Histriobdella homari – a minute ectocommensal of lobsters putatively belonging to the Eunicida – using different comparative morphological approaches, including SEM, TEM, CLSM and subsequent 3D reconstruction. The H. homari jaw apparatus is composed of ventral paired mandibles and dorsal symmetrical maxillae consisting of numerous dental plates, ventral carriers and an unpaired dorsal rod, and the general assemblage and arrangement of the different parts are highly comparable to those of other eunicid families. The jaw ultrastructure of histriobdellids resembles that of the families Dorvilleidae and (juvenile) Onuphidae. Furthermore, our data reveal that in the process of development of the jaw apparatus, the mandibles, maxillae II and unpaired dorsal rod are formed first, and the remaining maxillae and ventral carriers appear later. Notably, the muscular apparatus differs from that in Dorvilleidae and Onuphidae in terms of the number and arrangement of muscle fibers encompassing the jaws – not only because of the very small size of Histriobdella but also because histriobdellid maxillary protraction occurs due to straightening of the dorsal rod and thus requires a different muscular scaffold. Based on our investigations, the arrangement of the muscular apparatus of the jaws, the presence of paired ventral carriers and the dorsal rod, and the morphology of the ventral pharyngeal organ represent a histriobdellid autapomorphy. Our datasets form a basis for further comparative analyses to elucidate the evolution of Eunicida and jaw-bearing Annelida.The jaw apparatus in several annelid families represents a powerful tool for systematic approaches and evolutionary investigations. Nevertheless, for several taxa, this character complex has scarcely been investigated, and complete comparative analyses of all annelid jaws are lacking. In our comprehensive study, we described the fine structure of the jaw apparatus and the ventral pharyngeal organ (VPO) in  Annelids, one of the largest groups of lophotrochozoan animals , exhibitHistriobdella Van Beneden, 1858 and Steineridrilus Zhang, 2014, as well as the most species-rich taxon, Stratiodrilus Menchini, 1900, comprising 11 species [Histriobdellidae is a scarcely studied family of extant Eunicida with a still-unresolved phylogenetic position , 19, 20  species –24. The  species . Since t species –31. HoweHistriobdella homari Van Beneden, 1858, and described the muscular system and the movement of the jaws. They showed that backward movement of the maxillae is ensured by muscle contraction , while forward movement of the maxillae is associated with straightening of the dorsal rod.In their outstanding paper, Jennings and Gelder  providedHistriobdella and other Eunicida has not been dubious since the paper by Mesnil and Caullery [While the homology of mandibles and maxillary plates (mxI – mxIV) in Caullery , the queCaullery ) in otheCaullery  suggesteCaullery  did not Caullery , and no Histriobdella homari to place the histriobdellid jaw apparatus within the accepted classification of eunicidan jaws. Such comprehensive analyses and resulting hypotheses concerning putative homologies between different eunicidan jaw apparatuses will help elucidate the evolution of the jaw-bearing Eunicida. Furthermore, detailed analyses of underinvestigated annelid groups will deepen our understanding of Annelida character evolution scenarios in general.Hence, the present study aims to broaden our knowledge concerning detailed features of the histriobdellid jaw apparatus. Using a comparative set of morphological methods, we investigated the fine morphology and ultrastructure of the ventral pharyngeal organ and jaw apparatus in Histriobdella homari were collected from their hosts, Homarus gammarus , which were obtained from the central fish market in Bergen (Norway) in December 2016 and June 2017 and originated from the northern Atlantic. The adult and juvenile individuals were washed from their hosts using 7% MgCl2 in seawater (1:1). Histriobdellid eggs attached to the host were collected using dissecting needles.Specimens of H. homari were investigated in whole animal preparations using standard immunohistochemical staining protocols and phalloidin-rhodamine as a muscular marker. The staining was carried out using 15–25 specimens of each stage. The individuals were relaxed in a 7% MgCl2 seawater (1:1) solution and subsequently fixed in 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline with Tween  for 2 h at room temperature. After fixation, the specimens were stored in PTW containing 0,005% NaN3 until usage at 4 °C.Anatomical details of adults and developmental stages of For investigations, specimens were rinsed 2x for 5 min in PTW at RT (room temperature) and transferred into 10 μg proteinase K/ml PTW for 10 min. After 2 short rinses in glycine (2 mg glycine/ml PTW) and 3 5-min washes in PTW, the specimens were refixed using 4% PFA in PTW containing 0.1% Tween for 20 min at RT. Subsequently, the samples were rinsed 2x for 5 min in PTW, rinsed 2x for 5 min in THT  and incubated with rhodamine-labeled phalloidin in THT  overnight at 4 °C.Subsequently, the specimens were dehydrated in an ascending isopropanol series, cleared using Murray’s clear  and embedded between two cover slips using DPX mounting medium . The specimens were analyzed with a Leica TCS SP5 confocal laser-scanning microscope . The confocal image stacks were processed with Leica AS AF v2.3.5 (Leica Microsystems) and Imaris 9.3 .For electron microscopy (EM), the specimens were fixed in a 2.5% glutaraldehyde solution buffered in 0.05 M phosphate and 0.3 M saline (PBS) (pH 7.2) at 4 °C for 1 h and kept in the same buffer. For scanning electron microscopy (SEM), the specimens were postfixed in 1% OsO4 buffered in 0.05 M phosphate and 0.3 M saline at 4 °C for 30 min and then immediately dehydrated in an ascending ethanol series. The samples dehydrated in 100% ethanol were transferred into microporous specimen capsules  for subsequent critical-point drying. The jaws were freed from the surrounding tissue using proteinase K in PTW (overnight at RT), rinsed in distilled water, transferred to coverslips, and air dried. Animals were mounted on conductive carbon adhesive tabs on pin stubs . Both the jaws and the specimens were subsequently sputter-coated with 40% gold:60% palladium (Polaron SC502 Sputter Coater) and examined under a Zeiss SUPRA 55VP field emission scanning electron microscope. Image acquisition was performed using a secondary electron detector at a 3 kV accelerating voltage with a 30 μm aperture. The final images were processed using Adobe  Photoshop CC and Illustrator CC.For transmission electron microscopy (TEM) and the preparation of semithin section series, the fixed specimens were dehydrated in an ascending ethanol series, transferred to acetone and embedded in Spurr’s resin. The series of semithin and ultrathin sections were obtained with DuPont MT 5000 and Leica EM UC6 ultramicrotomes. The semithin sections were subsequently stained with 1% toluidine blue and 1% methylene blue in 1% sodium tetraborate. The ultrathin sections were stained with uranyl acetate  and lead citrate  and examined with a Jeol JEM 1011 transmission electron microscope. 3-D reconstructions were performed from a series of semithin sections. Based on the image stacks that were aligned into stacks using the software AMIRA 5.2.2 , the ventral pharyngeal organ and its musculature were reconstructed using the software Imaris 7.0.0 .H. homari is based on the terminology suggested by Tzetlin and Purschke [The description of the general anatomy of the stomodeum in Purschke , 18. ThePurschke .H. homari, the jaws are relatively large in comparison to the body size of the adult worms (approximately 1.3–1.5 mm for adult specimens investigated in this study). The entire ventral pharyngeal organ (VPO) in the respective specimens ranges from 70 to 80 μm in length and approximately 40–45 μm in width , which are located directly behind the mouth opening in the oral cavity  of muscle cells Figs. c. These The jaws in histriobdellids – as well as in other eunicids – comprise ventral mandibles and dorsal maxillae Figs. b. Every The paired mandibles are fused along the mandibular plates and attached to each other in the median part of the mandibular shafts Figs. b, d-f. TH. homari. The thickness of the mandibular plates and the mandibular shafts reaches 2.5 μm, whereas the margins of the mandibular complex are much thinner  bears four large teeth (up to 3 μm in height). The tooth located close to the basal part of the plate is the smallest Fig. c. The leThe maximal thickness of the maxillae reaches 0.5–1.0 μm Fig. a-c. The In the marginal areas of the maxillary plates, it is obvious that the solid, hard body of the maxillae represents a rapidly growing epicuticular part of the cuticle. The layer of living microvilli is preserved underneath Figs.  a-f. A lIn addition to the mandibles and maxillae, the histriobdellid jaw possesses additional supportive structures – the paired ventral carriers and the unpaired dorsal rod. The ventral carriers are represented by paired hardened structures with a complex (intricate) shape and a length of approximately 20 μm Fig. b, c. TheThe unpaired dorsal rod, the second prominent structure supporting the mandibles and maxillae, has a length of 70–75 μm and is formed in a deep invagination of the gnathoepithelium Fig. a. The paHistriobdella, the gnathoblasts surrounding the dorsal rod no longer exhibit prominent microvilli. No distinct features supporting the synthetic activity of the respective cells can be identified. The gnathoblasts have elongated nuclei up to 5 μm in length and approximately 1 μm in thickness. Furthermore, tonofilaments are well developed and provide a tight connection of the dorsal rod with the dorsal rod flexors  and adult specimens. Both cell types, i.e., the pharyngeal epithelial cells as well as the gnathoblasts, are underlain by a thin basal lamina (approximately 0.1 μm) Fig. ; Fig. 11The pharyngeal epithelium is covered by the cuticle and has no distinct ciliation, except for the heavily ciliated epithelial cells of the esophagus Fig. a. The cuHistriobdella jaw apparatus. In summary, our results largely corroborate the findings of [The combination of four different methods  employed here allowed an in-depth description of the anatomical features of the dings of  but alsodings of . Followidings of , the supdings of .Based on our investigations, the histriobdellid dorsal rod is histologically and positionally similar to the “carrier-like structure” found in Dorvilleidae  and in lDespite the fact that the ventral carriers move along the mandibular shafts, they are part of the maxillary apparatus since they are connected to the bases of maxillae I and II. The gnathoepithelium producing the ventral carriers is located on the lateral sides of the median invagination, where the maxillary gnathoepithelial zone is located.We suggest that the evolution of the unique paired ventral carriers is the result of the miniaturization of Histriobdellidae. A dramatic decrease in the number of VPO muscle cells led to extension of the spring-like dorsal rod to support the functionality of the entire structure. Simultaneously, the ventral carriers sliding along the shafts of the mandibles stabilize the feeding process and direct the movement of the whole maxillary apparatus. Interestingly, paired ventral carriers appear to be an autapomorphy of Histriobdellidae and have not been observed in any other extant Eunicida – at least not on the same level of structural complexity. Such paired structures are also unknown in the fossil record. Notably, in the process of stomodeum organogenesis, the ventral carriers appear latest of all elements of the maxillary apparatus.All other components of the maxillary apparatus – four pairs of maxillae and an unpaired supporting element represented by the dorsal rod – resemble the ctenognath jaw apparatus characteristic of Dorvilleidae  Fig. 7,. AccordiFurthermore, our investigations indicate that a significant part of the jaw apparatus – the maxillae II, mandibles and the dorsal rod – are fully developed in juveniles that have not yet emerged from the egg envelope. The remaining components of the jaw apparatus, namely, maxillae I, III and IV and the ventral carriers, develop shortly after juveniles hatch from the egg, since all free-living individuals  had a definitive and fully developed adult-like jaw apparatus. Such presence of an adult-sized jaw apparatus in juveniles hints at a pedomorphic developmental mode for the mentioned structure in histriobdellids. Whether pedomorphosis is responsible for the observed developmental characteristics must be investigated in the future, but comparable processes are well documented for several annelid families –34.To provide an in-depth comparative analysis of recent and fossil jaws in different families of Eunicida, more investigations dealing with the fine structure and development of the jaw apparatus are needed. This information is currently unavailable for two major eunicidan families, namely, Lumbrineridae and Oenonidae. For other eunicid groups, detailed investigations are also quite limited .In regard to the mechanical principles of the histriobdellid jaw apparatus, our results confirm and supplement the data of Jennings & Gelder . Thus, tHistriobdella’s VPO is rather small and strongly constant. The whole muscular apparatus of the VPO consists of only three muscle cells of the bulb, one pair of retractor cells of the carriers and a total of 18 cells (left and right) of flexors of the dorsal jaw apparatus, findings supporting those of earlier investigations by Jennings and Gelder [Histriobdella, the VPO muscle cells do not form a continuous muscle layer, as in most other polychaetes with a VPO . Insteamuscles, , 36. NevH. homari stomodeum provide further support that the jaws of these animals are very similar to the jaws of Dorvilleidae [Adercodon pleijeli [The ultrastructural data on the fine morphology of the illeidae , 18, as illeidae . In all pleijeli , 39 alsoHistriobdella, the gnathoepithelial cells forming the jaw elements underlie the jaw plates along their inner surface. They are actively functioning cells equipped with mitochondria and a distinct synthetic cellular apparatus in the basal zone. In contrast, the gnathoepithelium in the distal parts of the jaw plates is represented by somewhat degenerated parts of cells lacking mitochondria and endoplasmic reticulum but filled with an unstructured gray mass. This is very similar to the state of gnathoepithelial cells in the distal maxillary parts in Dorvilleidae [In illeidae , 18 and illeidae . Our datilleidae , 18, 19)illeidae .The stomodeum epithelium does not bear cilia, while the ciliature of the esophageal epithelium is very well pronounced – a condition that is similar to that in Dorvilleidae and Onuphidae as well , 13, 18.Our comparative and comprehensive investigations using a set of different morphological methods highly support the phylogenetic placement of Histriobdellidae within the Eunicida.Mooreonuphis stigmatis (Onuphidae) [Histriobdella homari are of the ctenognath jaw type, with an unpaired dorsal carrier-like element , unique ventral carriers and the lack of prionognath-like dorsal carriers. Comb-shaped ctenognath jaws have also been described in Dorvilleidae [[The quite simplified muscular scaffold surrounding the histriobdellid jaw apparatus might be the result of body miniaturization and adaptive evolution. Nevertheless, the ultrastructural characteristics of the histriobdellid jaws are very similar to those of Dorvilleidae jaws  and the uphidae)  in termslleidae [, Table 1


In recent years, transfer learning has been widely applied in fault diagnosis for solving the problem of inconsistent distribution of the original training dataset and the online-collecting testing dataset. In particular, the domain adaptation method can solve the problem of the unlabeled testing dataset in transfer learning. Moreover, Convolutional Neural Network (CNN) is the most widely used network among existing domain adaptation approaches due to its powerful feature extraction capability. However, network designing is too empirical, and there is no network designing principle from the frequency domain. In this paper, we propose a unified convolutional neural network architecture from a frequency domain perspective for a domain adaptation named Frequency-domain Fusing Convolutional Neural Network (FFCNN). The method of FFCNN contains two parts, frequency-domain fusing layer and feature extractor. The frequency-domain fusing layer uses convolution operations to filter signals at different frequency bands and combines them into new input signals. These signals are input to the feature extractor to extract features and make domain adaptation. We apply FFCNN for three domain adaptation methods, and the diagnosis accuracy is improved compared to the typical CNN. Modern machinery and equipment are widely used in industrial production, and their structures are sophisticated and complex. They are usually operated in a high-intensity working environment. Among them, rotating machinery plays an essential role in modern mechanical equipment, and is fragile and vulnerable to damage, significantly affecting the entire system’s stability. Therefore, fault diagnosis of rotating machinery is vital in the modern industry. To get better diagnosis results, it is critical to extract significant features. Traditional data-driven fault diagnosis methods extract features artificially from raw signals, namely handcraft features ,2,3. TheIn recent years, deep learning has been applied in fault diagnosis ,10,11, wWhile deep learning has achieved good performance in fault diagnosis, two problems need to be solved: (a) Exiting deep learning models require a lot of labeled data. However, sensors of industrial devices will produce a lot of unlabeled data in a short time, and labeling data is very time-consuming and labor-intensive . (b) Opesource domain, and the target domain is the object that we want to transfer knowledge to [domain adaptation, which is to learn common feature expressions between two domains to achieve feature adaptation [To solve the above problems, transfer learning, a branch of machine learning, has been employed in fault diagnosis . In tranledge to ,18. Baseledge to . In thisaptation ,20. Domaaptation . Howeveraptation ,22,23 toWe design the network architecture for fault diagnosis from the perspective of frequency-domain characteristics of convolution kernels. The motivation for network design has a clear physical meaning.For the first time, we use the amplitude-frequency characteristic curve to describe the frequency domain characteristic of the convolution kernels. This provides a new idea for analyzing the physical meaning of the convolution kernels.the proposed FFCNN is suitable for various domain adaptation loss functions, and can significantly improve the performance of domain adaptation for fault diagnosis without increasing the complexity of the networks.Dilated convolution is used in domain adaptation and fault diagnosis. Dilated convolution can improve the receptive field without increasing the number of parameters.While some papers have proposed similar network architectures of multi-scale convolution ,25,26,27The rest of this paper is organized as follows. In Deep learning for fault diagnosis. a variety of deep learning methods have been successfully applied in fault diagnosis in recent years. Jia et al.  proposesDomain adaptation for fault diagnosis. Domain adaptation method can use the unlabeled data for transfer learning. In the work of Li et al. , the muldomainZ is the learned features representation. Given two domains with different distributions named source domaintarget domaintransfer learning is to improve the performance of target domain using the knowledge of source domain, where We consider a deep learning classification task Homogeneous transfer learning. The input spaces of the source domain and target domain are similar and the label spaces are the same, expressed as Heterogeneous transfer learning. Both the input spaces and the label spaces may be different, expressed as From the perspective of input spaces and label spaces, transfer learning can be divided into the following two types:Supervised transfer learning. All data in the target domain have labels.Semi-supervised transfer learning. Only part of the data in the target domain have labels.Unsupervised transfer learning. All data in the target domain have no labels.Besides, according to whether the target domain contains labels, transfer learning can also be divided into following three types:Most of the research in recent years has focused on unsupervised homogeneous transfer learning , which iget risk :(1)errDTTo adapt the feature space of source domain and target domain, a specific criterion N is the number of channels, M is the dimensional of feature maps. The kernel of C is the number of channels in the output feature maps, H is kernel size. So the output of s is the stride step, and p is padding size to keep the input and output dimensions consistent. After convolution layer, a down-sampling layer is connected to reduce the number of parameters and avoid overfitting [s is the pooling step, and L is pooling size. Repeat convolution layer and pooling layer several times to deepen the network. Then the feature maps are flattened into one-dimension to connect a fully-connected layer. Finally, the softmax layer outputs the predicted classification probability:In this paper, a one-dimensional convolutional neural network is built to extract features and classify fault types. a typical CNN consists of convolution layers, pooling layers and a fully-connected layer. Let  follows :(2)xi,(orfitting :(3)xi,HdilaSo the dimension of output The standard convolution is the dilated convolution of The vibration signal is time domain signal, and most deep learning methods are designed from the perspective of time domain. But vibration signal can be composed of a series of sine wave signals with different frequencies, phases, and amplitudes, which are the frequency domain representations of the vibration signal. The vibration modes of different fault types are different, and the FFT spectrograms are also different, as shown in Let the input signal is Then a series of corresponding outputs the convolution kernels can be regarded as a series of filters, which can suppress signals in some single frequency bands.Different dilation rates have different AFC curves. Convolution kernels with a dilation rate To explore the meaning of the convolution kernel from a frequency domain perspective, we trained four CNN with different kernel sizes . The output of signal after the first convolution layer, AFC curve of one of the convolution kernels and FFT spectrogram of output are drawn in The above findings motivate us to design the network architecture from the perspective of the frequency domain. We change the first layer of CNN to a multi-scale convolution kernel fusion method. The input signal is preprocessed in multiple frequency bands before entering the next stage of feature extraction. Compared with single-scale CNN, the improved CNN can extract richer frequency domain information to improve CNN’s feature extraction ability.The architecture of the proposed FFCNN is shown in C channels and N dimensions, and the three feature maps are connected to a feature map with the shape of For FFL, there are three convolutional branches with different dilation rates in the first convolution layer. They can preprocess signals on multiple scales and produce feature maps with the same number of channels and dimensions. Then the three feature maps are connected in the channels axis and followed by a pooling layer. For example, there are three convolution layers with dilation rate For domain adaptation, the source data Let c is the number of labels. Here we only classify the labeled source feature representations Next a fully-connected layer with parameters To measure the discrepancy between the source and target feature representations, a certain criterion essed as :(15)minθAlgorithm 1 The algorithm of FFCNN back-propagation.Input: Labeled source domain samples   Output Network parameters   domain samples.Begin:  Initialization for while stopping criteria is not met do for each source and target domain samples of mini-batch size do     Calculate output    Equation (9).   Connect    to Equation (10).   Calculate features representations    Equation (11).   Calculate loss    Upgrade end for  end while To optimize the network, we calculate the gradient of objective function with respect to network parameters and upgrade parameters according to the backpropagation (BP) algorithm and mini-batch stochastic gradient descent (SGD) algorithm :(16)θclfi is the source domain, and working condition j is the target domain Bearing Data Center . Four diPaderborn dataset. This bearing dataset is provided by the Chair of Design and Drive Technology, Paderborn University . There aBoth above datasets are one-dimensional vibration signals, the example signals of CWRU and Paderborn dataset is shown in FFCNN is a method to improve the architecture of the domain adaptation network used in the feature representation based domain adaptation methods. These methods extract latent feature representations of the source domain and target domain, and reduce the discrepancy between them. Here we use three different discrepancy criterions: Maximum Mean Discrepancy (MMD), CORrelation ALignment (CORAL), and Central Moment Discrepancy (CMD).MMD: MMD criterion maps features to a Reproducing Kernel Hilbert Space (RKHS) to measure the discrepancy between source and target domain . It is dCORAL: CORAL criterion measures the discrepancy using the second-order statistics of source and target domain feature representations . It is dCMD: CMD criterion matches the domains by explicitly minimizing differences of higher order central moments for each moment order . It is dFor FFCNN, we use two dilate rate settings to evaluate the influence of dilate rate, one is The diagnosis results using CWRU dataset are shown in The effectiveness of domain adaptation. These tables show that source-only, without domain adaptation, performs poorly. In comparison, domain adaptation methods greatly exceed source-only in most tasks. For example, in task The effectiveness of FFCNN. FFCNN used different dilation rates to extract features at different scales, so that it may extract better features. Compared with ordinary CNN, FFCNN is more effective in most tasks. In some tasks, the effect of using FFCNN can be greatly improved. For example, in task The influence of dilation rate. To clearly illustrate the effect of dilation rate, the average accuracy of FFCNN with different dilation rates on all tasks is shown in Equation , the kerDilated convolution v.s. common convolution. Dilated convolution expands the receptive field by expanding the convolution kernel. According to Equation , the recFFCNN extracts features from multi scales using dilated convolution without increasing computational complexity, and different dilation rates represent different scales of the receptive field. To show the effect of frequency-domain fusing convolution, the performance of different single scale CNN is shown in Domain adaptation aims to align features of different domains. That is to say, domain adaptation will reduce the classification loss of source domain as well as the discrepancy between the source domain and target domain . So the features of different categories from the same domain can be dispersed as much as possible, and features of the same category from different domains can be gathered as much as possible. To illustrate the effectiveness of FFCNN from this perspective, we use task lgorithm  in FigurFFCNN is a unified domain adaptation architecture for fault diagnosis, it can also be applied to other CNN structures, domain adaptation methods or datasets.Which dilation rates are used to construct a FFCNN need to be determined according to the specific task, not necessarily AFC curve can be considered as a general CNN analysis method. It provides a new perspective for describing the characteristics of the convolution kernel.Multi-scale convolution kernels are generally applied in the first layer, and using multi-scale convolution in the middle layers has not been studied to prove its effectiveness.This paper has proved the effectiveness of FFCNN with a large number of experiments and explained it from multiple perspectives. For the application of FFCNN, we have the following suggestions:While FFCNN can improve the effect of domain adaptation, if the source domain and target domain are too different, FFCNN will also fail. How to further enhance the effect of domain adaptation still needs to be further studied .We explained the FFCNN from the perspective of frequency domain. How to improve the interpretability of deep learning methods for fault diagnosis is a more challenging task .While FFCNN is effectively applied in domain adaptation for fault diagnosis, we still face the following challenges regarding transfer learning and fault diagnosis:In this paper, a unified CNN architecture for domain adaptation named FFCNN using dilated convolutions with different scale is proposed. Experiments on two bearing datasets have proved the significant effect of FFCNN. Based on the results and analysis, three main significances of this paper can be concluded. First, the proposed FFCNN is driven from the perspective of frequency-domain characteristic. This inspires researchers to combine frequency-domain analysis with neural networks. Second, the frequency domain characteristic is described by the AFC curve, providing a new means to understand CNN. Third, results on different domain loss functions show that FFCNN is suitable for various domain adaptation losses. Thus, FFCNN provides an example for unified domain adaptation network design. While the proposed FFCNN has certain interpretability, it still does not fully explain the working principle of CNN. Further understanding of CNN to improve the effectiveness of fault diagnosis will be future work.


The internet has changed the way of people acquiring health information. Previous studies have shown that Wikipedia is a reasonably reliable medical resource, and it has been ranked higher than other general websites in various search engines. Baidu Encyclopedia is one of the most popular encyclopedia websites in China. However, no studies have shown the quality of the content provided in the Baidu Encyclopedia.This study aimed to evaluate the quality of liver disease information provided by Wikipedia (in English) and Baidu Encyclopedia (in Chinese) and to perform a comparison of the quality and timeliness of the articles published in these two encyclopedias. Moreover, a 3-year follow-up study was conducted to compare if the information in both these websites was updated regularly over this period.We searched for information on liver diseases by using the International Statistical Classification of Diseases and Related Health Problems 10th Revision Version 2016 codes on Wikipedia (in English) and Baidu Encyclopedia (in Chinese). The quality of the articles was assessed using the DISCERN instrument, which consists of 3 sections. We recorded the latest editing date of the webpages and calculated the date interval to evaluate the update timeliness of these websites.We found 22 entries on liver diseases in Baidu Encyclopedia and 15 articles in Wikipedia between September 15, 2016, and September 30, 2016, and we found 25 entries in Baidu Encyclopedia and 16 articles in Wikipedia between September 15, 2019, and September 30, 2019. In section 1 of the DISCERN instrument, the mean (SE) scores of Baidu Encyclopedia entries were significantly lower than those of Wikipedia articles. In section 2 and section 3 of the DISCERN instrument, the DISCERN scores of Baidu Encyclopedia entries were lower than those of Wikipedia articles, but the differences were not statistically significant. The total DISCERN scores of Baidu Encyclopedia entries were significantly lower than those of Wikipedia articles. The update interval of the entries in Baidu Encyclopedia was found to be significantly longer than that of the articles in Wikipedia.This study shows that the quality of articles and the reliability of the research content on liver diseases in Wikipedia are better than those of the entries in Baidu Encyclopedia. However, the quality of the treatment choices provided in both Wikipedia and Baidu Encyclopedia is not satisfactory. Wikipedia is updated more frequently than Baidu Encyclopedia, thereby ensuring that the information presented has the most recent research findings. The findings of our study suggest that in order to find accurate health information, it is important to seek the help of medical professionals instead of looking for a prescription amid the confusing information provided on the internet. Over 4.5 billion internet users were reported worldwide in June 2019, with 854 million of them being in China ,4. A stuWikipedia is a web-based encyclopedia that provides valuable web-based health information; it contains more than 5,956,750 articles in English -10. PrevLiver disease is among the top 10 causes of death in middle-income and high-income countries . We haveThe articles analyzed in this study are available in Wikipedia  in EngliThe selection of topics was based on the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10) version 2016. ICD defines almost all of the health-related conditions, and it is the diagnostic classification standard for all clinical and research purposes. In the ICD-10 version 2016, diseases of the liver are classiﬁed under Chapter XI Diseases of the digestive system, with categories K70 to K77 . The BaiThe quality of the articles was assessed using the DISCERN instrument, which is used for judging the quality of health information on treatment choices . The useThe DISCERN instrument consists of 16 questions that are rated on a 5-point scale  . All theAll 3 authors have been engaged in hepatobiliary and pancreatic surgery for many years and are competent in the diagnosis and treatment of liver diseases. We have mastered the professional knowledge of liver diseases and can make professional evaluations. Sun and Yang used the DISCERN instrument to evaluate the articles in Baidu Encyclopedia (in Chinese) and Wikipedia (in English), respectively. The DISCERN score was determined through discussions. If no agreement was reached during the debate, Zheng was consulted to make the final judgment. Subsequently, Sun and Yang agreed on all the ratings.We recorded and analyzed the last editing date of the web page. In 2016, the update interval was calculated as the date interval between the updated date and September 20, 2016. In 2019, the update interval was calculated as the date interval between the updated date and September 20, 2019. The update interval reflects the update frequency of a webpage. We compared the update interval of each paper to evaluate the update timeliness of the website.P values less than .05 were considered signiﬁcant. Differences between groups were assessed using two-tailed Student t test with Welch correction. Statistical analyses were performed using GraphPad Prism 6 software (GraphPad Prism Software Inc).For the statistical analysis, means and standard errors (mean [SE]) were calculated. We searched Baidu Encyclopedia and Wikipedia with the entries in the ICD-10 diseases of the liver category. A total of 8 liver disease categories were classiﬁed, with categories K70 to K77. Only a portion of the entries were retrieved as articles in the search results. We found 22 entries in Baidu Encyclopedia and 15 articles in Wikipedia in 2016 and 25 entries in Baidu Encyclopedia and 16 articles in Wikipedia in 2019 . In geneP<.001 in 2016, respectively; 19.00 [SE 0.9110] vs 27.88 [SE 0.8892], P<.001 in 2019, respectively). In section 2 and section 3 of the DISCERN instrument, the mean DISCERN scores of Baidu Encyclopedia entries were lower than those of Wikipedia articles, but the differences were not statistically significant. Overall, the total scores of Baidu Encyclopedia entries were significantly lower than those of Wikipedia articles   .The total DISCERN scores were categorized. In Baidu Encyclopedia in 2016, 9 entries were found to be of fair quality, 8 were of poor quality, and 8 were of very poor quality. In 2019, 10 entries were of fair quality, 11 were of poor quality, and 4 were of very poor quality. In Wikipedia in 2016, 5 articles were of good quality, 5 were of fair quality, 3 were of poor quality, and 2 were found to be of very poor quality. In 2019, 6 articles were found to be of good quality, 4 were of fair quality, and 5 were of poor quality.Over time, the number of entries in Baidu Encyclopedia and Wikipedia showed an increase . HoweverP<.001). After 3 years of follow-up, it was found that 11 of the 25 entries in Baidu Encyclopedia were recently edited before September 20, 2016, which meant that they had not been updated in the last 3 years and were in a state of loss of maintenance. In 2019, the mean (SE) update interval of Baidu Encyclopedia entries was 1067 (131.2) days and the mean (SE) update interval of Wikipedia articles was 27.44 (11.98) days. The update interval of the entries in Baidu Encyclopedia is significantly longer than that of the articles in Wikipedia (P<.001)  . TherefoThe main findings of this study were that the reliability of the articles on Wikipedia is better than that of the entries in Baidu Encyclopedia. However, the quality of the treatment information in both the encyclopedia websites is not satisfactory. With the increasing popularity of the internet, increasing number of patients are seeking health information and even treatment plans for their diseases on the internet . HoweverWe evaluated the articles on liver diseases in Baidu Encyclopedia and Wikipedia in terms of quantity and quality. The Baidu Encyclopedia has more overall entries than Wikipedia; therefore, it is reasonable for Baidu Encyclopedia to have more entries in the category of liver disease than Wikipedia. In Wikipedia, terms can be easily linked to each other, making it easier for readers to build connections of knowledge ,30. In BWe used the DISCERN instrument to evaluate the quality and reliability of the articles in Baidu Encyclopedia and Wikipedia. The DISCERN scores of Wikipedia entries were significantly higher than those of Baidu Encyclopedia entries. Consistent with that reported in previous studies, Wikipedia is a good resource of health information on the internet ,14. In sIn terms of the update timeliness, Wikipedia was significantly better than Baidu Encyclopedia in 2016 and 2019. During the 3-year follow-up, all Wikipedia pages were updated on time. As for Baidu Encyclopedia, among the 25 entries, 11 entries were not updated in 3 years and the longest update time reached 2564 days. Wikipedia is updated regularly and it evolves continuously while the content in Baidu Encyclopedia has been rarely maintained since its generation. The low update frequency has caused some information to be outdated, and some outdated information may even be wrong. Therefore, the timeliness of information is also an important aspect influencing the quality of medical information.Our study uses the DISCERN instrument to evaluate the quality of Baidu Encyclopedia entries and compares the quality of these entries with that of Wikipedia articles. This is the first study to assess the quality of health information in Baidu Encyclopedia with the DISCERN instrument. Moreover, this is the first study to follow up on the quality of encyclopedia websites for 3 years. In 3 years, we found that the quality of the articles of both websites had not improved significantly.Our study design does have some limitations. First, the results of our research only reflect the information on liver diseases in Baidu Encyclopedia and Wikipedia on September 20, 2016, and September 20, 2019. Second, the DISCERN instrument is the only tool used to evaluate article quality in this study. Although the DISCERN instrument is widely used to evaluate website information, comprehensive use of more evaluation tools may make the results more convincible. The LIDA tool is a web-based validation instrument to measure the design and content of health information on the internet . We planAlthough Wikipedia is not as good as the Baidu Encyclopedia in terms of the number of entries, its reliability is better than that of Baidu Encyclopedia, thereby making it more helpful to web-based health information seekers. However, the quality of the treatment choices in both Wikipedia and Baidu Encyclopedia’s entries is not satisfactory. Wikipedia is updated frequently to keep the information up-to-date. The entries in Baidu Encyclopedia lack maintenance and are not updated on time; some information is outdated and some new content are lacking. In order to find accurate health information, people are advised to seek the help of medical professionals instead of looking for a prescription amid the confusing information on the internet.


BRG1/SMARCA4 and its paralog BRM/SMARCA2 are the ATPase subunits of human SWI/SNF chromatin remodeling complexes. These multisubunit assemblies can act as either tumor suppressors or drivers of cancer, and inhibiting both BRG1 and BRM, is emerging as an effective therapeutic strategy in diverse cancers. BRG1 and BRM contain a BRK domain. The function of this domain is unknown, but it is often found in proteins involved in transcription and developmental signaling in higher eukaryotes, in particular in proteins that remodel chromatin. We report the NMR structure of the BRG1 BRK domain. It shows similarity to the glycine‐tyrosine‐phenylalanine (GYF) domain, an established protein–protein interaction module. Computational peptide‐binding‐site analysis of the BRK domain identifies a binding site that coincides with a highly conserved groove on the surface of the protein. This sets the scene for experiments to elucidate the role of this domain, and evaluate the potential of targeting it for cancer therapy. For example, several tumor types present elevated levels of BRG1, and multiple studies have shown that targeting BRG1 suppresses cell proliferation.We have also started to understand that at different stages of certain tumors SWI/SNF complexes can act either as tumor suppressors or as oncogenes.Human exome sequencing and genome‐wide association studies have revealed that mutations in SWI/SNF‐complex subunits are also linked to several neurodevelopmental disorders.The mutually exclusive ATPase subunits BRG1/SMARCA4 and BRM/SMARCA2 are core components of SWI/SNF complexes. They are multidomain proteins that contain both DNA and protein interaction modules Figure . These p23C/15N‐labeled protein using conventional heteronuclear methods,Examining the sequence of BRG1/SMARCA4 and BRM/SMARCA2 in a range of species, we identified a highly conserved region between residues 610 and 659, encompassing the BRK domain that is flanked by less conserved regions that are predicted to be intrinsically disordered (InterPro database). We expressed and purified this fragment, obtained the NMR assignments of Alignments of the region used for structure determination for BRG1 and BRM proteins from vertebrates as well as homologues from fly and worm, which only contain one SWI/SNF ATPase subunit is shown in Figure The residues flanking the structured region are conserved from zebrafish to human in both BRG1 and BRM. These residues likely constitute short‐linear‐interaction motifs (SLIMs) either mediating intra‐ or inter‐molecular protein–protein interactions. These sequences appeared at the same time as the duplication of the SWI/SNF ATPase subunit into the BRG1 and BRM paralogs, and may reflect a more‐complex regulation of SWI/SNF complexes in higher vertebrates. Interestingly two serine residues 610 and 613 in the N‐terminal flanking region have been reported to be sites for phosphorylation in multiple studies  cells. Cells were grown in 2XTY media at 37°C to mid‐log phase and induced with 1 mM IPTG. The temperature was reduced to 22°C and the cells were grown for a further 16 hr. Isotopically labelled BRK domain was prepared by growing cells in K‐MOPS minimal media containing 5NH4Cl and/or [13C]‐glucose. Cells where lysed by sonication, and the fusion protein was purified by Ni2+‐NTA affinity chromatography and then dialyzed overnight over night at 4°C in in the presence of TEV protease, which cleaves the BRK domain from the lipoyl‐domain. A second Ni2+‐NTA affinity chromatography step was carried out to remove the lipoyl domain and the BRK domain was further purified by size‐exclusion chromatography (SEC).The DNA encoding residues 610‐659 of human SMARCA4 were amplified from human cDNA by PCR and cloned into a modified pRSETA (Invitrogen) expression vector that produces proteins fused to N‐terminally His3.213C/15N‐labeled, 15N‐labeled, 10%13C‐labeled, and nonlabeled BRG1 BRK samples.1H‐15N HSQC, HNCACB, CBCA(CO)NH, HNCACO, HNCO, HBHACONH, and 1H‐13C HSQC. Additional assignments were made using 2D TOCSY and DQF‐COSY spectra. A set of distance constraints were derived from 2D 1H‐ and 3D 1H‐15N NOESY spectra recorded from a 1.5 mM sample with a mixing time of 100 ms. Assignment were made using ANSIG 3.3.All spectra were acquired using either a Bruker DRX800 or DRX500 spectrometers equipped with pulsed field gradient triple resonance at 20°C, and referenced relative to external sodium 2,2‐dimethyl‐2‐silapentane‐5‐sulfonate (DSS) for proton and carbon signals, or liquid ammonia for that of nitrogen. Assignments were obtained using standard NMR methods using 3.31H‐15N‐HSQC spectrum recorded in D2O at 278 K (pH 5.0). Candidates for the acceptors were identified using the program HBPLUS for the hydrogen bond donors that were identified by the H–D exchange experiments. When two or more candidates of acceptors were found for the same donor in different structures, the most frequently occurring candidate was selected. For hydrogen bond partners, two distance constraints were used where the distance (D)H‐O(A) corresponded to 1.5–2.5 Å and (D)N‐O(A) to 2.5–3.5 Å. Torsional angle constraints were obtained from an analysis of C′, N, Cα Hα, and Cβ chemical shifts using the program TALOS+.β resonances and chi‐1 angles were determined from DQF‐COSY and HNHB spectra and were confirmed by analyzing the initial ensemble of structures. Stereospecific assignments of Hγ and Hδ resonances of Val and Leu residues, respectively, were assigned using a fractionally 3C–labelled protein sample.Distance restraints were obtained from the analysis of 2D 1H and 3D‐15N‐ NOESY spectra integrated according to the cross‐peak strengths and calibrated by comparison with NOE connectivities obtained for standard inter‐residue distances within an α‐helices. After calibration, the NOE constraints were classified into the following categories: strong, medium, weak, and very weak, corresponding to interproton distance constraints of 1.8–2.8, 1.8–3.5, 1.8–4.75, and 2.5–6.0 Å, respectively. Hydrogen bond constraints were included for a number of backbone amide protons whose signals were still detected after 10 min in a 2D 3.4The atomic coordinates have been deposited in the Protein Data Bank, ID 6SY2.The authors declare no competing financial interests.G.Z. and M.B. designed this study. M.B. and M.D.A. performed the experiments. G.Z. and M.B. analyzed the data, drafted the main manuscript, and prepared all of the figures. All authors reviewed the manuscript.


Acrylic pressure-sensitive adhesives (PSAs) are used as fixatives between layers of a display. PSAs’ function is an important factor that determines the performance of the display. Of the various display types available, the touch screen panel (TSP) of smart devices is firmly related to the relative permittivity of the elementals. Therefore, adjusting the relative permittivity of the PSA is indispensable for driving the TSP. Accordingly, selected acrylic pre-polymers were polymerized and the pre-polymer was blended and cross-linked with monomers with different chemical structure to adjust the relative permittivity. The monomers were hexametyldisiloxane (HMDS), N-vinylcaprolactam (NVC), tert-butyl acrylate (TBA), and isooctadecyl acrylate (ISTA). The gel fraction and transmittance as a function of the monomers show a similar result to the pure acrylic PSA. However, the gel fraction value decreased to about 90% and the transmittance decreased to about 85%, due to the immiscibility between nonpolar HMDS and acrylic PSA. On the other hand, the adhesion properties were improved when NVC was added because of the polarity of the nitrogen group. In addition, the relative permittivity of the PSA decreased regardless of the monomer chosen. There was, however, a difference in the optimal content of each monomer, and NVC decreased from 4 phr content to about 3.4 in reducing relative permittivity. Through the above results, it was confirmed that NVC having a nitrogen group is most advantageous in lowering adhesion properties and relative permittivity, and necessitates further research based on the findings. Pressure-sensitive adhesives (PSAs) are widely used in various industrial fields, such as medical products, aircraft, space shuttles, electrical devices, optical products, and automobiles. In particular, the demand for functional PSAs is rapidly increasing in the display market . DisplayOptical clarity means a transmittance of 90% or more in the range of 400 to 800 nm and PSA satisfies this criteria defining the utility of optical clear adhesives (OCAs). PSA includes rubber, acrylic, silicone, and urethane, and acrylic PSA is widely used in terms of their optical clarity. Moreover, the process time can be significantly reduced, and UV crosslinking is considerably economical as a technique. As the transmittance of PSA is a very important factor in display quality, a low transmittance may limit its application in the display industry .A touch screen panel (TSP) is a major technology for increasing input convenience and reducing product weight by replacing the keyboard in the display of electronic devices. The demand for TSP has soared owing to the spread of smart phones and tablet PCs, and it is essential to improve touch sensitivity. A capacitive touch screen panel is composed of a top and bottom plate, on which a transparent electrode  is deposited and each layer is fixed using PSAs see . As partγ, dielectric constant) refers to the ratio of the permittivity of the material base relative to the permittivity in a vacuum. In other words, the relative permittivity is determined by the permittivity of a substance . The relative permittivity  that make-up electronic devices has been improved owing to an increase in transistor speed and a decrease in size. ICs have become faster and more complex because smaller transistors run faster. However, these efforts are causing a decrease in the speed of signal propagation. The signal delay can increase with the size of the electronic device, which can degrade the performance of the final product. In other words, continuous shrinkage increases the speed of the transistor, but decreases the interconnection between the transistors. A way to improve this is to use a non-conductor with a low dielectric constant . Non-conlectrics . In addilectrics . Therefolectrics ,21,22,23lectrics ,27,28,29k monomer in controlling the relative permittivity, adhesion performance, and optical property of the acrylic PSA for TSP. In addition, it was attempted to secure a reliable relative permittivity value of semi-solid samples at room temperature as a result of a low glass transition temperature (Tg).In this research, the possibility of adjusting the relative permittivity of monomers composed of various chemical structures was analyzed and mapped to behavioral changes in adhesion properties. An acrylic pre-polymer was synthesized via UV polymerization with acrylate monomers. In addition, four types of monomers were chosen and mixed with the pre-polymer. An acrylic PSA sheet was prepared via photo-crosslinking as a function of the monomer content. The crosslinking degree of the PSAs was confirmed via gel fraction and the transmittance was measured using a UV/vis spectrophotometer. The adhesion property was measured via the peel strength, probe tack, and lap shear strength. The relative permittivity was measured using a micro vacuum probe station to obtain reliable data as a function of chemical structure and content of the monomer. Through these results, we tried to confirm the applicability of the low-Reactive acrylic monomers, 2-hydroxyethyl acrylate , 2-ethylhexyl acrylate , and isobutyl acrylate , were purchased from Samchun Pure Chemical (Republic of Korea). They were used as received without further purification to synthesize the acrylic pre-polymers. Hexamethyldisiloxane , N-vinylcaprolactam , tert-butyl acrylate , and isooctadecyl acrylate  were selected as the monomers to adjust relative permittivity of the acrylic PSA via introduction of free volume see . 2-hydro2 purging tube  under a N2-rich atmosphere until the temperature of the mixture rose by 5 °C. The above process was iterated five times and the product was stored in a wide-mouth bottle to protect the acrylic pre-polymer from light and air.The acrylic monomers were blended with an Irgacure 1173 inside a 500 mL four-neck round-bottomed flask equipped with a stirrer, thermometer, and N2.PSA sheets with monomers were prepared by blending 100 wt.% of the synthesized acrylic pre-polymer with 1 part per hundred resin (phr) of HDDA and Irgacure 2100 as a function of the monomer content see . The mix0 and W1 are the weights before and after soaking and filtration, respectively.To confirm the influence of monomers on the crosslinking degree of the PSA, the gel fraction was measured as a function of the monomer content. The gel fractions of cross-linked PSA as a function of HDDA content were determined by soaking about 5 g of the PSA in toluene for 24 h at room temperature with shaking. The soluble part was removed by filtration and dried at 80 °C for 6 h to obtain a constant weight. The gel fraction was calculated by the following equation:Visible light transmittance of the specimens as a function of monomer type and content was measured in the wavelength of 400 to 800 nm using UV/vis spectrophotometry . The PSA specimens were prepared to a thickness of approximately 400 μm.The peel strengths of the PSAs according to the type and content of monomer were investigated using a texture analyzer . Firstly, 25 mm width PSA films were attached to a stainless steel (SUS) substrate and pressed twice by a 2 kg rubber roller. The peel strength was determined at an angle of 180° with a crosshead speed of 300 mm/min at room temperature based on ASTM D3330. The specimens were pressed onto stainless steel (SUS) substrates by two passes of a 2 kg rubber roller and then stored at room temperature for 24 h. Each sample was repeatedly measured three times, and the average value was calculated with N/25 mm.2 and a 2 kg rubber roller was passed over the film surface three times). The lap shear tests were performed at a crosshead rate of 5 mm/min. The shear strain rate values were calculated using the following equation:Lap shear testing was investigated using a texture analyzer. The tested specimens were cut into smaller pieces with a width of 25 mm. After being removed from a silicone release film, each PSA film was attached to another PET substrate (the adhesion cross-sectional area was equal to 25 × 25 mmThe shear stress property is one of the important factors of PSAs for display applications. Therefore, researching the relationship between the shear strain rate and the thickness of the applied PSA film is imperative for future use in the display industry .The cross-linking degree of the acrylic PSA was indirectly confirmed by calculating the insoluble portion in toluene. The underlying behavior of PSAs is related to adhesion, cohesion, and tack-related performance. Adhesion refers to adhesion to the substrate, cohesion refers to internal bonding strength, and tack refers to contact adhesion to the substrate . Thus, iThe relative permittivity in the low frequency region according to the type and content of the monomer was investigated as shown in In the PSA display, the value of the relative permittivity at a particular frequency is very important. In particular, the relative permittivity that accompanies the touch sensitivity of the TSP is related to a low frequency region. In this region, a degree of variability in the relative permittivity is expected and deviation from the required value varies with frequency. A study was conducted on the relative permittivity of PSA, which is closely related to the touch sensitivity of TSP. To lower the relative permittivity of PSA, four types of monomers were selected. The gel fraction value and transmittance according to the chemical structure and content of the monomer did not show a significant difference compared with the pristine acrylic PSA. However, at 10 phr of HMDS comprising a silane group, the gel fraction value decreased to about 90% and the transmittance decreased to about 85%. As a result, the adhesion performances deteriorated owing to immiscibility between the nonpolar HMDS and the acrylic molecular chain. In contrast, the adhesion properties improved as a result of the polarity reaction by the nitrogen group of NVC, TBA, and ISTA. Branched and long alkyl chain grouped monomers did not significantly affect the adhesion properties. In addition, it was confirmed that the monomers devoid of functional groups capable of reacting with the acrylic pre-polymer remained generating bubbles by heat evaporation and impacting the adhesion properties. Regardless of the type of monomer, the relative dielectric constant of the PSA decreased and the relative permittivity began to rise above a certain content. This is because of the generation of air bubbles due to heat generation in the cross-linking process, likely resulting from monomer aggregation. In hindsight, the optimum content of the monomer was effective in lowering the relative permittivity as a result of the formation of free volume, but the adhesion performances and the relative dielectric constant were affected by bubbles caused by evaporation.As shown in this study, the adhesion performance changed according to the type of monomer, and as a result, the relative permittivity can be adjusted according to monomer content. In the future, we plan to further investigate the application methods for monomer control, adhesion properties, and relative permittivity of acrylic PSA. We aim to study the behavior of adhesion performance and relative permittivity when the dispersibility of monomer is enhanced by producing a pre-polymer by simultaneously reacting reactive acrylic monomer and monomer via in situ polymerization.


YrU1, a stripe rust resistance gene from the diploid wheat Triticum urartu, the progenitor of the A genome of hexaploid wheat. YrU1 encodes a coiled-coil-NBS-leucine-rich repeat protein with N-terminal ankyrin-repeat and C-terminal WRKY domains, representing a unique NLR structure in plants. Database searches identify similar architecture only in wheat relatives. Transient expression of YrU1 in Nicotiana benthamiana does not induce cell death in the absence of pathogens. The ankyrin-repeat and coiled-coil domains of YrU1 self-associate, suggesting that homodimerisation is critical for YrU1 function. The identification and cloning of this disease resistance gene sheds light on NLR protein function and may facilitate breeding to control the devastating wheat stripe rust disease.Perception of pathogenic effectors in plants often relies on nucleotide-binding domain (NBS) and leucine-rich-repeat-containing (NLR) proteins. Some NLRs contain additional domains that function as integrated decoys for pathogen effector targets and activation of immune signalling. Wheat stripe rust is one of the most devastating diseases of crop plants. Here, we report the cloning of  YrU1, a stripe rust resistance gene from Triticum urartu, a diploid progenitor of common wheat, and show it encodes a NLR protein with unusual domain architectureWheat stripe rust is a major disease of wheat caused by a fungal pathogen. Here the authors report the map-based cloning of  Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating diseases of crops, causing major production losses in hexaploid wheat (Triticum aestivum L.) around the world1. Stripe rust has become the most damaging of all the crop rusts due to its expanding range and the resulting increased production losses2. To date, over 80 stripe rust resistance genes have been identified and mapped in wheat3, but only a few, including Yr5, Yr7, YrSP, Yr15, Yr18/Lr34, Yr36, Yr46 and YrAS2388, have been cloned9. Among these, Yr18/Lr34 encodes a putative ATP-binding cassette (ABC) transporter; Yr36 encodes a protein with a kinase domain and a putative lipid-binding domain; Yr46 encodes a hexose transporter; and Yr15 encodes a protein with predicted kinase and pseudokinase domains. These genes encode different protein families and confer a relatively broad spectrum of resistance. In contrast, YrAS2388 encodes a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) proteins (NLRs), and Yr5, Yr7 and YrSP encode NLR with additional BED domain; all of these genes are race specific, being effective against only a subset of Pst isolates at the seedling or all stages9. The robustness of stripe rust resistance genes has been challenged by coevolution between wheat and stripe rust pathogens in which the pathogens have evolved the capacity to overcome the resistance conveyed by these genes10. For instance, Yr7, which confers resistance to many stripe rust isolates, has been overcome by newer isolates12.Wheat stripe rust, caused by 13. Most NLRs contain three domains: an N-terminal variable domain, NBS domain and C-terminal LRR14. Several also have additional functional domains, which are hypothesised to act as integrated decoys. For instance, the Arabidopsis thaliana (Arabidopsis) resistance protein RRS1 has a C-terminal WRKY domain; the rice (Oryza sativa) resistance protein RGA5 has a C-terminal RATX1 domain; the wheat stripe rust resistance proteins Yr5, Yr7 and YrSP have an N-terminal BED domain; and the tomato resistance proteins Sw-5b and Prf have an extra N-terminal domain18. Among these, the RATX1 domain of RGA5 directly binds to a Magnaporthe oryzae effector, and activates RGA4-mediated resistance and cell death upon recognition19. The WRKY domain of RRS1 functions as a decoy to bind the pathogen effector and triggers immune signalling mediated by its neighbouring NLR RPS421. The additional domains in other NLRs are not well characterised.Plants use intracellular NLRs to detect pathogens by direct or indirect recognition of pathogen effectorsT. urartu is the progenitor species of the A genome of common wheat and a potential source for stripe rust resistance genes23. The high-quality genome sequence of T. urartu has been published and can be used for map-based cloning of genes25. In this study, we map-based cloned a stripe rust resistance gene from the resistant T. urartu accession PI428309. The gene encodes a CC-NBS-LRR protein with an N-terminal ankyrin-repeat (ANK) domain and a C-terminal WRKY domain. The ANK-NLR-WRKY domain is rare, and this type of protein/gene structure is found only in the genomes of wheat and its relatives. The identification and cloning of this type of stripe rust resistance gene will not only help to uncover new mechanism of disease resistance but also facilitate breeding for disease resistance in wheat.The diploid wheat species T. urartu, we infected a large number of T. urartu accessions at the seedling stage with the Puccinia striiformis f. sp. tritici (Pst) isolate CYR33 and observed the disease severity 14 days later. G1812, the accession used for the reference genome sequence24, was highly susceptible to Pst CYR33, displaying a large number of visible uredia (spore-bearing pustules characteristic of rust disease) and lacked visible cell death (which is indicative of a resistance response) on the leaves at 14 days post inoculation  staining, fluorescence micrographs of the fungal structures showed that substomatal vesicles (SSV), primary infection hyphae (PH) and haustoria mother cells (HMCs) were formed in both PI428309 and the susceptible accession G1812, and haustoria were already found in G1812, but not in PI428309  markers on the common wheat chromosomes (1A to 7A) to analyse the polymorphisms between PI428309 and G1812. Eighty-two polymorphic markers were used to screen the susceptible plants of the F2 population derived from the cross between PI428309 and G1812. We discovered that marker Xgpw7007-5A , SCF13 (from scaffold 21752), SCF19 (from scaffold 9979) and SCF20 (from scaffold 49434)  markers with those sequences  in the deletion bins 5AL12-0.35-0.57 and 5AL10-0.57-0.78 to search the genome sequences of G1812 using BLASTN. We then used the scaffolds of G1812 in the deletion-line bins to develop SSR markers. Through this method, we developed several markers closely linked with the stripe rust resistance locus 5AL Fig. . This reTuG1812G0500003718, TuG1812G0500003719 and TuG1812G0500003720 all encode NLRs. We obtained two candidate genes in PI428309 allelic to TuG1812G0500003718 and TuG1812G0500003719, which we designated YrU1 and CG2 based on subsequent analysis . The expression of YrU1 was only slightly affected by infection -induced gene silencing system. This silencing impaired the plants’ resistance to stripe rust Pst CYR33. At 14 dpi, the leaf surfaces of BSMV:YrU1-treated plants displayed numerous visible uredia, in contrast with the BSMV:GFP control plants, which had few uredia , YrU1 has integrated domains at both its C and N termini. The domain structure of the YrU1 protein is very unusual: we identified only three ANK-NLR-WRKY genes across the genomes of all species from the public reference proteome database UniProt. Those three ANK-NLR-WRKY genes are from T. urartu (YrU1), T. aestivum and emmer wheat (Triticum dicoccoides). We also performed a BLASTP analysis with the YrU1 protein in T. durum Svevo whole genome and did not find any ANK-NLR-WRKY protein, indicating that YrU1-type genes exist only in Triticum species  and 17 NLR-WRKY genes across the wheat genomes and the genomes of the related grass species Aegilops tauschii, Triticum urartu, Hordeum vulgare (barley), Brachypodium distachyon, Oryza sativa (rice) and Zea mays (corn)  Fig. . Sequencrn) Fig. . The seqT. urartu genome  show that WRKY domains are diverse, but are split from NLR-WRKY proteins and non-NLR proteins , did induce cell death in N. benthamiana leaves  race CYR33, indicating that the allelic YrU1 in Chinese Spring is nonfunctional against Pst CYR33. The high level of identity of the three Triticum ANK-NLR-WRKY proteins suggests that they derive from a common evolutionary origin and that the YrU1 variant in Chinese Spring lost the resistance to stripe rust during the evolution of the rust strains. This type of NLR protein is unique to wheat-related families and may reflect the existence of a specific NLR-mediated disease resistance mechanism in wheat relatives.YrU1 is a NLR protein that contains an N-terminal ANK and a C-terminal WRKY domain. ANK-NLR-WRKY proteins are rare: we identified only three ANK-NLR-WRKY proteins from the public reference proteome databases UniProt and Ensemble. All three are found in Arabidopsis resistance protein RRS1, with a C-terminal WRKY domain; rice blast resistance protein RGA5, with a C-terminal RATX1 domain; tomato resistance protein Sw-5b has an extra N-terminal domain; and wheat stripe rust resistance proteins Yr5, Yr7 and YrSP, with N-terminal BED domains19. YrU1 is the first NLR protein reported with extra domains at both the N and C termini. YrU1’s N-terminal ankyrin repeats, a 33-amino-acid sequence motif, are generally involved in protein–protein interactions33. The best-characterized ANK protein from plants is Arabidopsis NPR1, which is a master regulator of the salicylic acid (SA) signalling pathway and functions as a transcriptional coactivator to regulate plant defence response35. The SUMO-interaction motif (SIM3) located in the ANK domain of NPR1 is modified by SUMO3 to regulate the defence response36. Pseudomonas syringae type III effector AvrPtoB targets NPR1 and subverts plant innate immunity by repressing NPR1-dependent SA signalling37. ACD6 and BDA1 are also ANK proteins and function as positive regulators of SA signalling in defence responses40. Although YrU1 contains an ANK domain, it does not show high similarity to NPR1, ACD6 or BDA1. YrU1 is an NLR protein, whose ANK domain may interact with other NLR proteins or act as an integrated decoy domain to interact with the effector of the pathogen, thereby inducing plant immunity.Several NLR resistance proteins reported previously have additional domains, such as the Arabidopsis resistance protein RRS1-R is a TIR-NBS-LRR protein with a C-terminal WRKY domain that acts as an integrated decoy to detect and bind the bacterial effectors PopP2 and AvrRps4 to trigger the plant immunity21. Thus, the YrU1 protein, which has a WRKY domain similar to that of RRS1-R, may recognise and bind stripe rust effector through that domain, thereby activating immune responses.The C-terminal WRKY domain of YrU1 is a putative transcriptional domain. The N. benthamiana leaves. NLR oligomerization via N-terminal domains, which are commonly coiled-coil or TIR domains in plants, is thought to initiate disease resistance signals by recruiting or activating downstream signals41. Indeed, previous studies of several NLR proteins, such as MLA10, Sr33, Sr35, N, L6, RRS1-R/RPS4 and RGA4/RGA5, indicate that their N-terminal domains can self-associate42. MLA10, Sr33, Sr35 and L6 can induce an effector-independent cell death response in planta29. Recent structural work has also shown that oligomerisation is critical for NLR activation44. However, RRS1-R or RGA5 triggering of cell death requires their partners RPS4 or RGA4 and pathogen effectors20. YrU1, like RPS4 or RGA4, may require other NLR proteins and pathogen to trigger the defence responses.Here, we showed that the ANK and CC domains of YrU1 self-associate. However, neither the individual YrU1 domains nor full-length YrU1 induced cell death in It is possible that YrU1, like previously identified NLR proteins (such as RRS1 and RGA5), initiates the resistance response by recognising and binding a stripe rust effector through integrated decoy domains. However, YrU1 differs from these other NLR proteins in having integrated domains at both its C and N termini, and YrU1-type proteins exists only in wheat and its relatives. These two domains may thus play different roles in activating resistance responses than other NLR proteins do, and YrU1-mediated disease resistance may represent a mechanism present only in wheat. Activation of YrU1 may represent a unique molecular mechanism in NLR-mediated plant immunity. Based on current information, we present a working model of how YrU1 functions  was used to maintain the Pst race used in the experiments. The susceptible spring common wheat cultivar Bobwhite was used to generate the transgenic plants.Pst races CYR17, CYR31, CYR32, CYR33 and V26 were used in the experiments. We inoculated the stripe rust by spraying the mixture of urediospores and talcum powder at a ratio of 1:2 at the two-leaf stage, and talcum powder was used as an indicator of the urediospores on the leaves during the inoculation9. Immediately after inoculation, plants were incubated in a greenhouse at 10 °C with 100% relative humidity in the dark for 24 h. The plants were then moved to a greenhouse at 16–18 °C under a 14-h-light/10-h-dark cycle. The disease infection type (IT) was evaluated at 14 dpi. Stripe rust infection types were assessed based on a 0–4 scale45. In details, IT 0: immune, no visible uredia and necrosis on leaves; IT: nearly immune, no uredia with hypersensitive flecks on leaves; IT 1: very resistant, few small uredia with distinct necrosis on leaves; IT 2: moderately resistant, few small- to medium sized uredia with dead or chlorosis on leaves; IT 3: moderately susceptible, a lot of medium-sized uredia, no necrosis, but with chlorosis on leaves; IT 4: highest susceptible, a large number of large-sized uredia without necrosis on leaves.47, with minor modification. Briefly, the leaves were cut into 2-cm pieces and placed in a 10 ml centrifuge tube with 5 ml of 1 M KOH and 0.05% Silwet L-77. After 12 h, the KOH solution was gently poured off and washed with 10 ml of 50 mM Tris (pH 7.5). This solution was then replaced with another 10 ml of 50 mM Tris (pH 7.5). After 20 min, the Tris solution was removed and replaced with 5 ml of 20 μg ml-1 WGA-FITC. Tissue was stained for 15 min and then washed with 50 mM Tris (pH 7.5). The WGA-FITC-stained tissue was examined under blue light excitation with a Zeiss LSM 880 confocal microscope.To visualise the average infection area, the infected leaves were detached at 2 and 14 dpi and stained with WGA-FITC  as described previouslyYrU1. First, the SSR markers were used to screen the polymorphisms between PI428309 and G1812, and then eighty-two polymorphic markers were used to construct linkage map using the susceptible plants of the F2 population derived from the cross between PI428309 and G1812. The genetic map was constructed by software JoinMap 4.0. Closely linked flanking markers were developed from the scaffolds of G1812 and designed using SSR Locator software48 and dCAPS Finder 2.0 (http://helix.wustl.edu/dcaps/dcaps.html). The polymorphic markers were used to screen the susceptible plants of the F2 population for the recombinants. In total, 3304 susceptible plants were used to map the resistance gene.The simple sequence repeat (SSR) markers on hexaploid wheat chromosomes (1A to 7A) were used to generate the initial genetic map of Total RNA was isolated using TRIzol reagent (Invitrogen). First-strand cDNA from total RNA was synthesized using murine leukemia virus reverse transcriptase (Promega) and quantitative reverse transcription PCR (qRT-PCR) was performed with SYBR green kit (Takara) following the manufacturer’s instructions.TuG1812G0500003718, TuG1812G0500003719 and TuG1812G0500003720. We obtained the DNA sequences of two candidate genes in PI428309, which we designated YrU1 and CG2 based on subsequent analysis, by PCR amplification using gene specific primers YrU1-C and CG2-C according to the sequences of TuG1812G0500003718 and TuG1812G0500003719, respectively. We obtained the coding sequence of YrU1 by PCR amplification from the cDNA of PI428309 using the primer YrU1-C, however, we could not obtain the coding sequence of CG2 as expression of CG2 was not detected. We also validated the coding sequence of YrU1 by rapid amplification of 3’ and 5’ cDNA ends.In order to clone the candidate genes in PI428309, we designed primers based on the sequences of http://www.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind) and translated using the DNAMAN tool (v9.0.1.116). The YrU1 encoded an ANK-NLR-WRKY protein based on the functional annotation using the SMART program (http://smart.embl-heidelberg.de/), the LRRsearch program (http://lrrsearch.com/) and the CD-search online tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).The predicted open reading frame of the coding sequence based on the sequence of 3’ and 5’ RACE was performed with the online programs  (UniProt references proteomes) to identify conserved domains in protein sequences from the T. urartu genome. We applied default parameters to filter out any unrelated identified domains. ANK domains of the corresponding proteins in the output from hmmer were verified on the CD-search database. We divided the group between ANK-NLRs and non-NLRs based on the presence of NB-ARC domains. The phylogenetic tree analyses were performed in MEGA (v7.0.26). We used the same method to generate a phylogenetic tree of WRKY domains.We used the neighbour-joining method implemented in MEGA (v7.0.26) to analyse the relationships between ANK domains from ANK-NLR and non-NLR proteins. The phylogenetic tree analyses were performed as described previouslySaccharomyces cerevisiae strain AH109.Yeast two-hybrid assays were performed in the Matchmaker GAL4 Two-Hybrid System 3 (Clontech). To examine the self-association of the ANK and CC domains of YrU1, the coding sequences of YrU1-ANK and YrU1-CC were cloned into pGADT7 or pGBKT7. Different pairs of constructs were co-transformed into Agrobacterium tumefaciens GV3101 were suspended in infiltration buffer as previously described50 to OD600 = 1.5. Transient expression, total protein extraction and immunoblot analysis were performed as described51, with minor alterations. Total protein extracts (1 ml) were incubated with 20 μL agarose-conjugated anti-GFP antibody  at 4 °C for 4 h with gentle rotation. After incubation, the agarose beads were washed four times with phosphate-buffered saline (PBS) containing 0.008-0.01% (v/v) Triton X-100 and resuspended in 80 μL PBS; 20 μL 5× SDS/PAGE loading buffer was added and then the mixture was boiled for 5 min. The proteins were detected with anti-MYC , anti-GFP  and anti-HA  immunoblot.Different plasmids transformed into N. benthamiana by infiltration with Agrobacterium GV3101. After 2 days, 1 mM precooled luciferin was sprayed onto the leaves, and the samples were incubated in the dark for 5–10 min. LUC images were captured using a cooled CCD imaging apparatus52.To examine the self-association of the ANK and CC domains of YrU1, the coding sequences of YrU1-ANK and YrU1-CC were cloned into pCAMBIA1300-nLUC or pCAMBIA1300-cLUC. YrU1-ANK-nLUC/YrU1-ANK-cLUC or YrU1-CC-nLUC/YrU1-CC-cLUC were cotransformed into Primers used in this study are listed in Supplementary Table Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1


Reciprocating Upsetting-Extrusion (RUE) deformation process can significantly refine the grains size and weaken the basal plane texture by applying a large cumulative strain to the alloy, which is of great significance to weaken the anisotropy of magnesium (Mg) alloys and increase the application range. In this paper, the Mg-8.27Gd-3.18Y-0.43Zr (wt %) alloy was subjected to isothermal multi-passes RUE. The microstructure and texture evolution, crystal orientation-dependent deformation mechanism of the alloy after deformation were investigated. The results clearly show that with the increase of RUE process, the grains are significantly refined through continuous dynamic recrystallization (CDRX) and discontinuous dynamic recrystallization (DDRX) mechanisms, the uniformity of the microstructure is improved, and the texture intensity is reduced. At the same time, a large number of particle phases are dynamically precipitated during the deformation process, promoting grain refinement by the particle-stimulated nucleation (PSN) mechanism. The typical [10-10] fiber texture is produced after one pass due to the basal plane of the deformed grains with a relatively high proportion is gradually parallel to the ED during extrusion process. However, the texture concentration is reduced compared with the traditional extrusion deformation, indicating that the upsetting deformation has a certain delay effect on the subsequent extrusion texture generation. After three or four passes deformation, the grain orientation is randomized due to the continuous progress of the dynamic recrystallization process. Magnesium (Mg) alloys as the first choice for lightweight materials have attracted wide attention in the aerospace and military fields due to their high specific strength, specific stiffness, excellent thermal conductivity ,2,3. HowSevere plastic deformations (SPDs) can effectively refine grains and weaken texture through multiple-passes repeated deformation of a certain volume of metal to obtain a large cumulative strain , thus atIn addition to weakening the texture and improving the forming performance of the alloy from the perspective of the deformation method, another important way is to add rare earth elements for alloying. For instance, the addition of Zr element could refine the grains size and the Gd element was beneficial to the formation of precipitation phase with strong thermal stability ,17,18,19In this paper, the Mg-8.27Gd-3.18Y-0.43 Zr (wt %) alloy was subjected to isothermal multi-passes RUE process. The microstructure and texture evolution during RUE process were investigated. Further, the relationship between texture evolution and dynamic recrystallization behavior were also examined in detail. Understanding the relationship between microstructure evolution and texture change during the deformation process is conducive to obtaining a reasonable texture type and intensity by control the deformation mode or deformation parameters.The Mg-8.27Gd-3.18Y-0.43 Zr (wt %) alloy used in this research is a cylindrical bar with 50 mm diameter and 220 mm height turned from the cast rod. Before RUE deformation, the material was homogenized at 520 °C for 16 h to eliminate dendrite segregation that occurs during the casting process, followed by air cooling. The RUE processing was carried out by pushing a specimen into the cavity with the diameter of D for upsetting firstly and then extruded it with the diameter of d, as shown in The microstructure of RUE samples is characterized by optical microscope  and scanning electron microscope  under an accelerated voltage of 20 kV. Specimens for OM measurement are mechanically polished and chemically etched using a solution with 1 g picric acid, 2 mL acetic acid, 2 mL distilled water and 14 mL alcohol. X-ray diffraction (XRD) was used for phase identification during RUE process. Furthermore, the SEM equipped with electron back-scattered diffraction (EBSD) was used to observe the evolution of grain orientation. TSL OIM analysis version 7.3  was used to quantify the details on the grain misorientation angles, grain size, micro-texture, kernel average misorientation, and Schmidt Factor.The microstructure evolution of the samples is shown in Study  had showIn order to further understanding the deformation mechanism and lattice orientation texture during RUE process, the microstructures after RUE were further investigated using EBSD. When the alloy is continuously deformed, the texture will change significantly due to the formation of preferential orientation, especially in the Mg alloy with less slip system ,32,33. FAfter cumulative strain up to 2.7, the (0001) pole figure b shows aIn order to further reveal the influence of different dynamic recrystallization mechanisms on orientation evolution in unDRXed regions and DRXed regions during RUE process, typical unDRXed regions R1 and DRXed regions R2 were selected from 1, 2, passes, respectively, and analyzed in detail. The results are presented in It can be found from Due to the different initial orientations, the order of the grains forming the basal plane orientation with the extrusion deformation is different. Some grains with preferential basal plane orientation continue to deform due to stress in the subsequent deformation, and gradually change the grain orientation. The LAGBs represented by the white line in G2 are located at the position separating the region of different orientation. As the amount of strain increases, the previously formed sub-grains or LAGBs will absorb the new dislocations and transform into HAGBs, completing the typical CDRX process. In order to further understand the deformation mechanism of this transformation, During the subsequent upsetting deformation of the alloy after one pass, the c-axis of unDRXed grains to continue to deflect to ED, but initial grains (RUE1) orientation caused by extrusion weakens this deflection, which makes the angle between the c-axis of unDRXed grains and the equatorial axis decreases after extrusion during 2 passes, as shown in As the proportion of DRXed grains continues to rise, the grain orientation is gradually randomized after 3 passes. It can be seen from the previous description that CDRX process only rotates the crystal around the c-axis without changing basal plane orientation, so another DRX mechanism different from CDRX may have occurred during the later deformation. Chen et al.  performeThe texture intensity is further weakened accompanied by the dispersion of grain orientation after 4 passes. In addition to the increase in the proportion of DRXed grains formed by DDRX, it is also closely related to the deformation mode. With continuous grain refinement during RUE process, the texture formed by the single upsetting or extrusion deformation becomes more restrictive to each other, thus restricting the formation of a strong texture. It should be noted that the addition of rare earth elements also has an important effect on the texture weakening and the evolution of texture types. Compared with the texture obtained by repetitive extrusion deformation of pure Mg by Dong et al. , grain oThe RUE deformation has a significant effect on the grain refinement of the Mg alloy. The fraction of DRXed grains is significantly improved and homogeneous microstructure with statistically average grain size of 7.6 μm was obtained.5(Gd-Y) phase is dynamically precipitated during RUE process. With the increase of cumulative strain, the number of particle phases also increases, which is beneficial to promote the generation of DRXed grains through the PSN mechanism and accelerate the process of microstructure refinement. In addition, the precipitated particle phase greatly hinders the migration of GBs through the pinning effect, thereby hindering the growth of DRXed grains.MgRUE deformation can effectively weaken the basal plane texture through CDRX and DDRX mechanisms. In this deformation process, the upsetting has a certain effect on the deflection of the c-axis ED in the subsequent extrusion deformation, so the grain orientation is gradually dispersed with the increase of passes. At the same time, the rare earth elements are added to the alloy. It also has a certain effect on texture weakening.In this paper, Mg-Gd-Y-Zr alloy is used for multi-passes isothermal RUE deformation. The microstructure and texture evolution of materials during RUE process are studied. In addition, the relationship between the grain refinement mechanism and texture weakening is investigated. The main conclusions are summarized as follows: