four isoflavones is the same: the superoxide radical is redirected to H7 that is then captured, as shown in deposited The DFT scavenging activity of the four isoflavones was measured using a previously established cyclic voltammetry protocol . This me4; genistein: −6.4 × 104; daidzein: −7.0 × 104; biochanin A: −7.1 × 104). They are slightly weaker than DHDM, 2′,4′-dihydroxy-3,4-dimethoxy chalcone, which contains a 2′,4′-dihydroxy ring moiety responsible for scavenging. Butein, clovamide and quercetin are stronger scavengers than the isoflavones in this study and have catechol moieties, a structural feature associated with good scavenging activity. However, the best superoxide scavenger so far studied by the RRDE technique, galangin, has no ring B-hydroxyls.To better understand the interactions of FMNT as a possible PPARγ partial agonist, atomic coordinates from the crystal structure of FMNT were docked in the PPARγ ligand binding domain (LBD), PDB Code: 5UGM , which c2 with the Bruker incorporated ShelX programs [https://www.ccdc.cam.ac.uk/structures/? (accessed on 28 October 2022) using Identifier CCDC number 2216211.Formononetin was recrystallized from ethanol by slow evaporation. An APEX2 DUO platform X-ray diffractometer from Bruker Advanced X-ray Solutions was used to obtain X-ray data measurements at 125 K. Temperature was maintained using a cold liquid nitrogen stream from Oxford cryosystems; the X-ray source emitted MoKα radiation at 0.71069 Å. The crystal structure was solved and refined using full-matrix least-squares on Fprograms . We inpuprograms . Crystal© software. The antioxidant activity of the four isoflavones  was determined based on their superoxide radical scavenging ability that was measured using the protocol developed in our lab [Materials used to determine the antioxidant activity of the 4 isoflavones were tetrabutylammonium bromide  and 99.9% anhydrous dimethyl sulfoxide . The four isoflavones, FMNT, genistein, daidzein and biochanin A, were all obtained from Indofine Chemical Company . A 0.1 M TBAB/DMSO solution was used to produce electric current and enhance the occurrence of redox reactions. Antioxidant activity was measured via the hydrodynamic voltammetry technique with a rotating ring disk electrode (RRDE). The equipment used in this experiment was an MSR electrode rotator together with a WaveDriver 20 benchtop USB from Pine Instrumentation, Grove City, PA, USA. The main electrode tip was an E6RI ChangeDisk with a rigid gold ring and gold disk (Au/Au) insert. Before and after each experiment, 0.5 μm alumina suspension was used to clean the disk electrode tip  on a moistened polishing microcloth to eliminate potential film formation. A platinum (Pt) reference electrode and Pt counter electrode were also used in this experiment. All electrodes were obtained from Pine Research, Durham, NC, USA . Cyclic  our lab . Stock s2/N2 (35%/65%) gas mixture to establish its dissolved oxygen level. The Au disk electrode was then rotated at 1000 rpm while the disk was swept from 0.2 V to −1.2 Volts and the ring was held constant at 0.0 Volts, the disk voltage sweep rate was set to 25 mV/s. Several runs plus blank were performed in the RRDE experiment for the 4 compounds to determine their antioxidant activity with equal addition of 5 µL of each stock solution.For the experiment, the electrolytic cell was bubbled for 5 min with a dry OResults from each run were collected on Aftermath software and represented as voltammograms showing current vs. potential graphs that were later analyzed using Microsoft Excel. In an RRDE voltammetry experiment, the generation of the superoxide radicals occurs at the disk electrode while the oxidation of the residual superoxide radicals (that have not been scavenged by the scavenger) occurs at the ring electrode.Reverse Reaction 3: Oxidation of superoxide radicals at the ring electrodeThus, the rate at which increasing concentrations of antioxidant scavenged the generated superoxide radicals during the electrolytic reaction was determined by obtaining the ring current/disk current  at each concentration. These values were denoted as the Efficiency of the scavenger at different concentrations. Using Microsoft Excel, Collection Efficiency values were plotted against the corresponding concentrations of each analyzed compound to produce a graph illustrating the effect of their increasing concentrations on the scavenging of superoxide radicals in the electrolytic solution. Ultimately, the slope of the curves served as a quantitative measure of the antioxidant activity of each compound.−6 electron/Å3. The convergence criteria applied during geometry optimization were 2.72 × 10−4 eV for energy and 0.054 eV/Å for force. Calculations were generally performed with no solvent inclusion, those made in DMSO are specifically indicated. Docking studies were performed with the CDOCKER package in Discovery Studio 2020 version [Calculations were performed using programs from Biovia . Density functional theory (DFT) program DMol3 was applied to calculate energy, geometry and frequencies implemented in Materials Studio 7.0 . We empl version .2•– to H2O2 plus O2 through metal ion redox chemistry [The X-ray structure presents FMNT’s ability to have strong intermolecular interactions, such as hydrogen bonding and stacking, which are useful for antioxidant actions. Antioxidant activity measured using the RRDE method revealed the four isoflavones to share a similar pattern in the collection efficiency slopes. All DFT calculations confirm these findings and allow us to conclude that antioxidant activity is based on the familiar superoxide σ-scavenging mode involving ring-A H7(hydroxyl) capture and supplemented by the π–π (polyphenol–superoxide) scavenging activity. These results suggest the possibility of their mimicking SOD action and help to explain the ability of natural polyphenols to assist in lowering superoxide concentrations. The SOD metalloenzymes all dismutate Ohemistry , whereas


Precise regulation of the β2 integrin is critical for leukocyte adhesion and trafficking. In neutrophils, β2 integrins participate in slow rolling. When activated by inside–out signaling, fully activated β2 integrins mediate rapid leukocyte arrest and adhesion. The two activation pathways, starting with selectin ligand engagement and chemokine receptor ligation, respectively, converge on phosphoinositide 3-kinase, talin-1, kindlin-3 and Rap1. Here, we focus on recent structural insights into autoinhibited talin-1 and autoinhibited trimeric kindlin-3. When activated, both talin-1 and kindlin-3 can bind the β2 cytoplasmic tail at separate but adjacent sites. We discuss possible pathways for talin-1 and kindlin-3 activation, recruitment to the plasma membrane, and their role in integrin activation. We propose new models of the final steps of integrin activation involving the complex of talin-1, kindlin-3, integrin and the plasma membrane. Leukocytes are essential for host defense against bacterial and fungal infection. Upon infection or sterile injury, cytokines and chemokines are released that activate the vascular endothelium and recruit circulating neutrophils. Neutrophils are often the first cell type recruited to sites of inflammation. They arrive at the site of injury by a cascade-like process involving selectin-dependent rolling followed by β2 integrin-mediated cell arrest and trans-endothelial migration . This caNeutrophil rolling is mediated by the selectin family of CAMs which orchestrate the interactions between circulating cells and the endothelium. Neutrophils express P-selectin glycoprotein ligand-1  that binds all 3 selectins: P-selectin on endothelial cells and platelets, E-selectin on endothelial cells, and L-selectin on leukocytes . The intβ2 integrins  are expressed by all leukocyte types. They form a sub-class of the integrin family of CAMs. Integrins are heterodimeric adhesion receptors consisting of α- and β-subunits that are expressed on the plasma membrane. Each integrin recognizes, and connects to ICAM-1, ICAM-2 and ICAM-3 on endothelial cells and couples to the cells’ intracellular cytoskeletal and signaling machineries. There are four distinct α-subunits, αL, αM, αX and αD (or CD11a-d) that can pair with the same common β2 integrin subunit to form four distinct β2 integrin leukocyte receptors. All leukocytes express αLβ2 (CD11a/CD18), also known as lymphocyte-function-associated antigen 1, LFA-1). Myeloid cells also express αMβ2 . Some monocytes also express αXβ2 . Some activated lymphocytes express αDβ2. Over the years the names of these integrin receptors have changed but are standardized now as αLβ2, αMβ2, αXβ2 and αDβ2 and this nomenclature is used here. Neutrophils express mainly two of the four β2 integrins, αLβ2 and αMβ2 . These tBoth selectin- and chemokine-induced signals converge and trigger dramatic conformational changes that result in full β2 integrin activation by inside–out signaling ,8,9. IntResting β2 integrins exist in a closed, inactive conformation, that is neither extended (E-) nor high affinity (H-). Fully activated β2 integrins are E+H+ and can bind ligand in trans (on the surface of another cell), which can cause arrest from rolling. However, between the fully inactive, E-H- and fully active, E+H+ states, two intermediate states exist, E+H- which can bind ligand in trans with low affinity and high off-rate, and E-H+ which can bind ligand in cis (on the surface of the same cell) and therefore acts as an inhibitor of adhesion . SelectiIntegrin activation refers to conformational changes in the integrin extracellular domains, which are relayed from the binding of proteins to the integrin cytoplasmic tails. Many cytoplasmic proteins, including filamin , dockingAlthough integrins mediate bi-directional signaling, we will not discuss outside–in signaling in this review. This review focuses on the final steps of β2 integrin inside–out activation. Specifically, we focus on recent advances in understanding the structures of talin-1 and kindlin-3, the only talin and kindlin isoforms expressed in leukocytes. Both proteins are essential for effective β2 integrin activation.Talin-1 is a large cytoplasmic protein with a molecular weight of ~270 kDa. It contains an N-terminal head domain and a C-terminal rod domain . The talIn the context of integrin activation, a direct interaction between the talin FERM subdomain F3 and the cytoplasmic integrin β tail, binding to an asparagine, proline, any residue, tyrosine motif, the so-called NPxY domain (NPLF in β2) is critical for integrin activation ,33,34. AMany proteins are regulated by autoinhibition  and taliThe structures of FERM domains are defined by a classical clover leaf arrangement whereby the F1, F2 and F3 domains interact with each other. However, the multiple X-ray structures of the talin head domain have hinted at a plasticity of the talin head that makes it unique in the FERM domain family. In this section we briefly review the structure of the talin head as it impacts on the models of the regulation of integrin activation complex.In all structures of the talin head to date, including the structures of F0F1 and F2F3 double domain modules, the orientation of F0 relative to F1 and F2 relative to F3 is fixed. That means the F0F1 and F2F3 double domains have fixed orientations and behave as single units. The thing that varies between the structures is the orientation of the F0F1 module relative to the F2F3 module. In the full-length talin cryoEM structure, the F0F1 module is not resolved, indicating that it is flexible relative to F2F3.Crystallographic analysis of the truncated talin head domain  alone revealed that talin-1 head (with truncation of the F1 loop) can adopt a linear conformation  Fig top. HowThese findings suggest two models: either the clover-leaf structure is the correct structure and the others are artefacts, or the talin head has a unique propensity to adopt different conformations in response to different signals. The latter might allow different signaling pathways to modify the talin head conformation, thus integrating signaling pathways into the integrin activation process. The second model is more likely, supported by experimental evidence. As mentioned above, the talin FERM domain is atypical. The talin FERM domain is the only FERM domain from which the subdomains can be readily purified in isolation. By contrast, the kindlin, MyosinX and IDOL FERM domains cannot be subdivided because individual subdomains are insoluble. Third, the fact that F0F1-F2F3 has been crystalized in two conformations supports the notion of flexibility. Thus, we posit that the F1-F2 linker is a regulatory feature of the talin head. In particular, the inversion is of interest and might indicate a novel regulatory mechanism. There are significant spatial constraints, because the known ligands for the talin FERM domain are all membrane bound  or transmembrane (integrin β). If talin is bound to the membrane with the F0 and F1 aligned so that they are in an orientation to bind Rap1, then we predict the affinity for Rap1 will be high. However, if F0F1 is inverted its affinity for Rap1 will be markedly lower .The talin F0F1 domains are required for robust activation . Both F0It seems that the F1 loop, and the charged residues in F2 and F3 are also close enough to the plasma membrane to bind the negatively charged PIP2 phospholipids. When bound by talin, the β-integrin tail switches from a 25-degree tilt angle  and becoTwo pathways lead to talin-1 activation and Rap1 plays a central role in both, (1) direct activation of talin-1 by Rap1 through direct binding of Rap1 to talin-1 , and (2) indirect activation of talin-1 by Rap1 mediated by RIAM . Rap1 is targeted to the membrane by covalent geranyl-geranylation of a cysteine residue . Upon ac3mut) had only mild effects on platelet integrin activation in mice [NMR studies suggested direct but low-affinity interaction between Rap1b and the talin-1 F0 domain . However in mice ,57. Late in mice . The R35Full activation of talin-1 needs the cooperation of Rap1 binding and PIP2-mediated binding of talin-1 to the plasma membrane C 10,47,,47,58. T. The dimer formation is mediated by F2-F2 domain interaction and has very slow kinetics [Kindlin-3 is known to be required for β2 integrin activation ,63,64. Tkinetics . Whetherkinetics . Crystalkinetics . The F3 The kindlin-3 PH domain is necessary for recruitment of kindlin-3 to the plasma membrane ,72. To tBoth talin-1 and kindlin-3 bind to the integrin β cytoplasmic domain, which contains two NPXY sites. The membrane-proximal NPXY site (sequence NPLF in β2) is the primary talin-binding site . The memAs mentioned above, talin and kindlin autoinhibition provides a plausible mechanism for maintaining integrin in a low-activation state. In this E-H- state, the transmembrane helices blue in  of β2 anIn its resting state, integrins are bent  and low affinity  as shown in The talin head domain  has been crystallized three times, yielding different structures. The talin1 head extended conformation (PDB 3IVF, Given all the known structures and interactions, how might integrin actually be activated in a cell? We lay out a model for integrin activation by talin and kindlin binding. Both talin-1 and kindlin-3 are normally autoinhibited. How this autoinhibition is relieved is not known, but it is possible that formation of PIP2 and PIP3 patches upon chemokine receptor activation may be sufficient to initiate the process. Both talin-1 and kindlin-3 become active when recruited to the plasma membrane. Active Rap1 plays a central role and so the convergence of Rap1 and PIP2 provides a nexus at which talin-1 and kindlin-3 can help recruit and activate, or stabilize the integrin-activating form of talin. Both talin-1 and kindlin-3 can bind distinct domains in β2. In the absence of talin-1 or kindlin-3, β2 integrins cannot be activated. Talin-1 or kindlin-3 binding can release the inner membrane clasp and thus promote integrin leg separation and activation. Sophisticated mechanisms orchestrate the interactions between talin, kindlin, Rap1 and integrin, ensuring that the appropriate adhesion response occurs only in response to the correct signals.


The increasingly large structure of neural networks makes it difficult to deploy on edge devices with limited computing resources. Network pruning has become one of the most successful model compression methods in recent years. Existing works typically compress models based on importance, removing unimportant filters. This paper reconsiders model pruning from the perspective of structural redundancy, claiming that identifying functionally similar filters plays a more important role, and proposes a model pruning framework for clustering-based redundancy identification. First, we perform cluster analysis on the filters of each layer to generate similar sets with different functions. We then propose a criterion for identifying redundant filters within similar sets. Finally, we propose a pruning scheme that automatically determines the pruning rate of each layer. Extensive experiments on various benchmark network architectures and datasets demonstrate the effectiveness of our proposed framework. Compared with traditional machine learning methods, deep learning-based methods have greatly improved the performance of many computing tasks, such as image recognition , object Network pruning is mainly divided into unstructured pruning and structured pruning. Unstructured pruning prunes individual weights in the model to compress the DNN  . HoweverTypical filter pruning includes three stages : (1) traL1-norm , Taylor L1-norm , sparsitL1-norm , geometrL1-norm , high raL1-norm  and variL1-norm . Due to L1-norm  comparedWe propose a novel pruning scheme that does not depend on importance but is based on the similarity between filters for channel-level pruning.We introduce an effective method for measuring the relative importance of filters, avoiding the problems of over-pruning and under-pruning caused by threshold specification.The proposed scheme automatically determines each layer’s pruning rate according to each layer’s parameter distribution, which avoids the problem of unreasonable pruning structure caused by manually specifying the pruning rate.A large number of experiments prove the effectiveness of the algorithm proposed in this paper.In this paper, we propose a clustering-based dynamic pruning method considering the similarity between filters. Compared with existing importance-based methods, we analyze the relevant information on the representational power among all filters within a layer and remove filters with overlapping functions. The proposed scheme takes into account edge features that are ignored based on importance ranking. Specifically, we cluster all filters within a layer in units of filters and select one deletion in each group whose features can be replaced by other filters. Each layer automatically generates groups according to the parameter distribution to determine the pruning rate of each layer, avoiding the problem of manually specifying the pruning rate. To assess the similarity of the representational power of filters in each group, a criterion is defined to measure the relative importance of all filters within a group. This criterion is scoped to a group and avoids the problem of threshold specification for global or local pruning. Extensive experiments demonstrate that our proposed method is more general than importance-based methods. The research on network pruning originated from the 1989 paper on skeletonization . LeCun  in 1990 To overcome the above limitations, SSL  proposedThere are also other criteria based on the weights of the importance of ranking. He et al.  proposedOther types of DNN model compression techniques are also being explored. Quantization ,40 compru et al.  used a tu et al.  proposedu et al.  proposedv et al.  proposedThe current model compression methods through pruning still determine the importance of a single parameter or filter by looking for a criterion and combining the pruning method and processing to restore the performance of the pruned model. Different from previous methods, we use clustering to find filters with overlapping functions more effectively by comparing the similarity between filters.In this section, we propose a novel pruning scheme, which is the cluster similarity-based filter pruning method. We first introduce the overall framework and related notation definitions, and then describe the motivation and implementation details. Finally, we propose the corresponding pruning scheme.We show the flow of a single pruning of the proposed pruning scheme in One of the main problems of filter pruning is how to select effective filters and retain as much of the expressive power of the original network as possible. Current amplitude-based pruning algorithms all rely on the assumption that removing relatively insignificant weights in the network has little effect on the pruned network performance. Unlike current views based on parameter importance, we propose that the removal of any one of the filters will not significantly impair the representational power of the network as long as there are two sufficiently similar channels. This reduction also resonates with the well-known Hebbian principle, which roughly states that “neurons which fire together, wire together”. The visualization results of the filters and feature maps of the first convolutional layer of VGG16 are shown in Assuming that the neural network has We demonstrate that if there is a set of similar filters within a layer, pruning filters randomly or selectively in that set is better than pruning the least important filters within that layer. We assume that there is a set of similar filters Note that By Chebyshev’s inequality, for any real number From Equation (7) we can get:We assume that there are Available by Equation (8):This means By Equation (9) we can get:Letting Therefore, we have K-means clustering provides a solution for how to find similar convolution kernels within a layer. For the After getting sets containing similar information, the task is to find redundant filters in each set. The cluster center anchors the representative information of all filters in a similar cluster. After obtaining the cluster center of each similar cluster, it is intuitive to choose to keep the cluster center and delete other filters of the same cluster. However, this simple pruning method causes a large number of filters to be discarded, resulting in a sharp drop in performance. We look for a reasonable criterion for progressive pruning to determine redundant kernels in a cluster. In contrast to the intuitive pruning approach, we propose that cluster centers can be pruned. Since the cluster center is the mean value of all filters in clusters in each dimension, the cluster center of the remaining filters is still in the original cluster center position after removing the cluster center. This indicates that the information contained in all filters in the cluster is not lost after removing the cluster center.The above claim is in the ideal state, that is, the centroid of each cluster is exactly one element in the set. In experiments, it is difficult to find the element in the cluster set that happens to be the cluster center for each clustering. We find a compromise scheme, where each time we find the element closest to the cluster center, the information represented by this element can be transferred to other filters, and the original cluster set still retains the original information. First, we calculate the distance between all filters and cluster center in the In the above section, we propose how to identify redundant filters from a clustering set. However, for the number of hundreds or even thousands of filters in the current network structure, the number of redundant filters obtained by one clustering is far from enough. Therefore, we propose an iterative pruning scheme to identify more redundant filters to meet the compression requirements of large pruning rates and large network structures. After one pruning is completed, the next pruning continues to cluster to find a new similarity set and delete the redundant filters in the set. Compared with the current method of manually specifying the pruning rate of each layer, we only need to adjust one parameter to control the pruning rate of all layers. This is very efficient for large networks with hundreds of layers.For each convolutional layer, first initialize each cluster center For each cluster set After one pruning, calculate the pruning end condition, that is, the amount of computation The overall workflow of our cluster-based pruning algorithm is shown in Algorithm 1 and can be summarized as follows:Algorithm 1: Iterative pruning algorithm.Input: Training dataset /Output: The pruned model 1: while pruned rate = 0 to rate do2: for do3:      4:           initialize the clusters fordo5:           6:                7:                8:                end for9:           10:           11:           for do12:           fordo 13:                14:                     15:                     end for16:                end for17:           18:         end for 19:        20:       end while21: 22: In the network structure, the processing of special structures, such as dense block, residual block and inverted residual block, also has a greater impact on the final compression performance. In these structures, we process in blocks and perform pruning on the basis of satisfying the original relative relationship. For example, in a residual block, the number of input channels and output channels of each block is the same, and there are also constraints on the number of three convolutional layers in the block. According to previous work , the 3 ×We evaluate the effectiveness of our algorithm on CIFAR-10, CIFAR-100  and ILSVWe measure the complexity of the network using floating point operations (FLOPs) required for forward propagation. The computational cost of one convolutional layer is:We evaluate the effectiveness of the proposed framework using VGG16  and ResNIt can be seen from The results of ResNet with different depths on the CIFAR10 and CIFAR100 datasets are shown in In the experiments, we use ResNet-18/34/50 to demonstrate the proposed pruning performance on a large-scale dataset, ILSVRC-2012. All the baseline networks are obtained by training 100 epochs with a batch size of 256. We follow the same parameter settings as . We compThe results of ResNet with different depths on ILSVRC-2012 are shown in Aiming at the problems of ignoring edge features and manually specifying the pruning rate in current importance-based model pruning algorithms, this paper proposes a new model pruning framework based on similarity clustering. We reconsider the redundancy of neural network models from the perspective of similarity and find similar sets of filters through clustering and then propose a criterion for determining filter redundancy in similar sets. In order to solve the problem of a long compression period caused by excessive fine-tuning, we propose a corresponding iterative pruning scheme. Extensive experiments demonstrate the effectiveness of our proposed compression framework, while we cluster filters into multidimensional spaces and reconsider filter redundancy from a similar perspective, without specifying pruning rate. However, multi-dimensional values for clustering are still an unsolved problem. In the next step, we will combine this research with reinforcement learning and continue to mine redundant parameters in the network.


ADAMTS-13, a plasma metalloprotease, cleaves von Willebrand factor. ADAMTS-13 activity appears to be regulated through allosteric inhibition by its distal C-terminus.The objective of this study was to better understand how domain–domain interactions may affect ADAMTS-13 conformations and functions.We performed deuterium–hydrogen exchange plus mass spectrometry to assess the number and rate of deuterium incorporation into various peptides of full-length ADAMTS-13 and its truncated variants.Under physiological conditions, a bimodal distribution of deuterium incorporation was detected in the peptides from metalloprotease (217-230 and 282-304), cysteine-rich (446-482), and CUB  domains  of full-length recombinant ADAMTS-13, but not of truncated variants. These results suggest that the full-length ADAMTS-13 undergoes conformational changes. On removal of the middle and distal C-terminal domains, the number and rate of deuterium incorporation were increased in the peptides from cysteine-rich  and spacer domains (621-642 and 655-654) but decreased in the peptides from metalloprotease . Moreover, most peptides, except for 217-230 and 1357-1376, exhibited a pD-dependent deuterium incorporation in the full-length ADAMTS-13, but not in the truncated variant . These results further suggest that the bimodal deuterium incorporation observed in the peptides from the full-length ADAMTS-13 is the result of potential impact from the middle to distal C-terminal domains. Surface plasmon resonance revealed the direct binding interactions between the distal and proximal domains of ADAMTS-13.Our results provide novel insight on how intramolecular interactions may affect conformations of ADAMTS-13, thus regulating its proteolytic functions.  •The mechanism underlying allosteric regulation of ADAMTS-13 activity is not fully understood.•Biophysical study reveals conformational changes of ADAMTS-13 after truncation or pH changes.•Biophysical study also detects direct binding between distal and proximal ADAMTS-13 domains.•Our findings provide insights into how interdomain interactions affect ADAMTS-13 conformation. The digested peptides were than eluted from the C-18 column and electro sprayed into an LTQ Orbitrap XL mass spectrometer for a second dimension of separation. For different pD experiments, a full-length and a truncated variant was diluted into a 100% D2O buffer at 2 different pDs (6.0 and 8.0) with 10 mM sodium phosphate in a final volume of 32 μL. After an indicated incubation time (60 seconds for pD8.0 vs. 6000 seconds for pD6.0), an ice-cold quench buffer  was added. The protein was allowed to pass through the protease column and was later electro sprayed onto a mass spectrometer as described above  was determined by Prism 8.0 software (GraphPad) using the maximal response unit (RU) as a function of increasing concentrations of the ligands [A purified CUB domain fragment was immobilized on a CM5 sensor chip  through amine-coupling. A N-terminal ADAMTS-13 fragment  at various concentrations were perfused over the CUB-immobilized surface at 30 μl/min for 120 seconds. Protein dissociation was followed for 300 seconds. The surface was regenerated with 10 mM of glycine at pH2.0 for 30 seconds, and followed by perfusion with a re-equilibration buffer for another 30 seconds prior to next round of binding assay. An equilibrium dissociation constant  for 10, 60, 600, and 6000 seconds at pD7.0. The reaction was terminated by lowering the pD and chilling at 0 ºC. Deuterium and hydrogen exchange rate is the lowest around pH/pD2.5 and low temperature. Addition of quench buffer stops the deuterium labeling and minimizes the back exchange. Moreover, at pH >2.5, the rate of hydrogen and deuterium exchange increased logarithmically as the pH/pD increases. Because of this, the rate of exchange at pH6.0 is 10× slower than that at pH7.0 and 100× slower than that at pH8.0. Following the protease digestion, peptides were separated with liquid chromatography and identified by mass spectrometer. As shown, a time-dependent bimodal change of mass spectra was detected in various peptides of the metalloprotease domain (217-230 and 282-304)  B, the Cy282-304) . The res3.4In addition to the difference in the bimodal deuterium incorporation into the peptides in the full-length and the truncated ADAMTS-13 variant (MDTCS), a significant increase in the initial deuterium incorporation or the rate of deuterium incorporation as a function of time in the peptides, exemplified in 3.5Further analysis using the overlapping peptides allowed us to identify the specific amino acid residues (highlighted in red or underlined) in the CysR and/or spacer domains of the MDTCS fragment with an increased or accelerated rate  of deuterium incorporation compared with those in the full-length ADAMTS-13 protein at neutral pD  and 6.FiTogether, these results demonstrate dramatic alterations of deuterium accessibility, indicative of conformational changes in various discrete regions of ADAMTS-13, after its being split into 2 fragments. The findings suggest that direct domain–domain interactions may occur between the proximal and the distal segments of ADAMTS-13, which contributes to the overall stable ternary structure under physiological conditions.3.6Changing pH from 8.0 to 6.0 results in the activation of plasma ADAMTS-13, which increases its proteolytic activity by approximately 3-fold . This de3.7. To demonstrate the direct interactions do occur between the proximal and distal segments of ADAMTS-13, we performed a binding assay using surface plasmon resonance. As shown, following an injection of a purified recombinant ADAMTS-13 fragment  over a CM5 surface coupled with a CUB fragment, the RUs (or binding) increased as a function of time during an association phase and reduced during a dissociation phase. On fitting the data of maximum RUs as a function of concentration of an ADAMTS-13 variant, we were able to obtain the binding affinity. As shown, recombinant ADAMTS-13 variants MDTCS and DTCS, but not MDT (not shown), bound to immobilized CUB domains with dissociation constants at equilibrium (KD) of 0.33 μM and 0.46 μM, respectively  also show a pH-induced change, which may affect the overall proteolytic efficiency.Consistent with the biochemical and functional data in the literature, our HX-MS results further reveal the bimodal deuterium incorporation behavior in various peptides of the full-length ADAMTS-13, which is absent in the truncated ADAMTS-13 variant . More interestingly, most of these peptides derived from the CysR and spacer domains, as well as from the CUB domains in the truncated fragments, exhibit an increased initial and/or accelerated rate of deuterium incorporation as a function of time and lost pD-dependent deuterium incorporation. These results clearly indicate that in the absence of domain–domain interactions between the proximal and distal domains of ADAMTS-13, these peptides are more exposed to the surface and more accessible to the deuterium. Such a conformational change may explain why MDTCS has more proteolytic activity toward VWF substrates .Moreover, overall folding of the CUB domain is highly dependent on amino acid residues such as L1221, E1267, V1270, G1302, F1304, and G1305 . SimilarIt remains to be determined where exactly the distal domains interact with the proximal domains. It is possible that the distal CUB interacts strongly with the CysR and spacer domain, but weakly with other proximal domains including the metalloprotease domain. Evidence to support this hypothesis includes an increase in deuterium accessibility in the CysR and spacer domain in the MDTCS fragment with the distal domains being removed; the CUB domains bind directly to MDTCS and DTCS with a high affinity, but only weakly to MDT; site-directed mutation in the spacer domain (R568K/F592Y/R660K/Y661F/Y665F) appears to disrupt the distal domain interaction with the proximal domains, resulting in increased proteolytic activity ,37; moreComparison of truncated and full-length variants, as well as the bimodal distribution pattern, suggests the interaction of distal C-terminal domains with multiple discrete sites in the proximal domains of ADAMTS-13, resulting in protection from hydrogen–deuterium exchange. Increased deuterium uptake in the truncated variants at pH7.0 indicates quite exposed regions in them. ADAMTS-13 spacer exosite3  interact with VWF-A2 domain , whereas the exosite in the CysR and disintegrin domains (G471-V474) and (R349 and L350) may play a central role in recognizing VWF. These residues in the full-length ADAMTS-13 are not fully accessible to deuterium exchange while truncated variants  shows a relatively higher deuterium uptake than the full-length in various peptides.Our HX-MS findings provide a model structure where no experimental structures are available. The results provide further structural insight into ADAMTS-13 functions, which are essential for our understandings of the protease action in normal and pathological conditions. In addition, it supports further development of rationally designed therapeutics for TTP. Our future goal is to use this technique and information to elucidate the structure of specific ADAMTS-13 epitopes prone to the development of autoimmune responses and /or mapping of antibody epitopes. Highly sensitive and accurate HX-MS technique is compliment to other structural approaches and needs very little sample volume and has no limitation in protein size or class. It can be effectively applicable to challenging protein systems such as ADAMTS-13.Although HX-MS/MS can theoretically provide the best resolution and fastest analysis, there are a few drawbacks associated with: (1) it needs low temperature separation and high chromatography efficiency; (2) it is necessary to obtain high overlapping sequence coverage; (3) it needs a more efficient fragmentation system to obtain high resolution of peptide identification; (4) additionally, the glycosylation in the truncated vs. full-length ADAMTS-13 protein may be subtly different because they are expressed from 2 different cell lines; and (5) finally, certain areas without sufficient peptide recovery are difficult to assess for the deuterium incorporation, and pH change may result in protonation of certain residues within the domain, leading to conformational changes that are not entirely affected by the distal domain truncation.Nevertheless, this is for the first time that we are able to provide direct evidence that conformational changes occur at various proximal regions of ADAMTS-13 following its removal of the distal domains or lowering the pH (or pD). Such a conformational change may help expose the cryptic binding sites of ADAMTS-13 for substrate recognition and may alter its catalytic site in the metalloprotease domain, thus resulting in an enhanced proteolytic activity. Our results provide novel insight into the molecular mechanism underlying how domain–domain interaction or changes in pH environment may regulate ADAMTS-13 function.