Augmented reality (AR) is an emerging technology in which information is superimposed onto the real world directly in front of observers. AR images may behave as distractors because they are inside the observer’s field of view and may cause observers to overlook important information in the real world. This kind of overlooking of events or objects is known as “change blindness.” In change blindness, a distractor may cause someone to overlook a change between an original image and a modified image. In the present study, we investigated whether change blindness occurs when AR is used and whether the AR presentation method influences change blindness. An AR image was presented binocularly or monocularly as a distractor in a typical flicker paradigm. In the binocular presentation, the AR image was presented to the both of the participants’ eyes, so, it was not different from the typical flicker paradigm. By contrast, in the monocular presentation, the AR image was presented to only one eye. Therefore, it was hypothesized that if participants could observe the real-world image through the eye to which the AR image was not presented, change blindness would be avoided because the moment of change itself could be observed. In addition, the luminance of the AR image was expected to influence the ease to observe the real world because the AR image is somewhat translucent. Hence, the AR distractor had three luminance conditions , and we compared how many alternations were needed to detect changes among the conditions. Result revealed that more alternations were needed in the binocular presentation and in the high luminance condition. However, in all luminance conditions in the monocular presentation, the number of alternations needed to detect the change was not significantly different from that when the AR distractor was not presented. This result indicates that the monocular presentation could attenuate change blindness, and this might be because the observers’ visual attention is attracted to the location where the change has occurred automatically. Augmented reality (AR), in which the information is superimposed onto the real world directly in front of an observer, is one of the most promising information presentation technologies to emerge in recent decades . AR is vHowever, AR has not only tremendous merits but also some problems. AR superimposes information onto the real world directly, which means that there is a risk that AR information presented over real-world objects will prevent users from observing those objects , 2015. TTo address this problem, monocular AR presentation has been proposed , 2015. ANevertheless, although the advantage of the monocular AR presentation was revealed in previous studies, AR has been barely researched in psychology because it is just emerging and has been studied mainly in computer science and engineering. One problematic psychological phenomenon that may occur in actual AR use is “change blindness” . A changOne of the most typical experimental procedures for change blindness is the “flicker paradigm” . In the There are two major explanations of why change blindness occurs. One claims that attention must be directed to the place the change occurs at the moment the change occurs to form robust visual representation . The othThis theory is called “coherence theory”  and propThe flicker paradigm and coherence theory imply that AR presentation may lead to change blindness. For example, in a driving scenario, an AR image may be presented in front of a driver. In this situation, a child may jump into the road simultaneously as the AR image is presented. This situation is very similar to the flicker paradigm procedure: the situation before the AR image is presented is an original image, the AR image itself is a distractor, and the situation after the emergence of the child is a modified image. Therefore, the driver may overlook the child because the AR image behaves as a distractor that prevents the driver from directing his/her attention to where the child is, and the result may be a fatal accident. Because of the concept of AR, the AR image is always inside the field of view of a user . TherefoAs mentioned above, the flicker paradigm and the situations in which AR is used share some similarities. Hence, change blindness when the AR is used should be investigated. In related work, However, in their study, Therefore, comparison between the binocular and monocular AR presentations in change blindness has still not been investigated enough.To tackle this problem, the present study has two objectives: (1) to investigate whether change blindness occurs during AR use and (2) to investigate whether the AR presentation method influences change blindness. For the second objective, we focused on the observation condition (binocular and monocular) and luminance  of the AR image as the factors that influence change blindness in AR use. The reasons we chose these as important factors are explained as below.The difference between the binocular and monocular presentations might lead to different result in terms of change blindness. In the binocular presentation, the AR image is presented to both of the observer’s eyes, so it is a very similar situation to the typical flicker paradigm. This means that change blindness cannot be avoided in the binocular presentation. On the other hand, in the monocular presentation, the AR image is presented to only one of the observer’s eyes, so the observer can see the real world through the eye to which the AR image is not presented. In the flicker paradigm, if there is no distractor between the original and modified images, an observer hardly ever misses the change. This is because an abrupt onset of a stimulus, like the change, attracts attention automatically and forcibly , hence, When extremely different images are presented to each eye, like in the monocular AR presentation, the images compete for dominance, and when one image is perceived, the other image is suppressed. This ongoing perceptual alternation is known as binocular rivalry . In binoIn the typical change blindness flicker paradigm, the original and modified images are real-world pictures, whereas a distractor or blank is static. In the present study, we replicated this setting; hence, the AR image as a distractor was a static gray rectangle. Given the characteristics of binocular rivalry , the AR In addition, given actual AR use scenarios, the luminance of AR images might influence change blindness. As mentioned above, in optical see-through AR, AR images are superimposed onto the real world directly but are somewhat translucent. Therefore, users can see the real world through the AR images. The higher the luminance of the AR, the higher the visibility of the AR image itself. Of course, higher visibility is desirable for actual use. However, higher visibility of the AR images means that the images cover the real world more strongly and that the real world becomes less visible . TherefoMoreover, as mentioned above, the AR distractor would not evoke change blindness in the monocular presentation because the AR perception would be suppressed in the flicker paradigm due to binocular rivalry. Therefore, the luminance might not influence how often change blindness occurs in the monocular presentation. In other words, in the monocular presentation, change blindness would not occur in any luminance conditions.In summary, we investigated two hypotheses. The first hypothesis is that change blindness would occur less in the monocular presentation than in the binocular presentation, because in the monocular presentation, participants seem to be able to see the real world through the eye to which the AR image is not presented. The second hypothesis is that, in the binocular presentation, change blindness would occur less frequently in the low luminance condition than in the high condition, because the visibility of the real world would be higher in the low luminance condition.For both hypotheses, to investigate whether change blindness occurs or not, monocular and binocular presentations must be compared with a situation in which change blindness does not occur. Hence, participants were assigned one of three observation conditions: binocular, monocular, or none. In the none condition, no distractor appeared between the original and modified images. In this condition, participants detect the change easily, and change blindness hardly ever occurs. We compared the binocular and monocular conditions with the none condition.All these experiments were approved by the Behavioral Research Ethics Committee of the Osaka University School of Human Sciences. Written informed consent was obtained from all participants. No participants were under the age of 16. After the experiment was finished, they were paid money for their participation.In the present study, the observation condition  was a between design, and the luminance condition  was a within design. In change blindness, the same stimuli image pairs cannot be used twice for a particular participant, because once participants have seen a pair of images, they learn where the change occurs . TherefoSix students  at Osaka University participated in the preliminary experiment. Their mean age was 21.67 [standard deviation (SD) = 0.75] years old. All participants had normal or corrected-to-normal vision . By using Ishihara color test II (24 plates), all participants were certified to have normal color vision.Polarized filter holders , a semi-transparent mirror, a pen-tablet monitor , a liquid crystal display , a computer , and a mouse  were set as shown in Although polarized filter holders and the semi-transparent mirror were not needed to control the stimuli in the preliminary experiment, they were located in front of the participants to make the apparatus arrangement the same as in the main experiment.The pen-tablet monitor was located at 50 cm from the participants, and all stimuli, both image pairs for the flicker task and a blank, were presented on the monitor.° × 15.5° as visual angle) real-world pictures without living things. The modified images each contained one of three types of modifications: a change in color, a change in the location of an object, and a disappearing object. During the blank, no stimulus was presented, and the luminance of the display was 0.0 cd/m2.In total, 108 pairs of original and modified images were used. The images were 9.2 cm high and 13.6 cm wide  “How conspicuous was the location or object that changed?” and (2) “How predictable was the location or object that changed?” Participants answered these questions by using sliders below the questions, ranging from one [not at all conspicuous (predictable)] to seven [very conspicuous (predictable)]. After answering, participants clicked the button below the sliders, then the trial finished, and the next trial started.The 108 pairs of images were separated into three blocks, each consisting of 36 trials. For all participants, the same 36 pairs of images were included in a certain block. However, the order of the trial was random, and the order of blocks was counterbalanced between participants.Thirty-six pairs of images had to be selected for the main experiment, including 12 trials in each luminance condition. Each change condition  had 12 trials. The criteria for exclusion were as follows. First, if the participants failed to press the button, the trials were excluded . Next, if any participant could not detect the change within 60 s or click the location of the change correctly, the pair was regarded as too difficult and excluded . The clicked location was classified as an error if participants could not click within 10 pixels from the location or object that changed. Furthermore, if more than two participants responded earlier than 2,250 ms, the pair was regarded as too easy and excluded (24 pairs).Finally, 12 image pairs for which the average response times of six participants were from 3,250 to 10,250 ms were found in each change condition. We separated these 36 pairs of images into three stimulus sets. All three stimulus sets had similar average response times . Therefore, each set had similar difficulty even though the stimulus sets had different contents.In the main experiment, we conducted a flicker paradigm task almost the same as in the preliminary experiment. The difference between the preliminary experiment and the main experiment was that the distractor was presented as an AR gray rectangle to investigate change blindness when AR is used. The AR image was presented binocularly or monocularly and not presented in the none condition. Moreover, we controlled the luminance of the AR image to investigate its influence on change blindness.Thirty-six students at Osaka University participated in the main experiment. Twelve participants were assigned to each observation condition. In the binocular condition, participants were seven females and five males with a mean age of 23.17 (SD = 5.32). In the monocular condition, participants were six males and six females with a mean age of 21.42 (SD = 1.55). In the none condition, participants were six females and six males with a mean age of 20.75 (SD = 1.09). All participants had normal or corrected-to-normal vision . By using Ishihara color test II (24 plates), all participants were certified to have normal color vision.The apparatus and arrangement were the same as in the preliminary experiment.2), medium (5.4 cd/m2), and low (2.7 cd/m2). The size of the AR rectangle was the same in both the original and modified images . The AR image covered the pairs of the images entirely although participants could still observe the stimuli through the AR image.The 36 pairs of the images selected in the preliminary experiment were used. AR images as distractors were gray rectangles. The three luminance conditions of the AR image were high  × 12 , meaning 36 trials for each participant. The observation condition  was a between design.The time between the starting point of the stimuli presentation and pressing the 5 key was recorded as the reaction time. Subjective conspicuity and predictivity were also recorded.The binocular and monocular conditions cannot be directly compared with the none condition because there was no luminance condition in the none condition. Hence, first, we analyzed each data using a 2  × 3  mixed design analysis of variance (ANOVA). All degrees of freedom were adjusted by Chi-Muller’s ɛ, and Shaffer’s procedure was used in all multiple comparisons.We measured the dominant eye of participants, but p was below this value, we consider the result as significant.A Welch two-sample test was conducted to compare the none condition with the binocular-high luminance, binocular-medium luminance, binocular-low luminance, monocular-high luminance, monocular-medium luminance, and monocular-low luminance conditions. The level of significance was adjusted by the Bonferroni procedure. In other words, 0.05/6 = 0.00833… was treated as an adjusted level of significance, and when We did not conduct statistical analysis for the error rate or accuracy of clicked location for two reasons. First, participants pressed the key after they fully recognized where or what was changed, and in the phase of clicking, they could observe the stimuli image without any distractors with both eyes not only in the binocular condition but also in monocular and none conditions. Therefore, there must have been no difference among the conditions in terms of accuracy of the clicked place. Second, the error rate of the clicked location was extremely low , which revealed that participants could detect changes correctly in almost all trials at least before the end of the trial. These means that meaningful statistical analysis cannot be conducted.In the main experiment, the images were alternated every 1,000 ms. Therefore, we discarded the data in which the reaction time was less than 1,000 ms because it was a response made before the first alternation . The data in which the participant could not click the correct location of the change were also excluded from the analysis .The remaining data were transformed from the reaction time to the number of alternations. For example, if the reaction time was between 1,000 and 1,999 ms, the value was transformed to one alternation, and between 2,000 and 2,999 ms, the value was two alternations. After this transformation, we calculated mean alternation times and a SD for each participant and luminance condition, and we excluded the data that exceeded mean ± 2 SD .F = 41.68, p < 0.001, η2 = 0.503; F = 6.54, p < 0.01, η2 = 0.045] and the interaction between them  were all significant. The analysis (the observation condition × the luminance condition) showed that the simple main effect of the observation condition was significant in all luminance conditions . There were fewer alternations in all luminance conditions in the monocular condition than in the binocular condition. The simple main effect of the luminance was significant only in the binocular condition . Multiple comparisons showed that there were more alternations in the high luminance condition  than in the medium  and low conditions  (p < 0.05). The difference between the medium and low conditions was not significant (p > 0.05).The main effects of the observation and luminance conditions . The main effects of the observation condition and the luminance condition  were not significant.In terms of conspicuity, the interaction between the observation condition and the luminance condition was significant . The value of the subjective conspicuity was higher in the monocular condition than in the binocular condition. The simple main effect of the luminance condition in the binocular condition was significant . Multiple comparisons showed that the conspicuity in the high luminance condition was less than in the medium and low conditions (p < 0.05). The difference between the medium and low conditions was not significant (p > 0.05).The analysis (the observation condition × the luminance condition) showed that the simple main effect of the observation condition was significant in the high luminance condition .In the predictivity, the main effects of the observation and luminance conditions and the interaction between them were not significant [observation condition, p > 0.05).A Welch two-sample test was conducted to compare the none condition with each observation and luminance condition. The results are shown in In the present study, to investigate change blindness when AR is used, a flicker paradigm task was conducted while a distractor was presented binocularly or monocularly. We manipulated the luminance of the AR image to control the intensity of the distractor during the task.Comparison with the none condition  revealedRegarding the luminance condition, the number of alternations needed to detect the change was less in the monocular condition than in the binocular condition in all luminance conditions . Even inMoreover, in the binocular condition, change blindness occurred more in higher luminance conditions. This result implies that the real world became more difficult to observe due to the AR distractor in the higher luminance condition in the binocular condition because the AR image was less translucent, resulting in attention being less likely to be attracted to the place where the change occurred. In the monocular condition, by contrast, the number of alternations in all luminance conditions was no higher than in the none condition. This result indicates that participants could select information from the eye to which the AR distractor was not presented. Hence, this result supports the second hypothesis that the change blindness in the binocular condition would occur less frequently in the lower luminance condition than in the higher luminance condition.From the results of subjective conspicuity, the interaction between the observation condition and the luminance condition was significant . In the According to coherence theory , visual In the monocular condition, participants could detect the change as early as in the none condition in all luminance conditions. Therefore, it is supposed that visual attention was attracted when the change occurred as if the AR distractor was not presented, and the visual representation of the location of the change was similarly well organized to that in the none condition, so it was very easy to compare the visual representation with input from after the change. Thus, it is implied that the input from the eye to which the AR distractor was presented was suppressed by the other eye, in which only the stimulus in the real world was presented, and only input from the real world was processed to reach the visual representation.In the binocular condition, it is thought that the attention was hardly ever captured by the location of the change, especially in the high luminance condition, because the AR distractor itself was able to distract from the change. On the other hand, in the lower luminance condition, attention was relatively easily attracted by the location of change, because the AR distractor was more translucent than in the high luminance condition. However, it is supposed that even in the low luminance condition, attention was not as perfectly captured as in the none condition, because there were more alternations in the binocular-low luminance condition than in the none condition. Therefore, it is implied that even though attention was more likely to be captured in the low luminance condition than in the high luminance condition, still the AR image could behave as a distractor.Change blindness, which is one of the most critical issues in actual AR use, was effectively attenuated in the monocular condition in the present experiment. This characteristic is suitable for safety in actual AR use, so the monocular AR presentation might be an efficient solution to change blindness. However, the AR image used in the present experiment was very different from the AR images used in actual scenario. AR is technology for presenting information, so it basically requires images with meaning and rich colors. Even if a monocular AR image does not have any meaning, if it has rich colors and shapes, it might suppress the visual representation of the real world. In this case, the AR image will be noticed even in the monocular AR presentation, and as a result, change blindness may occur. Moreover, a user must look at the AR information in actual use, whereas the AR used in the present study was just a distractor, so it had to be ignored by participants. When participants have to direct their attention to an AR image, the results might be different, because it is thought that the AR distractor must attract more attention than in the present study. Furthermore, in actual AR use, the AR image may make sharp movements that themselves attract attention, so the influence of movement should be taken into account. In future research, various kinds of distractors  should be used to investigate whether the superiority of the monocular presentation is still observed in such situations.Moreover, In addition, considering binocular rivalry, unstable perception will occur in the monocular condition and may cause fatigue that is not felt in the binocular condition. This point was not considered in the present study, so it should be tackled in a future study. If the monocular condition has some disadvantages compared with the binocular condition, the device that can switch from the binocular presentation to the monocular presentation depending on a particular situation or user’s preference may be desirable in actual AR use.In conclusion, the monocular AR presentation demonstrated superiority in terms of attenuating change blindness. This result might be because participants could observe the stimuli when a change occurred by using the eye to which the AR image was not presented; hence, their visual attention was attracted to the location where the change had occurred automatically. However, more various AR distractors should be used in the same design experiments to investigate how stable the superiority is. Therefore, monocular AR presentation must be scrutinized more thoroughly in both engineering and psychology before applying the results in this paper to actual scenarios.All datasets generated for this study are included in the manuscript and/or the supplementary files.AK and KS contributed to the conception and design the study. AK contributed to the data acquisition. AK and YK contributed to the statistical analysis. AK wrote the first draft of the manuscript. All authors contributed to manuscript discussion, revision, reading, and final approval of the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The major landscape in the hilly-gully region of the Loess Plateau is greatly affected by vegetation rehabilitation on abandoned cropland. Although many studies have shown that the rehabilitation have greatly improved soil conditions and protected them from erosion, these effectiveness were not always in consensus possibly due to the land type of vegetation or to the rehabilitation time. To close this gap, we conducted a long term experiment as follows.In this study, we analysed four land types of vegetation rehabilitation  with different rehabilitation times and investigated the mechanical composition and erodibility of the soil. Areas of slope croplandand natural forest were selected as controls.The results showed that soil depth, rehabilitation time and rehabilitation land type had strong impacts on soil mechanical composition, micro-aggregation and erodibility. Following rehabilitation, naturally revegetated grassland and shrub land had lower fractal dimensions of particle size distribution , fractal dimensions of micro-aggregation, and erodibility (K factor) than did cropland. Compared to the positive effects of rehabilitation mainly happened in the topsoil layer at other rehabilitation land type, that of woodland happened in the deeper soil layer. Besides, the indispensable rehabilitation time for the significant improvement of soil condition was shorter at naturally revegetated grassland than that at shrub land and woodland.Although rehabilitation time was more influential than was rehabilitation land type or soil depth, the differences among the rehabilitation land types showed that naturally revegetated grassland with native plants is the most time-saving rehabilitation vegetation for the Loess Plateau in the conversion from slope cropland. The success of rehabilitation in this forestry practice was mainly contributed by the suited species of rehabilitation land type to the local climate and soil. Based on the differences of rehabilitation effectiveness resulting from land type, we should be cautious to choose land types for the rehabilitation of soil conditions in the Loess Plateau. Soil erosion initiated by either natural or human factors is a serious environmental problem in many parts of the world. It not only caused the degradation of soil quality but also the destruction of ecosystem function and safety . Severe Several studies had examined the effects of plant species changes, land preparation, rainfall intensity, anthropogenic disturbance, afforestation, and land abandonment on the mechanical composition and erodibility of the soil . Soil meFor the past fifty years, people pressurized by an increasing population into converting native grasslands into farmlands in the most parts of Loess Plateau of China, which caused the loss of most of the topsoil in many locations . The “GrThis study was conducted in Ansai County, Shannxi Province, China , which lies in the middle part of the Loess Plateau. This region has a typical semiarid continental climate with a mean annual temperature of 8.8 °C, meaning that monthly temperature ranges from 22.5 °C in July to 7 °C in January and an annual precipitation of 549.1 mm, which mainly occurs between July and September . The lanRobinia pseudoacacia), shrubland , artificial grassland (Medicago sativa) and naturally revegetated grassland to control soil erosion −2 yr−1 (Quercus liaotungensis Koidz (Q. liaotungensis) over about 150 years, based on previous research of secondary forests in this area. So, we considered it as representative of the soil-dominated climax community in vegetation rehabilitation to assess the effectiveness of vegetation rehabilitation. These selected sites offered representativeness, typicality and consistency and had similar slope gradients, slope aspects, and topography. The properties of the experimental sites are shown in Based on the investigation of the history of land use, we selected four types of vegetation rehabilitation of cropland abandoned between July 10 and September 10 in 2011 and 2012, the season in which plant community biomass peaks. The four types were naturally revegetated grassland , planted shrubland , planted woodland , and orchard land . All these sites suffering similar farming practices before conversion, and the farmlands had more than 200 years of cultivation history . Thus, sis Koidz , which wRobinia pseudoacacia), while the replicated plots of 10 × 10 m were established in each shrub land site  and in each orchard land site. The smaller replicate plots (2 × 2 m) were randomly established in each naturally revegetated grassland site . The plots were separated by at least 50 m.The size of the plots were varied with the plant communities to match their spatial distribution: the replicated plots of 20 × 20 m were established in each site of planted woodland  were separately collected with a soil drilling sampler (4 cm diameter). The soil samples from the same layer of the same plot were mixed to form one sample. The samples were divided into two parts and were passed through 2 mm screens for removing roots, gravel, and coarse fragments. Then each sample was brought to laboratory. One part was naturally air-dried to measure the organic carbon and analyse soil organic carbon (SOC), total nitrogen (TN), and total phosphorus (TP) contents, particle size distributions and micro-aggregates. The other part was stored in a refrigerator at 4 °C to analyse water-soluble amounts , microbial biomass , enzyme activity as well as other variables not reported in this paper.2O2 and 10% HCL to remove organic matter and carbonates and oxides and were soaked in distilled water for 24 h, then mechanically dispersed with 0.4% Calgon by an ultrasonic bath for 5 min. For micro-aggregate determination, the soil samples were soaked in distilled water for 24 h and mechanically dispersed in ultrasonication for 5 min (The soil bulk density (BD) of each soil layer was measured with the cutting ring method . SOC wasor 5 min . Soil PSr is the particle diameter, Ri is the particle size of subinterval i in the particle size grading, V(r < Ri) is the total volume of soil particles with diameter less than Ri, VT is the sum volume of soil particles, and Rmax is the maximum diameter of soil particles.The fractal dimension of the PSD and micro-aggregation were calculated by the following formula : \documeSoil erodibility was measured by the K factor in the EPIC model using SOC content and soil PSD  and was P < 0.05). Duncan’s (D) post hoc test was used to perform multiple comparisons when significance of the ANOVA was observed. The differences between the natural forest and various types of vegetation rehabilitation at each last restoration year were examined by student’s t test. Pearson correlation analysis was used to analyse the correlations among particle fractal dimension, micro-aggregate fractal dimension, erodibility and soil nutrients, soil texture, and recovery time. In addition, linear regression analysis was used to determine the relationships between each of particle fractal dimension, micro-aggregate fractal dimension, and erodibility and recovery time in each soil layer between 0 and 100 cm. All of the above statistical analyses were conducted using SPSS 17.0  and R version 3.4.4.Three-way ANOVA was performed to test the effects of rehabilitation land type , rehabilitation time (years since sloping cropland abandonment) and soil depth  on soil mechanical composition and erodibility. Significance was evaluated at the 0.05 level Fig. 8). hed away . Dense ched away .Vegetation rehabilitation type was the most influential factor in driving soil mechanical composition and erodibility  Fig. 8)Fig. 8). In addition, vegetation alleviates erosion of soil by its canopy effectively reducing water-induced soil erosion . HoweverIn the analysed four types of vegetation rehabilitation, soil depth had large influences on soil mechanical composition and erodibility  Fig. 8)Fig. 8) bOur study suggested that vegetation rehabilitation time, type and soil depth significantly affect soil mechanical composition and erosion. Following the conversion of sloping cropland to naturally revegetated grassland, shrub land or woodland, the soil structure gradually recovered, and the resistance of the soil against erosive forces gradually increased, primarily within the topsoil. For the conversion of sloping cropland, the natural restoration process of grass represents a more efficient rehabilitation practice than does the planting of other vegetation types. The key point of success rehabilitation project is whether the selection of species fit current climatic and geological conditions, such as the naturally revegetated grass in our study. Based on the differences of rehabilitation effectiveness among the rehabilitation land type, it is important to carefully select land types for the rehabilitation of soil conditions in the Loess Plateau. Our study, conducted at the regional scale, revealed the effects of vegetation rehabilitation on soil erosion in the Loess Plateau, China, but it strongly contributes to our understanding of the mechanisms through which rehabilitation improves soil quality and provides a suggestion for ecosystem management in arid and semi-arid regions.10.7717/peerj.8090/supp-1Data S1Click here for additional data file.10.7717/peerj.8090/supp-2Table S1P < 0.05), * means significant differences between the natural forest and various vegetation restoration patterns at each last restoration year (P < 0.05).Different lower-case letters above the bars mean significant differences among different ages within the same rehabilitation patterns , * means significant differences between the natural forest and various vegetation restoration patterns at each last restoration year (P < 0.05)Different lower-case letters above the bars mean significant differences among different ages within the same rehabilitation patterns , * means significant differences between the natural forest and various vegetation restoration patterns at each last restoration year (P < 0.05)Different lower-case letters above the bars mean significant differences among different ages within the same rehabilitation patterns  and after (B) revegetation on the hilly Loess Plateau.Click here for additional data file.


Caenorhabditis elegans, and the possible protective effect of this formulation against lipid peroxidation caused by paraquat (PQ).Melatonin has been described in the literature as a potent antioxidant. However, melatonin presents variable, low bioavailability and a short half-life. The use of polymeric nanoparticulated systems has been proposed for controlled release. Thus, the purpose of this study was to investigate the action of melatonin-loaded lipid-core nanocapsules (Mel-LNC) in the antioxidant system of C. elegans N2 wild type and transgenic worm CF1553, muls84  were obtained from the Caenorhabditis Genetics Center (CGC). The worms were divided into 5 groups: Control, PQ 0.5 mM, PQ 0.5 mM + Mel-LNC 10 μg/mL, PQ + unloaded lipid-core nanocapsules (LNC), and PQ + free melatonin (Mel) 10 μg/mL. The lipid peroxidation was assessed through thiobarbituric acid (TBARS) levels and the fluorescence levels of the transgenic worms expressing GFP were measured.The suspensions were prepared by interfacial deposition of the polymer and were physiochemically characterized. p < 0.001). Mel-LNC reduced TBARS levels to similar levels found in the control group. Moreover, only Mel-LNC significantly enhanced the SOD-3 expression (p < 0.05). Mel-LNC was capable of protecting C. elegans from lipid peroxidation caused by PQ and this was not observed when free melatonin was used. Moreover, Mel-LNC increased the fluorescence intensity of the transgenic strain that encodes the antioxidant enzyme SOD-3, demonstrating a possible mechanism of protection from PQ-induced damage.The LNC and Mel-LNC presented a bluish-white liquid, with pH values of 5.56 and 5.69, respectively. The zeta potential was − 6.4 ± 0.6 and − 5.2 ± 0.2, respectively. The mean particle diameter was 205 ± 4 nm and 203 ± 3 nm, respectively. The total melatonin content was 0.967 mg/ml. The TBARS levels were significantly higher in the PQ group when compared to the control group (C. elegans.These findings demonstrated that melatonin, when associated with nanocapsules, had improved antioxidant properties and the protective activity against PQ-induced lipid peroxidation could be associated with the activation of antioxidant enzymes by Mel-LNC in  Melatonin, a hormone produced by the pineal glandule, has a high number of effects that are described in the literature, such as sedative, analgesic, and anticarcinogenic effects . Also, iWistar rats. Recently, our research group reported that melatonin-loaded lipid-core nanocapsules (Mel-LNC), prepared with PCL, presented protective effects against cytotoxicity and genotoxicity caused by paraquat (PQ) in the A549 cell line  superoxide dismutase-3 (SOD::GFP) were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 °C.The C. elegans were rinsed off the plates into centrifuge tubes and were lysed with a bleaching mixture , followed by flotation in a 30% sucrose solution (m/v) to separate the eggs. The eggs were washed with M9 buffer  and allowed to hatch overnight on NGM agar plates without bacteria ) were subjected to acute exposure as described above. One thousand five hundred L1 worms were maintained in 100 μL of saline buffer and transferred to a 96 well plate, where total GFP fluorescence was measured after 1 h of treatment using 485 nm excitation and 530 nm emission filters and a microplate reader  at 20 °C. The results were expressed as the mean percentage of fluorescence intensity relative to control wells.p ≤ 0.05 were considered statistically significant.The results were expressed as the mean ± standard deviation (SD). All figures were generated using GraphPad Prism . The normality of data was tested by the Shapiro-Wilk test. The statistical analysis of significance was carried out using ANOVA, followed by the post hoc Bonferroni test in the SPSS Statistics software (version 22). Values of Both formulations, LNC and Mel-LNC, presented a bluish-white liquid macroscopic aspect, with pH values ranging from 5.56 to 5.69. Particle sizing analysis by dynamic light scattering showed hydrodynamic diameters (Z-average) ranging from 200 to 209 nm Table . The LNCAs according to previous studies by our research group, 30% of the worms exposed to PQ 0.5 mM were not alive 24 h after treatment, in contrast with the control group, where significant mortality was not observed. Pre-treatment with Mel-LNC, before PQ exposure, was capable of significantly increasing the survival rate of the worms (about 25%) when compared with the worms that did not receive the pre-treatment. The same was not observed when the worms were pre-treated with free melatonin.p < 0.001). When the worms were pre-treated with free melatonin and LNC, it was not possible to verify any protection from lipid peroxidation inflicted by PQ, whereas Mel-LNC significantly decreased the TBARS levels induced by this herbicide  presented mean particle sizes similar to other studies that have used nanocapsules prepared by deposition of pre-formed polymer , 27. The2O2) through modulation of the ErK/Akt/NFkB pathway [Melatonin and its metabolite are described as potent free radical scavengers . Recentl pathway . In addi pathway  showed tC. elegans it is possible to investigate the involvement of this antioxidant enzyme with strain CF1553. The imbalance between oxidant species and antioxidants results in cell damage, such as lipid peroxidation. Due to its antioxidant capacity, melatonin has been described in cases of acute PQ intoxication [The toxic mechanism of PQ is mainly the formation of ROS leading to oxidative stress. PQ is responsible for inhibition of the electron transport chain and reacts with NADPH forming a PQ-radical. This radical reacts with oxygen and generates superoxide radicals. The antioxidant enzyme involved in this detoxification is superoxide dismutase, which converts superoxide anion to a relatively lower active form of hydrogen peroxide . In C. exication –37. In txication –40. Therxication ; howeverC. elegans when exposed to PQ compared to the control group (not exposed to PQ), and this is a good way to evaluate lipid peroxidation in this model [All cellular components are susceptible to ROS action; however, the lipid membrane is one of the most affected. Higher levels of TBARS were observed in is model . The lipis model .2O2 [Melatonin is also described as a modulator of some antioxidant enzymes that help the body to eliminate reactive oxygen and nitrogen species . Our res2O2 .C. elegans [Wen et al. demonstrated that polydatin, a natural resveratrol glycoside, enhanced SOD-3::GFP expression in CF1553 worms, improving oxidative stress resistance in this model . Moreove elegans .C. elegans were exposed to stressors (H2O2 and juglone), and was responsible for activating genes involved in antioxidant defenses, such as SOD-3. It was demonstrated that Se- and Te-xylofuranosides did not present toxic effects in concentrations that prevent and/or reverse the oxidative damage induced by manganese, as well as increasing SOD-3 expression [Tambara et al.  reportedpression .C. elegans. These findings show that the protective activity against oxidative stress induced by PQ is closely associated with the increase of antioxidant enzyme SOD-3 by Mel-LNC in C. elegans.The better effects of Mel-LNC observed in this study could be due to the mechanism of distribution of melatonin when this molecule is associated with polymeric nanocapsules and the characteristics of LNC per se. About 30–40% of melatonin is incorporated into the nanocapsules and this leads to a large load arrival of this molecule in cells, in this case in lipid membranes, where each nanocapsule present in the suspension can carry about 1200 molecules of melatonin. To our knowledge, this is the first time that the involvement of nanoencapsulated melatonin in SOD-3 expression has been demonstrated in C. elegans treated with nanoencapsuled melatonin. Therefore, in this study Mel-LNC was more effective against paraquat damage, showing that nanotechnology presents a useful tool for improving the characteristics and biological properties of melatonin.Taken together, these results show that pre-treatment with MEL-LNC decreased lipid peroxidation probably through modulation of SOD enzymatic activity. Interestingly, these results were only observed in


Nrp1 for SMC function during development, we generated conditional inducible Nrp1 SMC-specific knockout mice. Induction of early postnatal SMC-specific Nrp1 knockout led to pulmonary hemorrhage associated with defects in alveogenesis and revealed a specific requirement for Nrp1 in myofibroblast recruitment to the alveolar septae and PDGF-AA-induced migration in vitro. Furthermore, SMC-specific Nrp1 knockout inhibited PDGF-BB-stimulated SMC outgrowth ex vivo in aortic ring assays and reduced pathological arterial neointima formation in vivo. In contrast, we observed little significant effect of SMC-specific Nrp1 knockout on neonatal retinal vascularization. Our results point to a requirement of Nrp1 in vascular smooth muscle and myofibroblast function in vivo, which may have relevance for postnatal lung development and for pathologies characterized by excessive SMC and/or myofibroblast proliferation.Neuropilin 1 (NRP1) is important for neuronal and cardiovascular development due to its role in conveying class 3 semaphorin and vascular endothelial growth factor signaling, respectively. NRP1 is expressed in smooth muscle cells (SMCs) and mediates their migration and proliferation in cell culture and is implicated in pathological SMC remodeling in vivo. To address the importance of  Nrp1-null mice are embryonic lethal between embryonic day (E) 10.5 and E14.5, dependent on genetic background, and display a spectrum of cardiovascular and neuronal defects (Neuropilin-1 (NRP1) is a transmembrane glycoprotein receptor essential for both vascular and neuronal development due to its role in mediating vascular endothelial growth factor (VEGF) and class 3 semaphorin signaling, respectively , 25, 36. defects , 23.Nrp1 were viable, displaying no overt early postnatal phenotype but exhibiting a late-onset defect in gastrointestinal motility due to loss of visceral SMC contractility  and display more severe defects in angiogenesis compared with single knockouts  and revealed an important role for NRP1 in vascular SMC migration , 30, 35.actility . The lacout mice , 42. NRPout mice , 30–32. nockouts . Moreoveema mice . These dNrp2 expression, we generated a conditional and inducible ERT2smMHC-Cre transgenic line to allow controlled Nrp1 knockout in SMCs when crossed to Nrp1 floxed mice, by administration of tamoxifen during development. Our results revealed a specific requirement for Nrp1 in myogenic cell lineages during pulmonary microvascular expansion and alveogenesis. Nrp1 was also necessary for pathological neointima formation in vivo, for PDGF-BB-induced SMC outgrowth ex vivo in aortic rings, and for PDGF-AA-induced pulmonary myofibroblast migration in cultured cells.To circumvent genetic redundancy effects due to possible compensatory upregulation of All procedures involving mice were conducted under a UK Home Office license and the approval of the University College London local ethics committee in accordance with the Animal Care and Ethics Guidelines and the 1986 United Kingdom Home Office Animals (Scientific Procedures) Act.Nrp1 knockout mice were generated by crossing floxed; R26RNrp1 mice to ERT2smMHC-Cre transgenic mice, on a C57BL/6 background. Genotypes of progeny were identified by PCR using the primers listed in Inducible SMC-specific Tissue was fixed in HistoChoice tissue fixative (Sigma) or zinc fixative (BD PharMingen) overnight and then processed to paraffin and cut into 10-µm sections, except for the retinas, which were fixed in 4% paraformaldehyde and stained as whole-mounts. Samples were subjected to hematoxylin-eosin histological staining, immunohistochemistry, or immunofluorescence using the antibodies and conditions listed in Nrp1  using RNAiMAX transfection reagent . Transfected cells were seeded at a count of ~2.5 × 104 cells/Transwell insert  and left to migrate for 20–24 h in response to either serum-free media or serum-free media with 50 ng/ml PDGF-AA . Nonmigrated cells were removed, and Transwell inserts were stained using the REASTAIN Quick-Diff Kit, according to manufacturer’s instructions , and migrated cells were counted under a Leica stereo microscope.Mouse pulmonary fibroblasts  were cultured in poly-l-lysine-coated dishes and treated with 10 ng/ml transforming growth factor-β1  for 48 h to induce differentiation to myofibroblasts. Cells were transfected with either scrambled siRNA or two different siRNAs targeting Western blot analysis was performed on transfected myofibroblast cell lysates as described previously , using tAortas from 8- to 10-wk-old male mice that had been treated with tamoxifen the week before harvest were dissected and cultured as described previously , 9 in th2, followed by cervical dislocation, and the cuffed femoral arteries and sham controls were harvested for analysis.Surgeries were performed as described . BrieflyPulmonary hemorrhaging was quantified by blinded measurements of the percentage of hemorrhaging per field of view from at least three different sections per sample. Statistical significance was calculated using the Kruskal-Wallis H test with post hoc Dunn’s multiple comparison test. Quantification of α-SMA expression and numbers of presumptive septal tips with α-SMA-expressing myofibroblasts was determined using the ImageJ Color Threshold function or the ImageJ Cell Counter plugin, respectively. Data were collected and analyzed blindly from at least three different sections/fields of view per sample.Outgrowth areas in aortic ring assays were quantified as described , using tt-test, except for the perivascular cuff intima/media ratio data, which were analyzed using the one-tailed unpaired t-test, and the myofibroblast Transwell migration data, which were analyzed using two-way ANOVA with Bonferroni post test.Unless specified otherwise, all data are presented as means ± SE, and statistical significance was calculated using the two-tailed unpaired Student’s ERT2mMHC-Cre mice 1–2 and monitored the effect of Nrp1 loss. The SMCiKONrp1 neonates appeared viable and healthy compared with their littermate controls; however, internal examination revealed hemorrhaging in the lungs of the SMCiKONrp1 neonatal pups from P8 . The number of alveolar septae was not significantly different between the SMCiKONrp1 mutants and controls when normalized to total tissue area, although a trend towards a reduction was observed . No significant changes in expression of the pericyte-specific marker neural/glial antigen 2 (NG2) were observed in the lungs of P14 SMCiKONrp1 neonates . Given the important role of myofibroblasts in alveolarization during the rapid early postnatal growth and remodeling of the lung, we hypothesize that the observed hemorrhaging resulting from SMC-specific Nrp1 knockout may be due to defective alveolarization consequent upon impaired myofibroblast recruitment, which in turn results in capillary loss and a reduced capacity of the developing lung to accommodate increased blood flow. Additionally, an impairment of capillary stability due to reduced smooth muscle cell recruitment to capillaries and small vessels may also contribute to the hemorrhaging phenotype in SMCiKONrp1 neonates. Further work is required to establish more precisely the causal links between defective myofibroblast recruitment and hemorrhaging in the lungs of SMCiKONrp1 mice.To determine the underlying cause of the pulmonary hemorrhaging, we examined SMC and myofibroblast coverage of the lung by immunohistochemical staining for α-SMA expression. Whereas SMCs are present in larger blood vessels and muscularized airways, myofibroblasts are specialized cells present on the alveolar septa in the developing postnatal lung and are identified by their expression of α-SMA and their location at septal tips , 45. Thital tips , 45, shoiKO mice F. In add mutants , G and Hobserved I. The exobserved A, which rization , 7, and  mutants C, we obscontrols , A and Gneonates B, suggesericytes , 43. Theericytes , B and CNrp1 loss on PDGF-induced myofibroblast migration, we performed in vitro Transwell migration assays. Pulmonary fibroblasts isolated from isolated from postnatal day 2 C57BL/6 mouse lung were differentiated into myofibroblasts in vitro based on cell plating density and treatment with TGF-β1, as previously described (Nrp1-specific siRNAs. The results showed a significant reduction in the migration of NRP1-depleted myofibroblasts in response to PDGF-AA (Platelet-derived growth factor subunit A (PDGF-A) signaling is required for pulmonary myofibroblast differentiation and migration, as evidenced by the absence of myofibroblasts from the primary alveolar septa in PDGF-A-null mice, resulting in severe alveolarization defects , 22, 26.escribed , and PDG PDGF-AA A. The ef PDGF-AA B. Nrp1 s PDGF-AA B.Nrp1 loss on PDGF-induced myogenic cell proliferation, migration, and recruitment to neovascular sprouts was determined ex vivo in aortic ring assays. A marked reduction in SMC outgrowth in response to PDGF-BB was observed in the SMCiKONrp1 aortic rings versus wild-type controls following treatment with tamoxifen (C and D). Although SMCs appeared to be recruited normally to the developing vascular sprouts in this model . Since Nrp1 loss in our model is restricted to SMCs and myofibroblasts, whereas pericytes are unaffected, it is possible that the mild retinal phenotype observed results from a restricted role of myofibroblast-mediated vascular remodeling in retinal vascularization.The impact of SMC-specific loss of illaries . Followicontrols A. Howeveaffected , B–D. SiNrp1 in pathological SMC accumulation giving rise to neointima formation and vascular remodeling, we utilized the mouse perivascular cuff model of vessel injury, in which neointima formation is induced by placement of a nonocclusive cuff around the femoral artery, without removal of the endothelial cell layer . Cell proliferation in the media in SMCiKONrp1 mice was decreased versus controls, as measured by ki67 staining (G–J), and the reduction in proliferation was statistically significant . Absence of alveolar myofibroblasts has been implicated in the pathology of BPD (Nrp1, Vegfr1, and Vegfr2 was reported in a baboon model of BPD (In conclusion, this study identifies a novel requirement for tal tips , as we ffunction . Given tfunction , the impy of BPD , whereasl of BPD . Conversl of BPD . NRP1 maNrp1 is largely dispensable for early postnatal vascular development in the retina and, since these mice appear normal and viable, is therefore seemingly not required for SMC maturation in arteriogenesis more generally. The apparent restriction of early postnatal defects in SMCiKONrp1 to the lung may be due to the important role of NRP1 in myofibroblast recruitment during postnatal alveolar development, as revealed in this study; in contrast, myofibroblasts may be less essential for postnatal retinal vascularization or in development and expansion of other vascular beds.Our findings demonstrate that SMC expression of Nrp1 in pathological SMC/myofibroblast proliferation and neointima formation in vivo in a mouse perivascular cuff model, in agreement with our previous findings demonstrating inhibition of neointima formation due to targeted shRNA-mediated knockdown of Nrp1 and Nrp2 in the rat balloon carotid artery injury model (Nrp1 in pathological neointimal SMC remodeling in response to vascular injury, findings that may be relevant for vasculoproliferative diseases such as atherosclerosis and arterial stenosis following angioplasty and transplantation.Our results also demonstrate a requirement for ry model . Taken tThis work was supported by British Heart Foundation program Grant RG/06/003.No conflicts of interest, financial or otherwise, are declared by the authors.I.Z. conceived and designed research; M.M., I.M.E., V.M., C.P.-M., and K.P. performed experiments; M.M. and I.Z. analyzed data; M.M. and I.Z. interpreted results of experiments; M.M. and I.M.E. prepared figures; M.M. and I.Z. drafted manuscript; I.M.E. and I.Z. edited and revised manuscript; I.Z. approved final version of manuscript.


Disorder of the sympathetic nervous system (SNS) is closely related to the pathogenesis of various autoimmune diseases (ADs). Catecholamine triggered beta2-adrenergic receptor (β2-AR) signaling is important in creating a bidirectional response in the progression of ADs due to factors including diverse expression patterns, single nucleotide polymorphisms (SNPs), biased signals, and desensitization of β2-AR, as well as different subtypes of Gα binding to β2-AR. In this review, we summarize the actions of β2-AR signaling in regulating the functions of immunocytes and in the pathogenesis of ADs, and the application of β2-AR agonists or antagonists in treating major types of ADs is also discussed. We suggest that restoring the immune balance via a soft regulation of the expression or activation of β2-AR is one of the promising therapeutic strategies for systematic ADs. Although the current understanding of the pathogenesis of autoimmune diseases (ADs) such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), myasthenia gravis (MG), and Grave’s disease (GD) is unsettled; many ADs hold in common the expression of autoantigens, abnormal immunoregulation, and shared genetic factors . Researcβ2-AR is widely expressed, with distinct densities, on various immune cells, including T cells, B cells, dendritic cells (DCs), and macrophages . Evidenc+ cytotoxic T cells (Tc) express a significantly higher level of β2-AR compared to CD4+ helper T cells (Th). Therefore, stimulation of β2-AR reduced the percentage of interferon-γ (IFN-γ) + Tc to a much higher extent than that of IFN-γ + Th  and the activity of protein kinase A (PKA), leading to reduced p38 mitogen-activated protein kinase (MAPK) activation. Subsequently, p38 MAPK inhibits CD3-induced CD25 expression and CD3-mediated IL-13, IFN-γ, and IL-2 production  reduced CD86 and MHC-II expression, enhanced antigen uptake and IL-10 production, and simultaneously prevented T cells activation and pro-inflammatory cytokines secretion such as TNF-α in a β2-AR dependent manner (+CD8a+ DCs impaired DC mediated CD8+ naïve T cell activation (Dendritic cells (DCs) express α1-AR, α2-AR, β1-AR and β2-AR. Nevertheless, β2-AR is the primary effecter of DCs for cytokine production and antigen presentation, with β2-AR able to exert either a stimulatory or inhibitory function that is dependent on the differentiation status of DCs and/or the activation time of the receptor . Activatt manner . In anottivation . Meanwhitivation . Stimulativation . TherefoAccumulating evidence shows that β2-AR signaling plays a pivotal role in macrophage activation and pro-inflammatory cytokine production. Some researchers reported that stimulation of β2-AR inhibits the activation of RAW264.7 (a mouse macrophage cell line) and THP-1 (a human monocytic cell line) induced by LPS through restraining ERK1/2, JNK and NF-κB pathways . Furtherβ2-AR exerts complicated effects on immune cells and influences the function of almost all kinds of immune cells. Stimulation of β2-AR on peripheral blood mononuclear cells (PBMCs) inhibits interferon alpha-1 (IFNA1) production mediated by Toll like receptor 9 (TLR9) . It drivThe SNS is activated in ADs and acts on humoral and cellular immunity primarily through β2-AR. Having a better understanding of β2-AR in the course and progression of ADs will help to develop β2-AR targeted drugs for the treatments of ADs .∗04 mutation have a positive correlation with the expression of anti-cyclic citrullinated peptide (CCP) and are strongly associated with RA  at codon 16 together with the HLA-DRB1 with RA . Similar with RA . General with RA . Therefo with RA . As a fu with RA .Results indicate that β2-AR desensitization of the β2-AR-AC-cAMP transmembrane signal transduction pathway plays a crucial role in the ongoing inflammation of RA . HoweverSLE is a very different disease from RA. Clinical observations indicate that psychological stress, which often stimulates the SNS, aggravates the activity of SLE . The up-Multiple sclerosis (MS) is a potentially disabling disease of the central nervous system . The mecMG has the characteristic of formation of autoantibodies to acetylcholine nicotinic receptors at the neuromuscular junction of skeletal muscles . The expGD is an AD that affects the thyroid. Although the pathogenesis of GD is still unclear, genetic predisposition, especially for SNPs within β2-AR is regarded to be important in the development of GD . MultiplGenomic variations contribute to the morbidity of various ADs besides those discussed above. The significant prevalence of homozygous Gly16Arg genotype in β2-AR has been found to also have a positive correlation with the predisposition to interstitial cystitis . ConversCatecholamine has both pro- and anti-inflammatory effects in patients with chronic inflammatory disease . Briefly+ Th0 cells to develop into Th1 cells by blocking the inhibitory effects of endogenous NE on Th1 cell development. β2-AR blockade effectively shifts the immune response to the arthritogenic antigen toward a cell-mediated response, that is beneficial for the processing of the arthritogenic antigen. Consequently, blocking β2-AR function during AA development can reduce pro-inflammatory cytokines production via inhibition of the expression and activation of α1-AR, which is elevated by chronic β2-AR stimulation. Therefore, β-blockers decrease arthritic severity in the active phase of AA , selective β2-AR antagonist butoxamine or ICI 118, 551 induces CD4se of AA .Likewise, the application of salbutamol is able to attenuate the rat model of experimental allergic encephalomyelitis (EAE) by decreasing the average neurologic function score of model rats, reducing calpain activity in brain tissues, and rescue the activities of the BB isoenzymes of creatine kinase and catalase . SimilarTaken together, β2-AR signaling is involved in the pathogenesis and progress of ADs, and is a potential drug target in treating these diseases. However, the genetic polymorphisms and the expression of the receptor is diverse from individual patients or different disease stages. Meanwhile, the function of β2-AR signaling is bidirectional between different species, different immune cell isotype, and different time point of diseases. In a particular situation, β2-AR aggravates inflammation, but in some cases, it reduces the production of pro-inflammatory cytokines and auto-antibodies, acts as an immunosuppressor. Therefore, there is a need for thorough elucidation of the precise regulation of β2-AR in each specific AD . In addiLW wrote the text. YT, MZ, and RW collected references. WZ and JT drew the cartoon. YH wrote the table. QW and WW revised the article. QW designed the topic.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In silico screening and the subsequent biological evaluation lead to the discovery of the novel lead compound IMB-A6 that binds to the AF2 site, which inhibits the activity of either wild-type (WT) or resistance mutated ARs. Our work demonstrates structure-based drug design is an efficient strategy to discover new antiandrogens, and provides a new class of small molecular antiandrogens for the development of novel treatment agents against PCa.Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer (PCa). Current antiandrogen therapies induce resistant mutations at the hormone binding pocket (HBP) that convert the activity of these agents from antagonist to agonist. Thus, there is a high unmet medical need for the development of novel antiandrogens which circumvent mutation-based resistance. Herein, through the analysis of AR structures with ligands binding to the activation function-2 (AF2) site, we built a combined pharmacophore model.  Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer death in men in the western countries . AndrogeAndrogen receptor antagonist prevents androgens from carrying out their biological activity by directly binding and blocking the AR ligand binding domain (LBD), or by inducing repressive activity  like bloBesides of HBP, several other binding sites on AR like activation function-2 (AF2), binding function-3 (BF3), the DNA binding site, and activation function-1 (AF1) have been attracted attentions in novel antiandrogen development . Among t1). Structure-based drug design (SBDD) has emerged as an effective way to obtain lead compounds targeting the AF2 site  based on the sequence alignment. The pharmacophore model was generated by pharmacophore module in DS. The pharmacophore based screening was performed through Screen Library protocol in Pharmacophore module of DS. The hits after pharmacophore filtration was screened using the docking module LIBDOCK in DS , and cultured with RPMI-1640 supplemented with 10% Fetal Bovine Serum (GiBCO). The PC-3 cell line was kindly provided by Dr J. H. Wu , and cultured with F-12K supplemented with 10% Fetal Bovine Serum.3 cells per well in 96-well plate in complete RPMI-1640 growth medium, or PC-3 cells in complete F-12K growth medium at the same density. After overnight incubation, 1 μL dimethyl sulfoxide  or 1 μL test compounds were added to each well at the designated concentration. After 72 h incubation , 20 μL of MTT (Invitrogen) solution (5 mg/mL in PBS) were added per well and incubated for another 2 h . The MTT formazan formed by metabolically viable cells was dissolved in 100 μL isopropanol. The absorbance was measured at 570 nm wavelength on a plate reader . Experiments were performed in triplicate. The value of DMSO group was defined as 100%.LNCaP cells were seeded at a density of 4–5 × 104 cells per well in 24-well plate and subsequently co-transfected with 100 ng of PSA-luc, 20 ng of F876L (20 ng T877A or 50 ng W741C+T877A), and 1 ng of Renilla plasmids using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s protocol. 24 h after transfection, the medium was changed to phenol red-free RPMI-1640 supplemented with 10% charcoal-stripped FBS, containing 1 nM of dihydrotestosterone (DHT) 1 μL and 1 μL test compounds at the designated concentration. After further 24 h, the cells were lysed in 100 μL per well passive lysis buffer, and 20 μL of the cell lysates was used for detection of the luciferase activity using Dual Luciferase Assay System (Promega) on a plate reader . All experiments were run in triplicate.Plasmid F876L is full length cDNA of mutant AR that harbors F876L mutation. Plasmid T877A is full length cDNA of mutant AR that harbors T877A mutation. Plasmid W741C+T877A is full length cDNA of mutant AR that harbors W741C and T877A mutations. The F876L and W741C+T877A plasmids were kindly provided by Dr J.H. Wu , and the plasmid T877A was constructed in house. For the reporter assays, 24 h before transfection, PC-3 cells were seeded at a density of 6–7 × 105 cells per well in 6-well plate. After overnight incubation, 1 μL DMSO (Sigma-Aldrich) or 1 μL compounds were added to each well at the designated concentration. After another 24 h incubation, the cells were lysed with RIPA. Then the protein lysis buffer was treated with 10% SDS–PAGE for western blot analysis. The following antibodies were used for the detection of proteins: rabbit anti-AR , goat anti-PSA , Mouse anti-actin  was used as a loading control. Proteins were visualized using anti-mouse or anti-rabbit HRP-conjugated secondary antibodies  and ECL-Plus (Millipore).LNCaP cells were seeded at a density of 3 × 10LNCaP cells were grown on the glass cell culture dish in phenol red-free RPMI-1640 medium containing 10% charcoal-stripped FBS for 24 h, and were then treated with DMSO, 1 nM DHT, 500 nM enzalutamide and 1 nM DHT, 5 μM IMB-A6 and 1 nM DHT, respectively, for further 24 h. Cells were fixed with 4% (vol/vol) paraformaldehyde and permeabilized with 0.2% Triton X-100. Then the cells were incubated with AR antibodies . The secondary antibodies, goat anti-rabbit with FITC (Santa Cruz) at 1:100 were used. The counterstain DAPI was used to visualize cell nucleus. The images were detected under UltraVIEW vox spinning disk confocal scanning system (Perkin Elmer) on an Olympus IX81 microscope.in vitro, relative to dihydro-testosterone , was evaluated by AR fluorescence polarization (FP) assay, using Polar-Screen AR competitor assay kit  on a plate reader .The binding assay of IMB-A6 to AR-LBD protein (HBP site) ∗, Invitrogen) was performed by FortéBio Inc. Company 2. The purified AR was biotinylated  by 3:1, and then incubated for 1 h at room temperature. Superstreptavidin biosensors  were coated in a solution containing 1 μM of biotinylated protein for 1 h at room temperature. Duplicated set of sensors were incubated in an assay buffer with 5% DMSO without protein for use as a background binding control. Both sets of sensors were blocked with a solution of 10 mg/ml biocytin for 5 min at room temperature. A negative control of 5% DMSO was also used. Binding of samples to coated and uncoated reference sensors was measured over 120 s. Data analysis on the FortéBio Octet RED instrument was performed using a double reference subtraction (sample and sensor references) in the FortéBio data analysis software.The Octet RED , equipped with super streptavidin biosensor chips (FortéBio), was used for the analysis of small molecule protein interactions in fluidics free system. The binding of IMB-A6 to the AR-LBD (A15675g for 10 min at 4°C and supernatant was transferred to a fresh 1.5 mL tube on ice. The cell lysates were incubated with anti-AR rabbit antibody for 3 h at 4°C. Thirty microliters of protein A-Agarose (Santa Cruz) was added and incubated overnight at 4°C. The beads were washed four times with lysis buffer (cold) and at last boiled in 40 μL sample buffer for 5–10 min. The samples were analyzed by western blotting.For AR and PELP-1 IP assay, LNCaP cells were seeded in 10 cm dishes. 48 h later, the cells were treated with DMSO or 1 μM, 10 μM IMB-A6 for 24 h. The cells were collected and lysed in NP-40-containing buffer for 30 min on ice. Cell lysates were centrifugated at 10,000 ×P-value of <0.05 was considered statistically significant. In all figures, error bars indicate the standard deviations for three independent samples. One asterisk (∗) indicate a P-value lower than 0.05, and two asterisks (∗∗) indicate a P-value lower than 0.01. All the experiments were repeated more than two times.Unpaired two-tailed Student’s tests or one-way analysis of variance with the Tukey post test were conducted to compare results obtained from the different experimental groups. Structure-based drug design has becoming an effective way to discover the lead compound. We herein planned to design the novel antiandrogens that bind to AF2 site using SBDD strategy. During structural analysis of the AR-LBD, we collected the structures with ligands binding to the AF2 site, and the seven structures were found and listed in Supplementary Table The schedule of virtual screen was shown in Figure To evaluate the biological activity of the 12 hits, we assessed their activity through MTT assay and dual-luciferase assay Figure . ImportaTo further evaluate the antiandrogenic potency of IMB-A6, we investigated antiandrogenic activity of IMB-A6 against WT and T877A (FLU resistant mutant), F876L (ENZ and ARN509 resistant mutant), and W741C + T877A (FLU and BIC resistant mutant) mutated ARs in presence of DHT at 0.1 μM, 1 μM, and 10 μM. As a result, IMB-A6 inhibited the AR transcriptional activity in a dose-dependent manner in LNCaP cells Figure . In PC-3We further investigated whether IMB-A6 inhibits AR activity through binding to AF2 site. Firstly, we performed the Octet binding assay using biotinylated AR-LBD on superstreptavidin sensors and IMB-A6 to test if IMB-A6 directly binds to AR-LBD. IMB-A6 had a dissociation equilibrium constant of 8.33 μM Figure , which dUpon activation by androgens, AR translocates into the nucleus where it binds to androgen response element (ARE) of DNA. We then evaluated if IMB-A6 could affect DHT-induced translocation of AR from the cytoplasm to the nucleus using the confocal assay. The real-time nuclear localization of endogenous AR after 1 nM DHT treatment in the presence of 5 μM IMB-A6 or ENZ was evaluated. As a result, IMB-A6 blocked DHT-induced translocation of AR to the nucleus compared with the DHT-only treated group, which was comparable to ENZ or DHT-free (DMSO) group  to furnish a new screening series of 248 compounds. These compounds were further screened through CDOCKER in DS and 14 compounds  of mouse mammary tumor virus (MMTV) contains an androgen hormone response element. As we previously only used PSA reporter assay to evaluate the activity of AR, we preferred to use MMTV-luc reporter to confirm the antiandrogen activity of the active compounds . As results, the compounds IMB-A6, IMB-B2, and IMB-B5 efficiently inhibited the DHT induced MMTV level . Two compounds, tolfenamic acid and 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole binding to AF2 site were found as top candidates for rescuing lethality, locomotor function and neuromuscular junction defects in SBMA flies .Besides of AF2 site, the sites on AR other than HBP include BF3 site, DNA binding site, and AF1 site. To date, several ligands have been identified to bind to these sites and exhibiting potent activities in antagonizing the AR signaling. For examples, Apart from the resistant mutations, other mechanisms were reported for the ENZ-resistance for CRPC. For instance, the expression of AR splice variants by CRPC like AR-V7 or AR-V567, which are missed the LBD domain, thus leading to the persistent AR signaling and resistance to ENZ therapies . Also, tYL, MW, and TW performed the experiments and analyzed the data. YX, XC, LH, and YH helped to set up the assay methodology. XL, ML, LH, and SC provided the resource and partial funding supports. JZ designed the experiments and wrote the original manuscript. JZ and SC reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nanomaterials Editorial Office has been made aware that the published paper [Results in Physics [The ed paper  was prev Physics  by the s Physics  will be Nanomaterials for any inconvenience caused, and regret that this issue was not detected earlier. The decision to retract has been taken in agreement with the authors. The manuscript [Results in Physics, and prior to its publication. The issue resulted from an incorrect version submitted by the authors during the editorial process. We apologize to the readers of nuscript  was submnuscript  in ResulMDPI is a member of the Committee on Publication Ethics, and takes its responsibility to enforce strict ethical policies and standards very seriously. To ensure the addition of only high-quality scientific works to the field of scholarly publications, reference  is retra


Stroke is a common disabling disease that severely affects the daily life of patients. Accumulating evidence indicates that rehabilitation therapy can improve movement function. However, no clear guidelines have specific and effective rehabilitation therapy schemes, and the development of new rehabilitation techniques has been relatively slow. This study used a text mining approach, the ABC model, to identify an existing rehabilitation candidate therapy method that is most likely to be repositioned for stroke. In the model, we built the internal links of stroke (A), assessment scales (B), and rehabilitation therapies (C) in PubMed and the links were related to upper limb function measurements for patients with stroke. In the first step, using E-utility, we retrieved both stroke-related assessment scales and rehabilitation therapy records and then compiled two datasets, which were called Stroke_Scales and Stroke_Therapies, respectively. In the next step, we crawled all rehabilitation therapies co-occurring with the Stroke_Therapies and then named them as All_Therapies. Therapies that were already included in Stroke_Therapies were deleted from All_Therapies; therefore, the remaining therapies were the potential rehabilitation therapies, which could be repositioned for stroke after subsequent filtration by a manual check. We identified the top-ranked repositioning rehabilitation therapy and subsequently examined its clinical validation. Hand-arm bimanual intensive training (HABIT) was ranked the first in our repositioning rehabilitation therapies and had the most interaction links with Stroke_Scales. HABIT significantly improved clinical scores on assessment scales [Fugl-Meyer Assessment (FMA) and action research arm test (ARAT)] in the clinical validation study for acute stroke patients with upper limb dysfunction. Therefore, based on the ABC model and clinical validation, HABIT is a promising repositioned rehabilitation therapy for stroke, and the ABC model is an effective text mining approach for rehabilitation therapy repositioning. The findings in this study would be helpful in clinical knowledge discovery. Stroke is a common disabling health-care problem, and it is the second-leading cause of mortality and disability worldwide. In the United States, nearly 0.8 million people have stroke annually, and the estimated direct and indirect cost of stroke was $95 billion in 2015 and is expected to rise to 185 billion in 2030  by systematically scrutinizing a vast number of abstracts or full-text versions of scientific publications . In particular, In via B terms whereas the dotted line is not of interest from the new discovery perspective.In order to identify new rehabilitation therapies for stroke, we developed a relation extraction method based on ABC model, with which new knowledge could be discovered from sets of disjointed scientific entities, as shown in The ABC model embodies the stages of retrieval, interpretation, and analysis. The retrieval phase investigates unstructured stroke-related information, designs text queries, and extracts the entity documents; the interpretation phase constructs a connected graph to represent A-B and B-C relationships between entities; in the analysis phase, annotation information between transitivity diffusion entities is used to sort the best entity that should be further verified. The ABC model starts with the theme “A” (stroke) in PubMed that collects scientific questions. The phrase “B”  is then listed, and a separate search is used for each “B” term; then, the phrase “C” (rehabilitation therapy) appearing in the code of the B is compiled; finally, with frequency criteria, the C terms are ranked such that a high ranked C term is used to represent the most promising hypothesis. For example, the term C may be the name of a therapeutic strategy that has not been tested for A (stroke) but has been demonstrated in other situations  with curative effects, suggesting that C may be explored as a new therapy.The overall flow chart is shown in To collect articles related to stroke with upper limb impairment, we searched PubMed with stroke-related keywords  and (“hand” OR “arm” OR “upper extremity” OR “upper limb”) in April 2016. We did not use Medical Subject Headings (MeSHs) terms because most specific assessment scales and therapies do not directly belong to MeSH terms. When we searched stroke-related rehabilitation items, the MeSH terms that we obtained were only the common words, which should be deleted as confounding factors. The NCBI provides the E-utilities API for data query and download; therefore, we utilized python to call the API to fetch all query-related data in PubMed of NCBI through the HTTP protocol, and the data were then stored in plain text files , and 26 scales were finally obtained . We rankAccordingly, in the potential therapy list, the following common words were deleted: “clinical practice,” “conventional therapy,” “medical therapy,” “physical therapy,” “specific training,” and “combined therapy”; in addition, the following drug therapies were deleted in the manual inspection process: “antiplatelet therapy,” “anticoagulant therapy,” “antihypertensive therapy,” and “antithrombotic therapy.” Subsequently, we obtained the stroke-related rehabilitation therapy dataset, Stroke_Therapies, which compromised 47 rehabilitation therapies . Two senUsing the extracted Stroke_Scales, we searched again in PubMed and retrieved 60,307 records, in which 60,202 records had abstracts. From these records, we extracted the rehabilitation therapies (All_Therapies dataset), removed those applied for stroke and listed in As shown in the upper left quadrant in We used these potential repositioning rehabilitation therapies with “stroke” in PubMed search to exclude the records that include the associations of those rehabilitation therapies with stroke. We found that except for “hand arm bimanual intensive training,” other NPs all co-occur with stroke. Thus, the “hand arm bimanual intensive training” was our target validation candidate.Hand arm bimanual intensive training (HABIT) is a bimanual intervention addressing the specific upper extremity impairments in pediatric congenital hemiplegia, which is the most common physical disability in childhood  and the HABIT groups. The same therapist who held national certification performed all treatments and training. The treatment duration was 2 weeks (10 working days), and the rehabilitation time was the same in both groups. In general, motor function and extremity activity were assessed using FMA and ARAT before and after therapy in these patients. Both groups showed statistically significant improvements of FMA and ARAT from baseline to post-treatment assessment. While more participates need to be enrolled to get a positive comparison between HABIT group and RRP group . More deStroke is caused by the sudden rupture of blood vessels in the brain or blood vessels that cannot flow into the brain owing to vascular occlusion. Pediatric congenital hemiplegia mostly occurs because of poor nutrition during pregnancy, anemia during pregnancy, encephalopathy, birth asphyxia, pathological jaundice, and intrauterine distress, causing immature fetal brain and other organ tissues. Furthermore, pediatric congenital hemiplegia can further damage the central nervous system, leading to cerebral palsy. Although the pathogenesis and clinical manifestations of stroke and pediatric congenital hemiplegia are not the same, the HABIT is effective for both conditions, indicating that the two diseases share similar rehabilitation mechanisms. Besides, patients who received HABIT showed significant improvement, indicating that HABIT has a positive impact on rehabilitation therapy for upper extremity impaired patients. The principle of HABIT includes motor learning , neuroplasticity, brain transformation upon increasingly difficult therapy and incentive reward Nudo, , and traAlthough the repositioning of rehabilitation therapy is common in clinical practice, the discovery is often based on the clinician’s personal experience or deduction from the medical community, without objective and systemic approaches with data mining application. For instance, VR, a rehabilitation intervention, was first applied to basic motor disability  interacted with each other on numerous occasions, constantly trying to adjust the parameters, and finally provided effective evidence for HABIT. Besides, we relied much on a manual check, more than half were deleted after the manual check.de facto approach to inform clinicians who are trying to master the exponentially rapid expansion of publications and datasets. As demonstrated in this study, the ABC model can be applied to new hypothesis generation for rehabilitation therapy repositioning.We understand that knowledge discovery is a process of trial and error, and it is an iterative procedure that domain scientists work closely with information scientists. The present study has shown the potential of applying text mining for new medical discoveries. Mining literatures to generate a hypothesis about rehabilitation therapy repositioning semi-automatically could be the In the present study, we proposed a text mining approach to mine terms related to disease, rehabilitation therapy, and assessment scale from literature, and a subsequent ABC inference analysis was used to identify relationships of these terms across publications. The clinical validation study demonstrated that our approach could be used to identify potential repositioning rehabilitation therapy strategies for stroke. In particular, we identified a promising rehabilitation method, called HABIT, which was previously used in pediatric congenital hemiplegia. A subsequent full clinical trial confirmed the HABIT as a highly promising rehabilitation therapy for stroke. Further clinical studies are needed to investigate the long-term effect of HABIT on stroke patients and identify optimal parameters of the therapy. In addition, further studies are needed to improve text mining and inference strategy and identify other clinical applications amenable to the technique.GM, XL, and MS designed the study. YH and YD performed the experiments and prepared the figures. QY, DW, and YZ analyzed the data. XL and YZ performed the manual inspection as senior domain experts. GM wrote and discussed all sections of the manuscript. All authors reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vibrio vulnificus and Vibrio parahaemolyticus strains, as well as a genomic fitness island found in a Vibrio diabolicus strain. All four islands had markedly different GC profiles than the rest of the genome, indicating that all of these islands were acquired via lateral gene transfer. Genes on the PAIs and fitness island were characterized. The PAI found in V. parahaemolyticus contained the tdh gene, a collagenase gene, and genes involved in the type 3 secretion system II (T3SS2). A V. vulnificus environmental strain contained two PAIs, a small 25 kbp PAI and a larger 143 kbp PAI. Both PAIs contained virulence genes. Toxin–antitoxin (TA) genes were found in all three species: on the V. diabolicus fitness island, and on the V. parahaemolyticus and V. vulnificus PAIs.Pathogenicity islands (PAIs) are large genomic regions that contain virulence genes, which aid pathogens in establishing infections. While PAIs in clinical strains (strains isolated from a human infection) are well-studied, less is known about the occurrence of PAIs in strains isolated from the environment. In this study we describe three PAIs found in environmental  Vibrio parahaemolyticus and Vibrio vulnificus can cause illnesses in humans, with an estimated 80,000 cases occurring annually in the United States  and septicemia (43%); only 5% are gastroenteritis  increases to 60%. Pathogenesis of both species is complex, and while some virulence factor genes have been implicated, the mechanisms underlying V. vulnificus and V. parahaemolyticus virulence are not well understood , meaning that a single PAI could make up as much as 4% of a Vibrio genome. PAIs are flanked by highly conserved tRNA genes that act as both integration and excision sites. The majority (~ 75%) of PAIs discovered have tRNA flanking sequences (Schmidt and Hensel Pathogenicity islands (PAIs), a subgroup of genomic islands that aid in and contribute to pathogenesis, have been found in clinical strains of both There is considerable evidence that PAIs are acquired horizontally via one or more lateral transfer events. Within some PAIs there is evidence of one large transfer event, while other PAIs are more “mosaic-like.” The “mosaic-like” composition of certain PAIs is caused by multiple, independent lateral transfer events  being the most studied  and type III secretion system 2 (T3SS2)  and nine V. vulnificus genomic islands (VVI–I to VVI–IX) have been found in these clinical strains. PAIs have not been detected in environmentally derived V. vulnificus strains  strains rarely considered. In this study, we characterized four genomic islands found in environmental Vibrio strains: a PAI within a V. parahaemolyticus strain, two novel PAIs within a V. vulnificus strain, and a novel fitness island found in a Vibrio diabolicus strain. Environmental Vibrio strains, and the PAIs within them, could serve as reservoirs for virulence genes.Work on V. parahaemolyticus and V. diabolicus strains were isolated previously  for isolation of V. vulnificus strains following the US Food and Drug Administration protocol . V. parahaemolyticus TS-8-11-4 and V. diabolicus JBS-8-11-1 were deposited into the DSMZ Public Culture Collection and were assigned their respective accession numbers: DSM 107522 and DSM 107521.Environmental ). After DNA was extracted, DNA quantity was measured via Quibit fluorimetry. Libraries were prepared and then sequenced using an Illumina MiSeq (V3 26300 base) at the Indiana University Center for Genomic Studies as a part of the Genome Consortium for Active Teaching NextGenSequencing Group (GCAT-SEEK) shared run . This tool displays GC content variation across a genome and can be useful for identifying genomic regions that differ from the rest of the genome in GC content (Gao and Zhang http://rast.nmpdr.org/). NCBI GenBank was also used to characterize genomic island genes (http://www.ncbi.nlm.nih.gov/genbank/). Gene sequences of interest were edited and maximum-likelihood trees were constructed using the Kimura 2-parameter model with Mega version 7. DNAPlotter was used to visualize the circular chromosomes of the Vibrio strains  to determine their GC profiles  of genes on the TS-8-11-4 PAI could not be assigned specific identities and were thus designated hypothetical. The genomic island of V. parahaemolyticus TS 8-11-4 was on the second chromosome of and it harbored virulence genes  contained two chromosomes, the first chromosome larger (2.96 mbp) than the second (1.99 mbp). The average GC content of V. vulnificus ranges from 46 to 48%   was found on this island, as well as genes involved in the type three secretion system II (T3SS2). Both the tdh gene and T3SS2 complex are the two major virulence factors implicated in V. parahaemolyticus pathogenesis   were also found on this PAI. Capsules aid pathogens in evasion of host immune defenses, establishing infections, and survival in harsh environments, such as the stomach. V. parahaemolyticus virulence is correlated with capsule production  is a main component of the outer membrane of Gram negative bacteria, and is a known pyrogen (fever-producing agent)  systems are found either on plasmids, genomic islands, or within the chromosome and are made up of closely linked toxin and antitoxin genes. The encoded labile antitoxin protects the host from the stable toxin, while competitor cells that do not have the TA system (and respective antitoxin) are eliminated  inhibits translation by blocking translation elongation at the 30S ribosomal subunit  were found on JBS-8-11-1’s fitness island. doc toxin genes and phd antitoxin genes are widespread in vibrios and were also found on V. parahaemolyticus strain TS-8-11-4’s PAI as well as V. vulnificus strain WR-2-BW’s PAI  in coastal ecosystems. Consequently, the pathogenicity loci  of naturally occurring environmental strains are clearly important.PAIs are present in environmental