In the digital era, tech devices (hardware and software) are increasingly within hand’s reach. Yet, implementing information and communication technologies for educational contexts that have robust and long-lasting effects on student learning outcomes is still a challenge. We propose that any such system must a) be theoretically motivated and designed to tackle specific cognitive skills  supporting a given cognitive task  and b) must be able to identify and adapt to the user’s profile. In the present study, we implemented a feedback-based adaptive system called A-book (assisted-reading book) and tested it in a sample of 4th, 5th, and 6th graders. To assess our hypotheses, we contrasted three experimental assisted-reading conditions; one that supported meta-cognitive skills and adapted to the user profile (adaptive condition), one that supported meta-cognitive skills but did not adapt to the user profile (training condition) and a control condition. The results provide initial support for our proposal; participants in the adaptive condition improved their accuracy scores on inference making questions over time, outperforming both the training and control groups. There was no evidence, however, of significant improvements on other tested meta-cognitive skills . We discussed the practical implications of using the A-book for the enhancement of meta-cognitive skills in school contexts, as well as its current limitations and future developments that could improve the system. The advent of increasingly accessible and cheaper digital information and communication technologies (ICTs) has raised the question about their role in the context of formal education. ICTs are seen by some as a natural, intuitive, and easy-to-use tool for mediated learning, and there are some examples of their successful application in education e.g., . There aIn the context of language education, ICTs have been predominately used as an aid for low-level language skills, such as decoding e.g.,  and lessA number of reviews suggest that there is virtually no evidence of the benefits that ICT could provide to reading comprehension during school years see . An exisOther studies have also failed to demonstrate the usability of ICT see . In contyes-or-no questions. Each of these questions related to a critical meta-cognitive ability, namely, inference making, comprehension monitoring and text structure knowledge. Our working hypothesis was that pertinent feedback  should prompt the young reader to begin to strategically apply these skills while reading  in particular when the system adapts to the users’ profile purposefully focusing on meliorating her weaknesses . Consequently, we predicted that both the training and adaptive conditions would in time produce better comprehension accuracy scores relative to the control condition. Furthermore, we hypothesized that the adaptive behavior of the system should benefit the user comprehension processes. Thus, children using the adaptive condition of the system should outperform the training condition group.In this article, we present the implementation and a preliminary assessment of an automatized feedback-based system we called A-book (assisted-reading book), designed to provide theoretically motivated user-based feedback during the process of reading. The A-book’s aim is to offer an adequate context for primary school students to develop meta-cognitive strategies at an early stage. In a between-subject design we contrasted three experimental assisted-reading conditions; a training condition, an adaptive condition and a control condition. In all three experimental conditions, readers were presented with stories (one page at the time) and three types of Ninety primary school students from 4th, 5th, and 6th grade (aged between 9 and 12 years) from a local school, who participated voluntarily on a session basis, were recruited to take part of the study. All children were monolingual Spanish native speakers.1. Each story was divided in a number of fragments  of about 100 words each  for their presentation. For each fragment, we wrote three questions, each of them related to a specific meta-cognitive skill . For each question, we then wrote two kinds of feedback  and each of them in two equivalent versions (one for correct answers and one for incorrect answers).We selected 10 stories from “Un cuento al día – Antología” , a book Questions aimed to capture the readers’ ability to make inferences were constructed to ask about information that was not explicitly given in the text. For instance, if the text said ‘the old man mixed the content of jars to make rain…,’ we asked whether ‘the old man knew a recipe for rain.’ The explanatory feedback alluded to such critical information, by stating for instance, ‘if the old man mixed the content of jars to make rain, he most probably knew a recipe for rain.’ Questions about the structure of the text directly asked the reader whether the story’s characters had been already presented, whether they already knew the scenario or context in which the story was taken place, or whether the story was about to end, or only at the beginning. Such questions did not include any content of stories, in other words they were story independent. The corresponding feedback was also story independent insofar it just reminded the reader the linkage between characters, scenario, story conflict and conflict resolution, and the structure of the text .No-answers. Feedback for correct Yes-answers consisted on a reinforcement sentence that referred to the concentration of the reader (‘Very good! It is clear that you are very concentrated’), while the feedback for correct answers No-answers always began by saying: ‘Of course not, because that never happened.’ and ended with the reinforcement sentence. When the readers responded incorrectly, the explanatory feedback consisted on a re-iteration of the text cited from the text plus the mentioning of the importance of paying attention to what one understands, as well as to what one does not clearly understand.Finally, questions intended to measure participants’ comprehension monitoring, were always built as ‘Did you realized that…’ and the sentence was completed by a literal phrase of the text or one slightly modified for Our study contrasted three assisted-reading experimental conditions, two of them with explanatory feedback  and one control condition. In the training and adaptive experimental conditions, the feedback the readers received after responding a question was aimed to encourage them to reflect and think over the question and the answer they chose, independently of whether the given answer was correct or not. Thus, the training/adaptive feedback provided the reader with an explanation for the answer of the question, while at the same time reassured readers when answering correctly  and inviting reconsideration , when the response was incorrect . The logic behind inviting reconsideration was to keep both training- and adaptive-feedback explanatory in nature. In other words, the feedback should point to the relevant information necessary to answer the question accurately, independently of whether the actual answer of the participant was correct or incorrect.The critical difference between the training and adaptive assisted-reading experimental conditions was the way in which the selection of the meta-cognitive skill, in other words the type of question , was presented to the reader in a particular moment. Presentation of questions in the training condition was counterbalanced: for each story, participants read the same number of questions on each meta-cognitive skill and their presentation was pseudo-randomized. The adaptive experimental condition, instead, selected the weakest meta-cognitive skill at the user level and prioritized its presentation. For this reason, the adaptive experimental condition required an individual profile as a starting point to work, and such data was obtained from participants’ session using the training condition (see Design). It also joined text fragments, first two, then three, for readers with accuracy higher than 75% (two fragments) and 85% (three fragments) in all meta-cognitive skills. The control condition also presented counterbalanced question types but the feedback consisted only in the word “Correct” or “Incorrect,” depending on whether the answer given by the reader was correct or not.Table 1). The first three sessions (week 1) formed the Exposure Block, while the subsequent three sessions (week 2) constituted the Testing Block. In the first block (week 1), the full sample of participants was randomly divided into two groups; one with approximately twice as many participants as the other (n = 34 and n = 58). The smaller group was assigned to the control condition, while the larger group was assigned to the training condition. During this block, participants were presented with five stories, each of which had nine fragments to be read, and 27 questions to be answer .We constructed a website we used for presentation and management of the stories and data. All the stories, questions and feedback were presented one at the time on the screen. In such way, the readers could concentrate on a single task at the time . Participants were given six 30-min reading sessions in two blocks (three sessions on each block) over a 2 weeks period  to enter the website and work individually on a computer. The number of participants per session varied between 10 and 15 students at the time. As participants logged in the website, they were first presented with written instructions. In these instructions, they were informed that they would read the stories fragment by fragment and receive questions about them. It also included words of encouragement such as ‘We want to invite you to read… but we want your reading to be as fun as possible.’ After reading the instructions, participants clicked on a ‘Next’-button and were presented with five pictures, each of them representing one story.On each reading session, the students were invited to participate voluntarily. No personal data from students was recorded and all of them participated voluntarily on a session basis. Students could read the titles and select any out of five stories to read. When they clicked on a picture they were presented with the title and first fragment of the story they selected. There was no time constraint and participants could read the story fragments at their own pace. When participants clicked on a ‘Next’-button, they were presented with a written question, plus a ‘Yes’- and a ‘No’-button. After they gave their response, the response feedback was presented on the screen plus a ‘Next’-button. When they pressed ‘Next’ a new fragment was presented, and the loop of fragment-question-feedback continued until the end of the story. When the story ended, participants read a message stating that they had finished the story and encouraged them to read other stories or even the same one again if they wanted. However, the icon of the most recently read story temporally disappeared and did appear again only after a different story was fully read. When an already-read story was chosen by the participant, the system would assign a new question for each fragment until there were no more questions to be answered. This meant that each story could be fully read up to three times, after which the icon for the story disappeared from the story selection screen.The procedure was approved by the Ethics Committee of the Campus Villarrica. All activities were performed in the school dependencies and during regular school hours as a complementary informatics and language activity. The participation of the students was approved by the Principal of the school and the head of the Technical Pedagogical Unit as the legally authorized representatives.Before inferential analysis, we examined individual participants’ responses and decided to exclude four participants since they gave only ‘Yes’ responses. All other data were included in the analysis. Our basic dependent variable was participants’ accuracy, but we were also interested in seeing how such accuracy developed in time and, particularly, as a function of the different assisted-reading experimental conditions. One clear candidate variable to evaluate such effect in time was the number of responses at the participants’ level. The more questions they responded, the more experience they are supposed to gain. If there are any differences in the effect of such experience on participants’ accuracy as a function of the assisted-reading experimental condition, we should observe them by contrasting the three experimental conditions across time (as reflected by the number of responses). However, due to the nature of the task, data were strongly unbalanced in the number of responses per participant, per condition and per meta-cognitive skill. The adaptive condition exhibits much less questions of text structure relative to inferences and comprehension monitoring, while the control condition exhibits overall much less questions per participant .Table 2 shows the cutting values that divided the percentile groups per condition and skill. It also shows the number of cases per group and the cumulative percentage this number meant for the total.Consequently, we decided to group the number of questions based on four quartiles per experimental condition and meta-cognitive skill. In doing so, we found a principled way to obtain a more balanced data set for comparison. z- and p-values.This grouping led to a more balanced data set for comparison, which we subsequently compared using a generalized linear mixed model approach, henceforth GLMM  and their interaction. They also included a random intercept for participants and fixed effects and interaction random slopes for the participant random intercept. To simplify the model and improve convergence, we did not include random correlations between predictors and the random intercept . To minimize collinearity between fixed factors, we centered the predictors’ values on a mean of 0 before analysis, using a scale function (base Package in R). The modelst = -2.04, p < 0.05) but no main effect of response percentile group. More importantly, it evidenced a reliable interaction effect between the condition and response group . Figure 2A, shows a graphic representation of the observed interaction pattern. Accuracy remained relatively similar for all experimental conditions within the first two quartiles, yet from the third quartile on, a distinctive pattern for each condition emerged: accuracy in the adaptive condition increased to 0.69 [CI95% ± 0.7] and then to 0.74 [CI95% ± 0.6] in the third and fourth quartile respectively, compared to the other conditions that remained at 0.64 [CI95% ± 0.8] and 0.65 [CI95% ± 0.8] for control and 0.55 [CI95% ± 0.8] and 0.58 [CI95% ± 0.8] for the training condition in the same quartiles.The results from the GLMM for the inference experimental condition showed a main effect of condition . Figure 2B illustrates the mean accuracy pattern across the response groups, showing no clear differences between conditions across time. Finally, the GLMM from the comprehension monitoring experimental condition detected a main effect of response percentile . As it is shown in Figure 2C, there is a tendency of a decrease in accuracy, in particular for the control condition. Such effect, however, was not modified by the condition as reflected by the absence of the predicted interaction between assisted-reading condition and the response percentile groups. Table 3 summarizes the results of the GLMMs analysis.In contrast to the results for the inference experimental condition, the outcome of the GLMM for the text structure experimental condition showed no main effect of condition or group, neither showed interaction between predictors , and those that concentrated in more high-level skills did so in older readers.Consequently, we designed and implemented a web platform that presented 4th, 5th, and 6th graders with a set of A-books, questions about them, and corresponding response feedback. Critically, we contrasted three assisted-reading experimental conditions to investigate our hypothesis, namely (1) a condition that supported specific cognitive skills  and that adapted to the users’ profile (adaptive condition), (2) a condition that supported the same specific cognitive skills but did not adapt to the users’ profile (training condition), and (3) a control condition that did not support cognitive skills nor adapted to the users’ profile.Figure 2A). Moreover, analysis of the accuracy for the text structure and comprehension monitoring did not reveal such advantage for the adaptive condition. In the text structure experimental condition, we observed a late advantage for the adaptive condition , which could be interpreted in favor of our predictions. This advantage was nevertheless not strong enough to bring about an interaction effect between condition and response percentile group that was statistically reliable. Finally, the results observed in the comprehension monitoring experimental condition are the most puzzling ones; we observed a reliable main effect that suggests that readers’ performance deteriorated over time. The accuracy pattern, however, suggests that effect is carried by the control condition, which evidence a drop of around 15% in the fourth quartile relative to the first three quartiles.Participants read stories in one of these three different experimental conditions, while we measured their accuracy on each meta-cognitive ability question. We predicted that the adaptive condition would produce over time better accuracy scores compared to the training and the control experimental conditions, and that the training condition would surpass the results of the control condition. Indeed, the analysis of the inference experimental condition showed a reliable advantage for the adaptive condition relative to both the training and the control experimental conditions. However, the participants in the control condition performed better relative to those in the training condition  needed to performed specific cognitive tasks , their accuracy increased over time. Yet, there are a number of issues worth addressing with regards to the data pattern observed in the study. First, the results pattern suggests that for the inference experimental condition as for the comprehension monitoring experimental condition, readers in the control condition overcame the performance of the readers in the training condition. This unexpected result might find an explanation on the literature about students’ self-regulation and behavior modulation . AccordiA second issue relates to the overall pattern observed for the comprehension monitoring experimental condition. The statistical analysis suggests a general tendency for a decrease in reader accuracy, in other words, the opposite to the intended effect. A potential explanation for the failure of this experimental condition could be that the questions intended to evaluate the comprehension monitoring of the comprehension process were not able to capture this meta-cognitive skill properly. Previous research has made used of the insertion of errors in the text. For instance, inconsistencies within the same paragraph were deliberately included to assess whether the reader was paying attention to the content of the story e.g., . We follThis discussion leads us to a final issue of the present study. The results (in particular from the comprehension monitoring skill) rise the question on whether our intervention can produce significant changes in different meta-cognitive skills, or such improvements are limited to a specific meta-cognitive skill, in this case, the capacity of the readers to make offline inferences about the text they are reading. Our findings speak against this possibility; however, there are some attenuating points that might prompt a more optimistic view. As we argue above, the comprehension monitoring questions were most probably not the most adequate ones. Moreover, recent research has also been unsuccessful in finding improvement in comprehension monitoring both in short and long term see , perhapsFigure 2). This meant that they received overall much more reinforcement in the inference condition relative to text structure condition , particularly in the adaptive condition (736 vs. 243 questions). There is nevertheless a (non-significant) trend of improvement in the last quartile in the text structure skill . Considering this  and the time readers spent using the system (only 1 week), the significant improvement observed at least for the most reinforced ability seems promising.With regard to the other two assessed meta-cognitive skills, participants were from the beginning less accurate on inference questions, relative to their performance on questions about the structure of the text  ICTs interventions for educational contexts. They show that a system (and perhaps any kind of instruction) that can adapt to the user’s profile is more effective compared to those that are less flexible in the assignment of a task. Such principles are not new in the context of school teaching e.g., , yet theOne challenge for personalized teaching is avoiding the overt separation of students in different groups in the classroom or in different classrooms see . In thisFigure 3 shows an example of an individual report  sent via email. This graphical information is accompanied by the exact score  and explanations and advice  for teachers and parents.Another practical implication is the potential use of the A-book as soft-assessment tool and guidance for teachers and parents. Students’ accuracy data are recorded online at the individual level. Every time a user reads a full story, an updated graphical profile is send automatically to the email address with which the user was created. In the present study, as testing phase of the system, we created all profiles prior testing and thus, we received all graphical reports. However, the basic idea is that teachers or parents create the children’s account and receive their progressive profiles. Indeed, the present study has limitations. Among them, we identify four, which we think can be corrected relatively easily and would mean a significant improvement for the system: first, the type of questions; second, adding more texts and of more diverse genres; third, adding a reaction time measure and minimal time for continuation; finally, making the A-book a multimodal platform. We are aware that closed-questions are not the best way to assess reading comprehension c.f. . HoweverFurthermore, in this first version of the A-book, we did not include a measure that could tell us how long students took to read each fragment , losing relevant data for exclusion of cases as well as for behavioral analysis. In this sense, including minimal time for continuation  would also mean an improvement. Finally, making the A-book a multimodal platform would make it not only more attractive for children and thus more likely to engage them in reading, but would also provide a much richer context, situating language within visual and auditory representations. Specifically, the insertion of illustrations accompanying text might allow the reader to construct a richer situation model of the narrative see .The present research started from the assumption that the interaction with the environment is of most relevance for the acquisition of language competencies see , withoutEG and GM developed the study concept, the experimental designed, prepared the materials and collected the data. GM implemented the website used for data collection. EG analyzed the data, interpreted the results and wrote 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.


Human menstrual blood-derived stem cells (MenSCs) are a novel source of MSCs that provide the advantage of being easy to collect and isolate. Exosomes contain some mRNAs and adhesion molecules that can potentially impact cellular and animal physiology. This study aimed to investigate the therapeutic potential of MenSC-derived exosomes (MenSC-Ex) on AML12 cells (in vitro) and D-GalN/LPS-induced FHF mice (in vivo).Transmission electron microscopy and Western blot were used to identify MenSC-Ex. Antibody array was used to examine cytokine levels on MenSC-Ex. MenSC-Ex were treated in D-GalN/LPS-induced AML12 in vitro. Cell proliferation and apoptosis were measured. MenSC-Ex were injected into the tail veins of mice 24 h before treatment with D-GalN/LPS. Blood and liver tissues served as physiological and biochemical indexes. The number of liver mononuclear cells (MNCs) and the amount of the active apoptotic protein caspase-3 were determined to elaborate the mechanism of hepatoprotective activity.Human menstrual blood-derived stem cell-derived exosomes (MenSC-Ex) are bi-lipid membrane vesicles that have a round, ball-like shape with a diameter of approximately 30–100 nm. Cytokine arrays have shown that MenSC-Ex expressed cytokines, including ICAM-1, angiopoietin-2, Axl, angiogenin, IGFBP-6, osteoprotegerin, IL-6, and IL-8. MenSC-Ex markedly improved liver function, enhanced survival rates, and inhibited liver cell apoptosis at 6 h after transplantation. MenSC-Ex migrated to sites of injury and to AML12 cells (a mouse hepatocyte cell line), respectively. Moreover, MenSC-Ex reduced the number of liver mononuclear cells (MNCs) and the amount of the active apoptotic protein caspase-3 in injured livers.In conclusion, our results provide preliminary evidence for the anti-apoptotic capacity of MenSC-Ex in FHF and suggest that MenSC-Ex may be an alternative therapeutic approach to treat FHF.The online version of this article (doi:10.1186/s13287-016-0453-6) contains supplementary material, which is available to authorized users. Clinical fulminant hepatic failure (FHF) causes relatively high mortality and affected patients present with very severe clinical symptoms, such as coagulopathy, jaundice, and multiorgan failure. However, the only clinical treatment for FHF is liver transplantation, which is limited by a shortage of donor livers .D-galactosamine  and lipopolysaccharide (LPS) co-induce  FHF in mice, producing a phenotype that copies clinical FHF. This mouse model is therefore commonly used as a test model . LPS is Human menstrual blood-derived stem cells (MenSCs) are human menstrual blood progenitor cells (MBPCs) that are isolated from menstrual fluids –6. MenSCExosomes are bi-lipid membrane vesicles that have a diameter of 50–100 nm and are secreted by various cell types. Exosomes carry a complex cargo load of proteins and RNAs that can potentially impact cellular and animal physiology. Studies have shown that exosomes are involved in complex physiological processes, such as intercellular communication, antigen presentation, and immune responses . High-peSilybum marianum, is the well-researched drug in the treatment of liver disease  using Western blot analysis.5 AML12 cells in one well of a 6-well culture plate, which was incubated for 24 h. AML12 cells were incubated with 100 μl of PBS instead of MenSC-Ex as the control. The cellular uptake of MenSC-Ex was observed under a confocal laser microscope .MenSC-Ex were labeled with a 1:10 ratio of Exo-Green . The exosomes solution was incubated at 37 °C for 10 min. A 100 μl volume of ExoQuick-TC was added to stop the labeling reaction. The labeled MenSC-Ex were placed on ice (or at 4 °C) for 30 min and centrifuged for 3 min at 14,000 rpm to remove the supernatant, which contained excess label. The pellet containing the labeled MenSC-Ex was resuspended in 500 μl of PBS, and at least 100 μl of the solution of labeled MenSC-Ex was added to approximately 1 × 10XenoLight DiR  was used to track exosomes in vivo. The solution of XenoLight DiR was diluted to 300 μM in PBS. We added the diluted XenoLight DiR solution to 10 μg of MenSC-Ex in 1 ml PBS to obtain a final concentration of 2 μM. The cells were incubated with MenSC-Ex at room temperature for 30 min. A 1/5 volume of ExoQuick-TC Exosome Precipitation Solution  was then added to the supernatant for 30 min. The solution was then centrifuged for 3 min at 14,000 rpm. The pellet, which contained the fluorescently labeled MenSC-Ex, was resuspended in 100 μl of PBS. We then systemically administered 50 μg of MenSC-Ex via the tail vein. IVIS analysis  and dissections were performed after 3 h and 6 h.p < 0.05) for samples with fluorescence intensity values that exceeded 300 (RayBiotech). Differentially expressed proteins were arranged using hierarchical clustering and represented as a heat map. The heat map was generated using R software (http://www.r-project.org/).Human Cytokine G1000 arrays  were used according to the manufacturer’s instructions to measure the expression levels of 120 cytokines in the MenSCs and MenSC-Ex. Positive signals were captured on glass chips using a laser scanner , and the observed fluorescence intensities were normalized to the intensities of the internal positive controls. These cytokines were screened using the following integrated conditions: the MenSC group compared to the MenSC-Ex group  was used to evaluate proliferation in MenSC-Ex-treated D-GalN/LPS-induced AML12 cells. After the cells were cultured for 24 h with 44 μg/ml D-GalN and 100 ng/ml LPS, the CCK-8 reagent was added to the chamber, and the cells were incubated for an additional 3 h according to the manufacturer’s protocol. The optical densities of the solutions were read at 450 nm (OD450) and measured using a multifunctional microplate reader .To analyze cell apoptosis, MenSC-Ex-treated AML12 cells that were cultured for 24 h with 44 μg/ml D-GalN and 100 ng/ml LPS were collected, centrifuged, and then stained with propidium iodide and Annexin V in the dark for 30 min at room temperature using a cell apoptosis Analysis Kit (Sigma-Aldrich). Cell apoptosis was then analyzed using flow cytometry.n = 10 per model group). To evaluate the therapeutic efficacy of MenSC-Ex on FHF, 1 μg/μl of MenSC-Ex in PBS or PBS alone was injected into the tail veins of mice 1 day before treatment. The animals were anesthetized using sodium pentobarbital  6 hours after treatment with D-GalN/LPS. The serum samples were centrifuged at 3000 rpm for 10 min to collect clear serum to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). A portion of liver tissue was stored in 4% paraformaldehyde for histological and immunohistochemical analysis. The remainder of the tissue samples was washed in cold saline and preserved at -80 °C for further analysis using reverse transcription-polymerase chain reaction (RT-PCR). The survival rates of the mice were determined for the 12 h period following D-GalN/LPS challenge.To induce FHF in mice, C57BL/6 mice (20 ± 2 g) were intraperitoneally injected with D-GalN (800 mg/kg) (Sigma-Aldrich) and LPS (50 μg/kg) (Sigma-Aldrich). Mice injected with an equal volume of PBS alone were used as the model group for the FHF model  and aspartate aminotransferase (AST) levels. ALT and AST levels were measured using commercial kits  according to the manufacturer’s instructions.The levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis alpha (TNF-α) were measured in serum samples using commercially available enzyme-linked immunosorbent assays (ELISAs) (RayBiotech) according to the manufacturer’s instructions.Liver tissues were harvested from mice 6 h after treatment with D-GalN/LPS or MenSC-Ex. Tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned to a 5-mm thickness, and stained with hematoxylin and eosin (H&E). The sections were imaged using an Olympus IX83 inverted microscope  equipped with Olympus cellSens software (cellSens Standard 1.9).2O2 for 10 min. The sections were then pretreated via heat-mediated antigen retrieval with sodium citrate buffer (pH6.0). The tissue sections were blocked in 10% FBS for 20 min at room temperature and then incubated with rabbit polyclonal antibodies against mouse caspase-3  overnight at 4 °C. The slides were washed three times with PBS for 5 min and subsequently incubated with secondary antibodies (Abcam). A solution of diaminobenzidine tetrahydrochloride  was used as the reaction substrate.To perform the immunohistochemistry test, peroxidase activity was blocked by incubating the sections in 3% HApoptosis was detected in liver cells in paraffin-embedded sections using a fluorescence terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay kit  according to the manufacturer’s instructions. Stained sections were observed and photographed using an Olympus IX83 inverted microscope equipped with Olympus cellSens software. The number of positive cells was counted in six randomly selected fields per slide.Genomic DNA was extracted from liver samples according to the instructions supplied by the manufacturer of the DNA Ladder kit . The DNA was then electrophoresed in a 1.5% agarose gel, which was stained with 0.1 g/ml ethidium bromide at 140 V for 20 min.Liver mononuclear cells (MNCs) were isolated and prepared as previously described . Briefly-ΔΔCT. GAPDH served as an internal control.Quantitative real-time RT-PCR (qRT-PCR) was used to analyze mRNA expression levels using a CFX96 Real-time PCR Detection System . These experiments were performed according to a previously described protocol . BrieflyAfter MenSCs were collected, the cells and MenSC-Ex were homogenized using cell lysis buffer  containing 100× phenylmethylsulfonyl fluoride (PMSF) . Twenty micrograms of total protein were obtained from MenSCs and MenSC-Ex and placed in separate lanes to be separated using electrophoresis on NuPAGE® Novex 10% Bis-Tris gels . The separated proteins were then transferred onto PVDF membranes (EMD Millipore). The membranes were blocked in 0.5% bovine serum albumin (BSA) for 1 h at room temperature and then incubated with primary antibodies at 4 °C overnight. They were subsequently incubated with HRP-conjugated secondary antibodies  for 1 h at room temperature. Immunoreactive bands were visualized using enhanced enhanced chemiluminescence (ECL) reagent (Bio-Rad) with a Tanon-4500 digital image system .P values < 0.05 (*) or < 0.01 (**) were considered to indicate statistical significance.All statistical analyses were performed using GraphPad Prism v5.0 . All data represent the means ± standard deviation. One-way analysis of variance (ANOVA) was used to determine differences between groups. MenSCs have morphologies and immunophenotypes that are similar to MSCs. The MenSCs exhibited a spindle-shaped, fibroblast-like morphology Fig. ; and theMenSC-Ex was prepared as previously described. Transmission electron microscopy (TEM) showed that MenSC-Ex displayed a round, ball-like shape and had diameters of approximately 30–100 nm Fig. . WesternTo identify molecules expressed on MenSCs and MenSC-Ex, an antibody array was used to examine cytokine levels Fig. . SeveralTo determine whether MenSC-Ex can be taken up by AML12 cells, we labeled MenSC-Ex with Exo-Green, a fluorescent cell linker compound that is incorporated into cellular proteins. When we incubated the Exo-Green labeled exosomes with AML12 cells, subsequently green fluorescence in the cytoplasm of almost every AML12 cell was observed Fig. . These rTo determine whether MenSC-Ex can be tracked in C57/BL6 mice, MenSC-Ex were labeled with XenoLight DiR. We administered the DiR-labeled MenSC-Ex into the tail veins of C57/BL6 mice, and then evaluated their distribution using in vivo imaging. At 3 h and 6 h after the injection, fluorescence was detected in the liver, the lungs, and the spleen Fig. . The in To measure the effect of D-GalN/LPS on the viability of AML12 cells, the ratio of its inhibitory effect was determined using a CCK-8 assay. Three different doses  of MenSC-Ex were found to inhibit the effects induced by D-GalN/LPS on AML12 cells. The data for five of the groups showed significant differences. Furthermore, the inhibitory ratio increased in a dose-dependent manner as the dose of MenSC-Ex increased. These results demonstrated that MenSC-Ex exert an anti-apoptosis effect on AML12 cells Fig. .Fig. 5MeTo determine the effects of MenSC-Ex on D-GalN/LPS-induced AML12 apoptosis, AML12 cells were pretreated with either exosomes or PBS (both in FBS-free medium) for 24 h. The cells were then co-incubated with D-GalN/LPS for an additional 6 h. Apoptosis was measured in the AML12 cells using Annexin V/PI Fig. . When ceMice began to die at 6 h after they were injected with D-GalN/LPS, and the mortality rate in these mice reached 80% within 12 h. However, mice pretreated with MenSC-Ex had significantly reduced mortality Fig. . Mice trp < 0.01). There was no significant difference between the MenSC-Ex group and the silymarin group    Fig. -F. Furth01) Fig. -I.To assess general morphological changes in the liver, liver tissue sections were mounted and stained with H&E Fig. . The norApoptosis was detected in hepatocytes using fluorescent TUNEL staining. A large number of TUNEL-positive hepatocytes were observed in liver tissues obtained from mice treated with D-GalN/LPS. However, few TUNEL-positive hepatocytes were observed in the livers of mice in the MenSC-Ex and silymarin groups. There were substantial differences in the number of apoptotic hepatocytes between the D-GalN/LPS and MenSC-Ex groups Fig. .Fig. 7MeGenomic DNA fragmentation was assayed to confirm that hepatocytes were undergoing apoptosis. DNA fragmentation was observed in the livers of mice treated with D-GalN/LPS, while no DNA fragmentation was observed in the livers of the MenSC-Ex group, and only a small amount of DNA fragmentation was observed in the silymarin group Fig. .p < 0.01), the PBS and silymarin livers exhibited lower levels than the D-GalN/LPS group  cells by the total number of lymphocytes per liver. NK cells (CD3−NK1.1+) and total T cells (CD3+NK1.1−) were also detected in the liver using flow cytometry. The results showed that treatment with D-GalN/LPS induced NK cells to accumulate in the liver (p < 0.01)  cells that were in the liver was calculated by multiplying the percentage of CD301) Fig. , C. Admip < 0.01)  Fig. , D. TherBecause they are easy to collect, isolate, and there are no ethical considerations associated with their use, MenSCs have become a useful tool for exploring how MSCs can be used to treat tissue injuries. Our group previously reported that MenSCs are a promising therapeutic method for treating some diseases, such as liver injury and type 1 diabetes , 31. BasSeveral articles have shown that MSCs may perform paracrine functions that might play therapeutic roles in disease states –35. It hWe evaluated liver cells in vivo and in vitro using fluorescence-labeled MenSC-Ex. Experiments in which treated cells or mice with MenSC-Ex provided initial insights into potential cellular therapies that might involve these structures. We observed clear fluorescence in AML12 cells and mice post-treatment with fluorescence-labeled MenSC-Ex. These data suggest that MenSC-derived exosomes are capable of entering into and performing functions within injured livers.We used a D-GalN/LPS-induced mouse model of FHF to explore the mechanisms underlying clinical liver failure. In the present study, administering an injection containing MenSC-Ex prior to D-GalN/LPS reduced the circulating levels of TNF-α, IL-6, and IL-1β, decreased hepatic apoptosis, improved liver function, and eventually lowered the fatality rate in FHF mice. Our findings also demonstrated that MenSC-Ex inhibited hepatocyte apoptosis and enhanced survival in mice treated with D-GalN/LPS. Specifically, an intravenous injection of MenSC-Ex reversed organ failure. All of our results showed that MenSC-Ex functioned to affect important pharmacological characteristics.According to the results of our experiments, the mechanisms that explain the therapeutic effect of MenSC-Ex on D-GalN/LPS-induced FHF involves the inhibitory immunomodulation of activated MNC proliferation or the activation of apoptosis-associated proteins. In the present study, one dataset suggested that there were more MNC in the D-GalN/LPS group than in the group treated with MenSC-Ex and the positive control group. One possible explanation for this result is that, in vivo MenSC-Ex may inhibit MNC proliferation, and MenSC-Ex may thereby act as crucial contributors to the observed reductions in FHF. The amount of cleaved caspase-3 expressed in the D-GalN/LPS group was lower than the amount expressed in the MenSC-Ex, but there was no difference with the positive control group. These data suggest that the activation of the apoptosis-related protein cleaved caspase-3 is another process that contributes to the mechanisms by which MenSC-Ex inhibit liver apoptosis in D-GalN/LPS-induced FHF.NK cells are innate immune cells. A variety of artificially synthesized and natural products have been tested to evaluate their ability to recruit and activate NK cells . MicrobiCaspase-3 is the most important apoptosis-associated protein . The actSilymarin has the therapeutic effect of restoring the normal liver functions . In our Although we demonstrated that MenSC-Ex ameliorated D-GalN/LPS-induced FHF in mice and explored mechanisms that may potentially underlie this effect in the present study, there are still many challenges to overcome before applications involving MenSC-Ex can be used in clinical medical practice. For example, FHF occurs rapidly, and when patients present at the hospital, they are almost always in the terminal stage of the disease. FHF causes the massive death of liver cells, and humans are much larger than mice and most other model mammals. Therefore, when MenSC-Ex are used to treat this disease, appropriate attention must be paid to the dose. In this study, MenSC-Ex reduced apoptosis in hepatocellular cells and enhanced longevity in FHF mice. However, treatments that do not renew hepatocellular tissues may be insufficient for treating FHF. Therefore, identifying methods to use MenSC-Ex in therapeutics remains a necessity for developing these cells into clinically applicable strategies.In this study, we present preliminary evidence demonstrating the potential of MenSC-Ex for reducing fulminant hepatic failure (FHF). These data suggest that MenSC-Ex transplantation deserves further study as an adjunctive or alternative approach for treating FHF.


Resveratrol (RSV) has been reported to stimulate osteoblast differentiation in which Wnt/β-catenin signaling pathway played a crucial role. However, whether and how RSV activated Wnt/β-catenin pathway in osteogenic differentiation still remained elusive.In vivo polymethylmethacrylate (PMMA) particle-induced osteolysis (PIO) mouse model and in vitro PMMA particle-stimulated mouse mesenchymal stem cells (mMSCs) experiments were established. Relative expression levels of lncRNA KCNQ1OT1, β-catenin, Runx2, Osterix and osteocalcin were determined using quantitative Real-Time PCR. Western blotting was used to measure β-catenin protein expression. In addition, the alkaline phosphatase activity and mineral deposition level using alizarin red S staining were performed to examine osteogenic differentiation status. The interaction between KCNQ1OT1 and β-catenin was confirmed by RNA pull down assay.RSV significantly attenuated PIO in vivo and PMMA-particle inhibition of osteogenic differentiation of mMSCs. Moreover, KCNQ1OT1 exerted the similar function in mMSCs by regulating β-catenin. Further study demonstrated that RSV exerted its effect on osteoblastic differentiation by regulating KCNQ1OT1. Consequently, RSV alleviated PMMA-particle inhibition of osteoblastic differentiation via Wnt/β-catenin pathway activation in vivo and in vitro.RSV accelerated osteoblast differentiation by regulating lncRNA KCNQ1OT1 via Wnt/β-catenin pathway activation, indicating the functional role of RSV in modulating osteogenesis. It has been well documented that bone remodeling was supported by dynamic equilibrium between bone resorption and bone formation which were regulated and maintained by osteoblasts throughout lifelong . ImbalanMultiple endogenous cytokines and growth factors have been identified to play crucial roles in regulating osteoblast differentiation. For instance, bone morphogenetic protein 2 (BMP2) potentiated osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) via BMP-2/Smad/Runx2 signaling pathway activation . In addiOn the other hand, resveratrol , a natural phytoalexin extracted from the root of veratrum grandiflorum, was widely found in a diversity of plants such as grape, peanut, hellebore and so on. Further, reports have shown that RSV had multiple pharmacological properties including antioxidant , anti-inTherefore, the objective of this study was to explore the underlying effect and the relevant molecular functions of RSV on osteogenic differentiation.Purified PMMA particles with the mean particle diameter of 0.330 ± 0.019 μm and 90% of the particles < 1 μm measured using scanning electron microscope were purchased from Polysciences . The particles were disinfected by torrefaction at 180 °C for 6 h, followed by treatment twice with 70% ethanol at room temperature for 24 h, then washed thrice with sterile phosphate-buffered saline (PBS) and finally desiccated under a bioclean bench. Only endotoxin-free particles were used in forthcoming experiments detected using a Limulus Amoebocyte Lysate assay .To investigate the role of RSV in osteogenic differentiation and osteolysis, mouse model of PIO was well established as previously described . In shorl-glutamine and 1% penicillin–streptomycin in a 5% CO2 incubator at 37 °C were treated with or without 20 or 40 μM RSV before incubation with 2 mg PMMA particles. MMSCs cultured in medium served as control. After 72 h, the expression levels of KCNQ1OT1, β-catenin, Runx2, Osterix and OCN were determined using qRT-PCR and western blotting. The ALP activity was also measured. In addition, ARS staining was used to examine the differentiation of mMSCs after 21 days.MMSCs  cultured in MSC basal media supplemented with MSC growth supplement (MCGS), 1% For further analyzing the biological functions of RSV in osteolysis, mMSCs treated with 40 µM RSV were incubated with or without ICG-001 (10 µM), a selective Wnt/β-catenin inhibitor  for 4 h prior to addition of 2 mg PMMA particles. In addition, mMSCs in media (namely control group) and mMSCs only treated with 2 mg PMMA particles (namely PMMA group) were used as negative controls. Three days later, ALP activity and the expression levels of Runx2, Osterix and OCN at mRNA levels were determined. The differentiation of mMSC was investigated using ARS staining after 21 days.7 cells/well in MSC basal media containing MCGS at 37 °C with 5% CO2 using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. After 48 h, mMSCs were respectively treated with 2 mg PMMA particles. Additionally, mMSCs in media (control) and mMSCs only treated with 2 mg PMMA particles (PMMA) served as negative controls.To explore the latent role of lncRNA KCNQ1OT1 in osteolysis, KCNQ1OT1 sequences were synthesized and subcloned into pCDNA3.1 vector  for KCNQ1OT1 overexpression. The pcDNA-KCNQ1OT1 or empty pcDNA vector were transfected into mMSCs seeded in six-well plates at a density of 10To clarify the interaction between RSV and KCNQ1OT1 in osteolysis, mMSCs were randomly divided into five groups: control, PMMA, PMMA + RSV, PMMA + RSV + sh-NC and PMMA + RSV + sh-KCNQ1OT1. Short-hairpin KCNQ1OT1 (sh-KCNQ1OT1) plasmids and its non-targeting sequence (sh-NC) as a negative control were transfected into mMSCs treated with or without 40 μM RSV in six-well plates with MSC basal media using Lipofectamine 2000 reagent and Opti-MEM I (Invitrogen) when the confluence reached 60%. After 24 h, mMSCs were finally treated with 2 mg PMMA particles.All mMSCs in above-mentioned groups were collected for ALP activity, qRT-PCR analysis of Runx2, Osterix and OCN expression and ARS staining.5/mL followed by transfection with pcDNA-KCNQ1OT1 or co-transfection with pcDNA-KCNQ1OT1 and siRNA-β-catenin  along with corresponding negative control (pcDNA) using Lipofectamine 2000 (Invitrogen) and then incubated at 37 °C with 5% CO2 for 24 h. MMSCs were used to detect the protein levels of β-catenin using western blot, ALP activity and the mRNA expression of Runx2, Osterix and OCN using qRT-PCR as well as mMSCs differentiation using ARS staining.To further understand the regulatory role of KCNQ1OT1 in osteolysis, mMSCs were then randomized to five groups: control, PMMA, PMMA + pcDNA, PMMA + pcDNA-KCNQ1OT1 and PMMA + pcDNA-KCNQ1OT1 + siRNA-β-catenin. Briefly, mMSCs were seeded in antibiotics-free medium in six-well plates at a density of 5 × 10The interaction between KCNQ1OT1 and β-catenin was further examined by RNA pull-down using a Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. The biotin-labeled KCNQ1OT1 was synthesized by transcription using reaction buffer containing Biotin RNA Labeling Mix and T7 RNA polymerase , treated with RNase-free DNase I (Roche) for 15 min and purified using an RNeasy Mini Kit . The protein extracts of mMSCs cells were incubated with purified biotin-labeled KCNQ1OT1 and streptavidin agarose magnetic beads , centrifuged at 1500 rpm for 30 min at 4 °C and mixed with PBS and protein loading buffer for 10 min at 98 °C. Eventually, the generated KCNQ1OT1-associated proteins and RNAs were boiled in SDS buffer for detecting β-catenin expressions at protein and RNA levels using IgG antibody as negative control (NC).For in vivo study of RSV, PMMA particle-induced mouse calvarial model was well established. Forty male 6–8 week-old C57BL/J6 mice were averagely distributed into four groups: PBS control , PMMA particles in PBS group , PMMA particles and RSV  as well as PMMA particles, RSV and ICG-001 . Mice in sham, vehicle group were daily injected with 20 μL PBS. Besides, mice in RSV and ICG group were treated with RSV at 10 mg/kg/day, while mice in ICG group daily received ICG-001 (10 μL). All mice were sacrificed after 2 week and craniums were separated for ALP activity and the mRNA and protein expressions of β-catenin, Runx2, Osterix and OCN using qRT-PCR.−ΔΔCt method. GAPDH was used as internal controls. Each measurement was performed in triplicate.Total RNA extracted from craniums and mMSCs following transfection was reversely transcribed to cDNA using Prime Script RT Reagent Kit with gDNA Eraser . RNA integrity was authenticated by the 260/280 nm ratio using spectrophotometer and electrophoresis method. Ultimately, the relative expression levels of target genes including LncRNA-H19, LncRNA NONHSAT009968, LncRNA KCNQ1OT1, LncRNA ANCR and β-catenin together with Runx2, Osterix and OCN were measured using a SYBR Green Real-Time PCR Master Mix-Plus  on an ABI 7500 Real-Time PCR system  and analyzed by 2Total protein was extracted by lysis buffer and isolated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and then blocked with primary anti active β-catenin antibody  overnight at 4 °C and incubated with anti-mouse horseradish-peroxidase (HRP) conjugated secondary antibody  after washed with Tris-buffered saline containing 10 mM Tris–HCl, 50 mM NaCl and 0.25% Tween 20 at 37 °C for 1 h. Protein quantification was performed using an enhanced chemiluminescence reagent . GAPDH was used as a loading control.ALP activity was considered as a specific marker of osteoblast differentiation which was detected using ALP activity colorimetric assay kit (Sigma-Aldrich). In the beginning, mMSCs incubated with PMMA particles following transfection or treated with different concentration of RSV for 3 days were harvested and rinsed twice with PBS followed by protein extraction using lysis buffer. The remaining supernatant was used to measure total protein content using a BCA protein kit  in accordance with the manufacturer’s instruction after centrifugation, and the absorption at 405 nm in each well was determined using p-nitrophenyl phosphate (PNPP) substrate (Sigma-Aldrich). However, craniums collected from mouse calvarial model were decalcified using nitric acid-paraformaldehyde solution for 3 days and sectioned followed by dewaxing using gradient ethanol. After that, clear sections were washed twice with PBS and incubated with chromogenic substrates at 37 °C for 1 h. ALP-positive cells were counted under a light microscope.Differentiated osteoblasts in vitro could induce cell mineralization after 21 days, which could be specifically stained with ARS. For ARS staining, mMSCs in each group were harvested after 21 days and stained with 40 mM ARS solution  at room temperature for 30 min, followed by lavement with double distilled water thrice and PBS for 15 min finally. Dye absorbance was determined at 570 nm after the addition of 10% cetylpyridinium chloride . Each group was performed in triplicate.All quantitative data were expressed as mean ± standard deviation (SD) and analyzed using SPSS version 22.0 . Statistical analyses were performed using two-tail Student’s T test for comparisons between two groups. P value < 0.05 indicated as significantly different.To identify the potential lncRNAs and the role of RSV in osteolysis in vivo, four previously reported lncRNAs, namely H19, NONHSAT009968, ANCR and KCNQ1OT1 were subjected to qRT-PCR analysis for relative expression in mouse model of PIO with or without RSV treatment. As shown in Fig. Next, we explored the effect of RSV on PIO in vitro. As shown in Fig. To evaluate the effect of KCNQ1OT1 in modulating osteogenesis, mMSCs were transfected with pcDNA KCNQ1OT1 for overexpressing KCNQ1OT1 and treated with RSV later. The results showed that PMMA particles treatment signally alleviated ALP activity (Fig. We further assessed the interaction between RSV and KCNQ1OT1 in mMSCs stimulated with PMMA particles. Our finding exhibited that ALP activity (Fig. We also evaluated the molecular mechanism by which RSV attenuated PMMA particles inhibition of mMSCs differentiation. Wnt/β-catenin signaling pathway was reported to play a key role in osteogenesis . In thisTo investigate the regulatory mechanism of KCNQ1OT1 associated with β-catenin, KCNQ1OT1 overexpression and β-catenin knockdown using siRNA were performed in mMSCs. Consistent with previous results, PMMA particles obviously downregulated β-catenin expression at mRNA and protein levels (Fig. Lastly, we verified whether RSV activated Wnt/β-catenin signaling pathway in vivo. As predicted, ALP activity (Fig. RSV was a naturally polyphenolic compound mainly isolated from grape and polygonum cuspidatum which was an effective therapeutic agent for osteoporosis  and osteGrowing evidence has manifested that osteogenic differentiation was a complicated physiological procedure involving a great deal of regulators containing ALP, Ruxn2, Osterix and OCN . ALP wasIn this study, KCNQ1OT1 and β-catenin expression levels were increased, moreover, ALP activity as well as Ruxn2/Osterix/OCN were obviously elevated after RSV treatment which also promoted the mineralization of mMSCs following PMMA particles stimulation. Besides, KCNQ1OT1 knockdown inhibited ALP activity and Ruxn2/Osterix/OCN as well as mineralization, however, RSV had the adverse effect, indicating that RSV accelerated osteoblastic differentiation in vitro by regulating KCNQ1OT1 and β-catenin.To further explore the molecular mechanism of KCNQ1OT1 in osteoblastic differentiation, PMMA particles-stimulated mMSCs were transfected with siRNA-β-catenin and/or pcDNA-KCNQ1OT1. The results showed that KCNQ1OT1 overexpression ascended ALP activity and Ruxn2/Osterix/OCN as well as extracellular matrix calcification, while simultaneously overexpression of KCNQ1OT1 and knockdown of β-catenin reversed the trend. These findings elucidated that KCNQ1OT1 alleviated PMMA particles induced-inhibition of osteoblastic differentiation by regulating β-catenin. Since KCNQ1OT1 was proved as the direct target of β-catenin signaling in colorectal cancer, the results indirectly revealed the potential interaction of KCNQ1OT1 and β-catenin.It was well known that Wnt/β-catenin pathway was an imperative positive regulator of bone homeostasis. In order to further discover the specified mechanisms of RSV in osteoblastic differentiation, its effects involving Wnt/β-catenin pathway were investigated. It turned out that RSV co-treated with ICG-001, a selective Wnt/β-catenin inhibitor decreased the ALP activity and Ruxn2/Osterix/OCN expression along with mineralization in mouse PIO model and mMSCs stimulated with PMMA particles. Consequently, the results confirmed that RSV alleviated PMMA particles-inhibition of osteogenic differentiation by activating Wnt/β-catenin in vivo and in vitro. Analogously, Ji et al. found that RSV exerted the anti-cancer effect via mediating Wnt/β-catenin pathway .In summary, our results uncovered that RSV alleviated PMMA particles-inhibition of osteogenic differentiation in mouse PIO model and mMSCs by upregulating β-catenin and KCNQ1OT1 expression. Meanwhile, KCNQ1OT1 also played similar effect on osteoblastic differentiation by mediating β-catenin. What’s more, RSV promoted osteogenic differentiation and suppressed osteolysis in vivo and in vitro via Wnt/β-catenin activation. Jointly, RSV could relieve osteolysis by promoting osteogenic differentiation through upregulating lncRNA KCNQ1OT1 via Wnt/β-catenin activation. This finding might serve as an essential pre-clinical evidence of RSV for prevention and treatment of bone diseases.


Our results provide the first proof of concept of targeting Fzd-TMD for the development of Wnt/Fzd modulators. The identified small molecular Fzd7 inhibitors can serve as a useful tool for studying the regulation mechanism(s) of Wnt/Fzd7 signaling as well as a starting point for the development of cancer therapeutic agents.Frizzled (Fzd) proteins are seven transmembrane receptors that belong to a novel and separated family of G-protein-coupled receptors (GPCRs). The Fzd receptors can respond to Wnt proteins to activate the canonical β-catenin pathway which is important for both initiation and progression of cancers. Disruption of the Wnt/β-catenin signal thus represents an opportunity for rational cancer prevention and therapy. Of the 10 members of the Fzd family, Fzd7 is the most important member involved in cancer development and progression. In the present studies, we applied structure-based virtual screening targeting the transmembrane domain (TMD) of Fzd7 to select compounds that could potentially bind to the Fzd7-TMD and block the Wnt/Fzd7 signaling and further evaluated them in biological assays. Six small molecule compounds were confirmed as Fzd7 inhibitors. The best hit, SRI37892, significantly blocked the Wnt/Fzd7 signaling with IC Frizzled (Fzd) proteins are a family of seven transmembrane receptors (7-TM) for secreted Wnt glycolipoproteins. The Fzd receptors can respond to Wnt proteins in the presence of the Wnt co-receptor low density lipoprotein receptor-related protein 5 (LRP5) or LRP6 to activate the canonical β-catenin pathway. Activation of Wnt/β-catenin signaling has been found to be important for both initiation and progression of cancers of different tissues , 2. ThusOf the 10 members of the Fzd family, Fzd7 is the most important member regulating cancer development and progression. Fzd7 is frequently up-regulated in a variety of cancers including colon cancer , 7, brea1-10) and the closely related Smoothened (SMO) have been listed by the International Union of Pharmacology (IUPHAR) as a novel and separated family of G-protein-coupled receptors (GPCRs) : the class-F (Frizzled) [The Fzd family (Fzdrizzled) . In addirizzled) , 28. Therizzled) , 30. Whirizzled) , 31, or rizzled) , 33 inteThe TMD of Fzd shares the same topology as classic GPCRs, including the conventional 7TM core, the intracellular C-terminal helix-8, the conserved cysteine residues in the extracellular loops as well as other common residues in the TMD of GPCRs , 35. TheThe recently reported crystal structure of the TMD of human SMO receptor bound to a small-molecule antagonist  further 1.35, Y2571.39), TH-III (L3433.32), TH-VI (Y4896.52), TH-VII  as well as a residue Y412 from EL-2. Results of multiple sequence alignment of the class-Frizzled showed that the properties of most of the binding site residues are well conserved  targeting the TMD binding site of the constructed Fzd7 model. 500,000 structurally diverse compounds were screened through a three-step docking/scoring process using the Glide program. The docked results of 738 top-scored compounds at the final docking stage were further visually inspected to select potential Fzd7 inhibitors that showed both shape and electrostatic complementarities to the receptor binding site. We hypothesized that occupying the two hydrophobic cores could be important for ligand binding and gave certain priorities to compounds that can fit into both hydrophobic regions, especially to those that fitted well to the bottom hydrophobic core. By taking into consideration structural diversity in our final selection, 67 structurally representative compounds were purchased and tested for their inhibitory effect on Wnt/β-catenin signaling in Wnt3-expressing HEK293 cells. At 10 μM concentrations, 3 of 67 compounds, SRI35959, SRI35961 and SRI35958, significantly ( > 50%) blocked Wnt3A induced Wnt/β-catenin signaling and their inhibitory effects were further confirmed in concentration dependent assays with ICM Figure .50 values of 0.66, 9.9 and 11.9 μM, respectively  possess a potentially metabolically oxidizable p-phenylenediamine-like unit. The most potent compound, SR37892, however, does not contain unfavorable chemical features and was therefore selected for further studies described below.50 values of 2.2 and 1.9 μM, respectively  protocol which allows binding site residues to be flexible. The two most potent hits, SRI35959 and SRI37892, were docked separately into the putative binding site of the Fzd7-TMD model. The docked models suggested that SRI37892 bound relatively tighter than SRI35959 with a docking score (which mimics the binding affinity) of -12.0 kcal/mol as compared to -10.3 kcal/mol of SRI35959, which is consistent with the observed experimental results that SRI37892 is a more potent inhibitor. Both compounds fit well into the Fzd7 binding site. In the docked models Figure , the phe2+ and Wnt/PCP signaling [In addition to the canonical β-catenin dependent signaling, there are also other noncannonical Wnt/Fzd signaling pathways that are independent of β-catenin, including Wnt/Caignaling . The mecignaling , other signaling . A possiignaling . NonetheAPC and CTNNB1 (β-catenin encoding gene) are two major factors for Wnt/β-catenin signaling overactivation in colorectal cancers [APC or CTNNB1 mutations [APC and CTNNB1 mutations.Wnt/β-catenin signaling is activated aberrantly in many human cancers, and FZD7 is frequently up-regulated in a variety of cancers including breast and colorectal cancer , 25. Whi cancers , 56. How cancers , 58, 59.Wnt/β-catenin signaling is a critical regulator of stem cells . Recent While increased Wnts expression has been reported in different forms of cancers, reduced expression of Wnts is associated with other disorders such as obesity . Recent Schrödinger, LLC, New York, NY, 2015).Structural model generation and molecular docking studies were conducted using the programs of the Schrödinger Suite 2015  complex (PDB ID: 4JVK) was used as the structural template. The sequences of SMO and frizzled receptors (Fzds1-10) was first aligned using ClusterX. The alignment results were then input into Prime to build the structural model of Fzd7-TMD. We also used the Ballesteros and Weinstein numbering system [1.50, Y2962.50, W3613.50, W3864.50, Y4325.50, A4876.50, I5427.50.Homology modeling was conducted using the g system  on the bGlide program with the default parameters. The 3D structures of ligands were prepared using the LigPrep program. The induced-Fit-Docking (IFD) protocol [Glide.Molecular docking studies were performed using the protocol , which iPipeline Pilot; [For the purpose of applying structure-based virtual screening, we assembled a virtual library consisting structurally diverse compounds selected from different commercial sources. Specifically, structures of a total of approximately eight million compounds were downloaded directly from the websites of twelve vendors . From the eight million commercially available compounds, we first identified the most diverse set of 100,000 structurally representative compounds using the clustering and diversity analysis protocols of e Pilot;  then formaestro, which adopts a three-step docking/scoring protocol implemented in Glide. 3D conformations of the 500,000 assembled compounds were prepared using LigPrep, and a total of 923,408 conformers were generated. All of the 923,408 conformers were first docked into the ligand binding site of the Fzd7-TMD model using the high-throughput virtual screening (HTVS) mode; the top 5% best-scored conformers were then re-docked and scored using the standard-precision (SP) mode; the top 10% of the SP docked conformers were further docked and scored using the extra-precision (XP) mode. Finally, the top 25% of the best XP-scored conformers were output for visual examination.Structure-based virtual screening was performed using the virtual screening workflow of Pipeline Pilot and the TOPOMER search module of SYBYLX program. The identified analogs were then docked to the binding site of the Fzd7-TMD model. The top-scored docked models were further visually examined for structural complementary in both shape and polarity to the receptor to select a final list of compounds for biological evaluations.Commercial analogs of confirmed active compounds were identified through substructure search and similarity search protocols of All cell lines were obtained from ATCC and grown under standard cell culture conditions at 37°C in a humidified atmosphere with 5% CO2. The cells were cultured in DMEM medium containing 10% of FBS, 2 mM of L-glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin.Plasmid pCS-Myc-hLRP6 containing the full-length human LRP6 cDNA was provided by Dr. Christof Niehrs , and the Super8XTOPFlash luciferase construct was provided by Dr. Randall T. Moon . Plasmid pcDNA3-Wnt3-HA was constructed as described previously . HEK293 Polyclonal anti-LRP6 and monoclonal anti-survivin were from Santa Cruz Biotechnology. Polyclonal anti-Fzd7 was from Novus Biologicals. Monoclonal anti-phospho-LRP6 and anti-axin2 were purchased from Cell Signaling Technology. Monoclonal anti-β-catenin was from BD Biosciences. Monoclonal anti-actin was from Sigma. Cells in 6-well plates were lysed in 0.5 ml of lysis buffer  at 4°C for 30 min. Equal quantities of protein were subjected to SDS-PAGE under reducing conditions. Following transfer to immobilon-P transfer membrane, successive incubations with a primary antibody, and a horseradish peroxidase-conjugated secondary antibody were carried out for 60-120 min at room temperature. The immunoreactive proteins were then detected using the ECL system. Films showing immunoreactive bands were scanned by HP Scanjet 5590 .Plasmid pGST-E-cadherin was provided by Dr. Gail Johnson (University of Rochester). The level of cytosolic free β-catenin was analyzed by the GST-E-cadherin binding assay as previously described . UncomplCells were seeded into 96-well tissue culture treated microtiter plates at a density of 4000 cells/well. After overnight incubation, the cells were treated with each individual compound for 96 h. Cell viability was measured by the CellTiter-Glo Assay (Promega).Cancer cells were seeded at a density of 800 cells/well into six-well plates. Sixteen hours after the plates had been set up, SR37892 was added, and media were replenished every 3 days. After being incubated for 12 days, colonies were fixed with 4% formaldehyde, stained with 0.5 mg/ml crystal violet, and imaged on a FluorChem HD2 Imager System (Alpha Innotech).


Aberrant expression of Fos-related antigen-1 (Fra1) is commonly elevated in various malignant cancers and is strongly implicated in invasion and metastasis. However, the molecular mechanisms underlying its dysregulation in human glioma remain poorly understood. In the present study, we demonstrate that up-regulation of Fra1 plays a crucial role in the glioma aggressiveness and epithelial–mesenchymal transition (EMT) activated by Wnt/β-catenin signal pathway. In glioma cells, activation of Wnt/β-catenin signalling by Wnt3a administration obviously induced EMT and directly activated the transcription of Fra1. Phenotype experiments revealed that up-regulation of Fra1 induced by Wnt/β-catenin signalling drove the EMT of glioma cells. Furthermore, it was found that the cisplatin resistance acquired by Wnt/β-catenin signalling activation depended on increased expression of Fra1. Analysis of clinical specimens verified a positive correlation between Fra1 and β-catenin as well as a poor prognosis in glioma patients with double-high expressions of them. These findings indicate that an aberrant Wnt/β-catenin signalling leads to the EMT and drug resistance of glioma via Fra1 induction, which suggests novel therapeutic strategies for the malignant disease. Among various human cancers, malignant glioma (MG) is one of the most aggressive tumours and occurs frequently in the central nervous system . MG ofteNumerous factors and signal pathways associated with the invasion and migration of glioma cancer have been found in previous studies. Among tc-Myc, cyclin D1 and matrix metalloproteinases (MMPs) [The Wnt/β-catenin pathway plays a critical role in many biological processes such as cell survival, proliferation, migration and polarity . Aberrans (MMPs) ,16. β-cas (MMPs) . Althougs (MMPs) .In the present study, Fra1 is identified as a target of Wnt/β-catenin signalling and then mediates EMT as well as cisplatin resistance in glioma cells. Further clinical analysis reveals that Fra1 is positively correlated with β-catenin and glioma progression, which confirms a critical role of Wnt/β-catenin/Fra1 signalling axis in glioma aggressiveness.2. For lithium chloride (LiCl) or CHIR99021 treatment, cells were cultured to approximately 40% confluence and then treated with 20 mM LiCl or 3 μM CHIR99021. Transfections of plasmids and siRNA were performed using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer’s instructions. Stable transfections of Wnt3a and shFra1 were maintained by G418.Human glioma cell lines U-251 and U-87 were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidified atmosphere with 5% COThe constructs encoding Flag-tagged Fra1 and Wnt3a were cloned into pcDNA3.0 vector. The siRNA (sc-35405) and shRNA (sc-35405-SH) targeting Fra1 were obtained from Santa Cruz Biotechnology. Antibodies against E-cadherin (sc-59780), Fibronectin (sc-69681), Vimentin (sc-73259), ZO-1 (sc-8146), Fra1 (sc-48424), Zeb2 (sc-271984), β-catenin (sc-31001) and GAPDH (sc-51907) were from Santa Cruz. Antibodies against Twist1 (ab50581), Zeb1 (ab124512), Snail (ab53519), Slug (ab27568) were from Abcam.Isolation of total RNA from cells was performed using RNAiso Plus (TaKaRa). The purity and concentration of RNA samples were determined by NanoDrop Spectrophotometer (NanoDrop). Reverse transcription was carried out with the PrimeScript RT reagent kit (TaKaRa). Real-time quantitative PCR was achieved using SYBR Select Master Mix (Roche) according to the manufacturer’s instructions. Target gene expression was normalized to actin levels in respective samples as an internal control and the results are representative of at least three independent experiments.Cells were harvested and lysed using Ripa lysis buffer . The protein concentration was determined with the Bradford method (Bio–Rad). The proteins were separated by 10% SDS/PAGE and transferred to nitrocellulose membranes (Whatman). The membranes were incubated with dilutions of primary antibodies followed by IRDye 800CW or IRDye 680–conjugated secondary antibodies and then detected by the Odyssey Infrared Imaging System (Li-COR).The promoter fragments of human Fra1 were amplified by PCR and cloned into the pGL3 vector. The reporter constructs containing different lengths of Fra1 promoter or mutated Tcf/Lef-binding sites were generated by subsequent PCR-based cloning. A pair of luciferase reporter constructs, TOPflash and FOPflash, was used to evaluate Tcf/Lef transcriptional activity. TOPflash contains three copies of TCF-4-binding sites and FOPflash contains mutated TCF-4-binding sites. The pRL-SV40 vector was cotransfected with the reporter constructs in each experiment as an internal control for transfection efficiency. After transfection for 24 h, cells were treated with 200 ng/ml Wnt3a for 12 h and luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). All experiments were performed in triplicate.Chromatin was cross-linked with 1% formaldehyde and sonicated to obtain a DNA fragment of 200–500 bp. After centrifugation, the supernatants were subjected to immunoprecipitation overnight at 4°C with antibodies against β-catenin or normal IgG. The DNA–protein complexes were isolated using Protein A/G PLUS-Agarose (Santa Cruz). The cross-linking was reversed and released DNA fragments were purified and quantified by QPCR using the following primer pairs for Fra1 promoter: (Forward) ACCGTAATGAAGATGGCG; (Reverse) TTAAATGTTTGCGATAAGG. Cmyc promoter :   was added and the ODCell culture and transfection were performed as described above. At 24 h after transfection, the monolayer was gently and slowly aspirated with a micropipette-like tip to create a 1 mm cell-free zone that was washed with serum-free medium or PBS to remove any remaining cells. The cell-free area was marked, serum-free medium was added, and the cells were returned to the incubator for 24–48 h. Images were acquired at different time points during the incubation. The wound was evaluated using ImageJ.5) were plated to a culture insert in serum-free medium, whereas complete medium with or without 200 ng/ml Wnt3a was applied to the lower compartment. After incubation for 48 h, the undersurface adherent cells that had invaded through the Matrigel were fixed in methanol and stained with 0.5% Crystal Violet. The air dried filter membrane was viewed under a microscope and four random fields were selected for cell counting.Cell invasion assay was performed in 24-well transwell plates (Costar) with 8 μm pore inserts coated with Matrigel (BD Biosciences). U-251 and U-87 cells  according to the manufacturer’s instructions. All the samples were assayed in triplicate.In vivo chemosensitivity experiment was performed by the subcutaneous transplantation of cells into BALB/c nude mice. The U-251 cells stably transfected with Wnt3a expression plasmids and shFra1 were suspended at a density of 107 cells/ml and 100 μl was injected into the flank of each nude mouse (n=6). Ten days post-injection, cisplatin (5 mg/kg) was intraperitoneally administered every 3 days for 18 days. Seven days after subcutaneous transplantation, we measured the sizes of the growing tumours every 3 days for 21 days using the formula (W2 × L). The mice were then killed and tumour weights were measured. All animal procedures were performed in compliance with the Guidelines for the Care and Use of Experimental Animals established by the Committee for Animal Experimentation of Hebei Medical University.In total, 87 glioma tissue specimens were collected for the present study from 2005 to 2012 from The Second Hospital of Hebei Medical University and The Third Hospital of Hebei Medical University . All specimens were fixed in 10% neutral formalin, embedded in paraffin and cut into 4-μm sections for immunohistochemical staining. The EnVision™ two-step method was used , as well as the following antibodies: antibodies against Fra1 and β-catenin, were purchased from Cell Signaling Technology . Prior to staining, sections were pretreated with microwaves for 18 min in a 0.01 M citrate buffer (pH 6.0) for antigen retrieval. 3,3′-Diaminobenzidine was used as a chromogen and 0.01 M PBS (pH 7.4) was substituted for primary antibodies as a negative control. To estimate the score for each slide, at least eight individual fields at 200× were chosen and 100 cancer cells were counted in each field. The immunostaining intensity was divided into four grades: 0, no expression; 1, mildly positive; 2, moderately positive and 3, markedly positive. The proportion of positive-staining cells was divided into five grades: 0, <10%; 1, 11–25%; 2, 26–50%; 3, 51–75% and 4, >75%. The staining results were assessed and confirmed by two independent investigators blinded to the clinical data. The percentage of positivity of the tumour cells and the staining intensities were then multiplied in order to generate the IHC score and graded as low expression (score 0–6) and high expression (score 7–12). Cases with a discrepancy in scores were discussed to obtain a consensus.t test or one-way ANOVA. Pearson’s correlation test was used in analysing the correlation. A value of P<0.05 was considered statistically significant.The software SPSS 13.0 was used in statistical analyses. Group distributions were performed with the Student’s It is noteworthy to determine the potential mechanisms of Wnt/β-catenin signalling in induction of glioma EMT. In U-251 and U-87 cells, decreased E-cadherin and ZO-1 accompanied by induction of Vimentin and Fibronectin at the mRNA level were observed in a Wnt3a dose-dependent manner A,B. ConsTo further study the relationship between Wnt/β-catenin signalling and Fra1, glioma cells were exposed to specific inhibitors (LiCl and CHIR99021) for GSK-3β. As a result, activating Wnt/β-catenin signalling by targeting GSK-3β increased the mRNA and protein levels of Fra1 A,B. ConsAs expected, overexpression of Fra1 decreased the expressions of epithelial markers and increased the expressions of mesenchymal markers, whereas Fra1 knockdown had the opposite effect A. Considin vitro and in vivo experiments. As shown in in vivo. The results showed that overexpressed Wnt3a increased resistance to cisplatin, and knockdown of Fra1 sensitized glioma cells to cisplatin. Notably, silence of Fra1 inhibited the protective effect of Wnt3a against cisplatin  but not with gender, age, tumour location, tumour diameter (P>0.05). Moreover, the Fra1 expression levels paralleled with the changes of β-catenin in the glioma cases as shown by immunohistochemical analysis (P<0.05) (P=0.003)  C. The 3-P=0.003) D. These cyclinD1 and c-myc are required for cellular proliferation in glioma [in vitro and in vivo. Further analysis of clinical specimens revealed that aberrant expression of Fra1 or β-catenin in glioma tissues indicated a poor prognosis.As the canonical Wnt pathway, β-catenin-dependent pathway plays an important role in many biological progresses such as tissue homoeostasis, embryonic development and tumorigenesis. In recent studies, it has been reported that aberrant activation of Wnt/β-catenin signalling contributed to glioma development . The evin glioma . Here, wThe activator protein-1 complex (AP1) consisting of homo- or heterodimers of Fos, Jun, ATF and MAF family members has been implicated as central activator of tumour cell invasion. Among them, Fra1 is one of the most frequently aberrant expressed AP1 proteins in a variety of tumour types including glioma –29. In pIn studies with GBM-derived cell lines, activation of the canonical Wnt/β-catenin pathway could modulate a set of EMT-TFs including Twist1 and Zeb1/2 . Twist1 In conclusion, our findings demonstrate that Wnt/β-catenin signalling can induce the EMT programme of glioma cells by up-regulating Fra1 expression. Further results reveal that Wnt/β-catenin signalling activates the transcription of Fra1 by directly binding β-catenin/Tcf/Lef transcriptional complex to the WRE located at the proximal region of Fra1 promoter. Phenotype experiments and clinical analysis support the critical role of Fra1 in mediating the glioma aggressiveness induced by Wnt/β-catenin signalling. Therefore, the correlation between Wnt/β-catenin signalling and Fra1 raises the possibility of developing novel biomarkers and therapeutic targets for glioma.


While women are generally underrepresented in STEM fields, there are noticeable differences between fields. For instance, the gender ratio in biology is more balanced than in computer science. We were interested in how this difference is reflected in the interdisciplinary field of computational/quantitative biology. To this end, we examined the proportion of female authors in publications from the PubMed and arXiv databases. There are fewer female authors on research papers in computational biology, as compared to biology in general. This is true across authorship position, year, and journal impact factor. A comparison with arXiv shows that quantitative biology papers have a higher ratio of female authors than computer science papers, placing computational biology in between its two parent fields in terms of gender representation. Both in biology and in computational biology, a female last author increases the probability of other authors on the paper being female, pointing to a potential role of female PIs in influencing the gender balance. There are fewer women than men working in Science, Technology, Engineering and Mathematics (STEM). However, some fields within STEM are more gender-balanced than others. For instance, biology has a relatively high proportion of women, whereas there are few women in computer science. But what about computational biology? As an interdisciplinary STEM field, would its gender balance be close to one of its “parent” fields, or in between the two? To investigate this question, we examined authorship data from databases of scholarly publications in biology, computational biology, and computer science. We found that computational biology lies in between computer science and biology, as far as female representation goes. This is independent of other factors, e.g. year of publication. This suggests that computational biology might provide an environment that is more conducive to female participation that other areas of computer science. Across all three fields, we also found that if the last author on a publication—usually the person leading the study—is a women, then there will also be more women in other authorship positions. This suggests that having women in leadership positions might be beneficial for overall gender balance, though our data do not allow us to uncover the underlying mechanism. There is ample literature on the underrepresentation of women in STEM fields and the biases contributing to it. Those biases, though often subtle, are pervasive in several ways: they are often held and perpetuated by both men and women, and they are apparent across all aspects of academic and scientific practice. Undergraduate students show bias in favor of men both when rating their peers  and theiWhen looking at research output in the form of publication and impact, the story is complex: Women tend to publish less than men , are undAlthough gender imbalance seems to be universal across all aspects of the scientific enterprise, there are also more nuanced effects. In particular, not all disciplines are equally affected. For instance, in the biosciences over half of PhD recipients are now women, while in computer science, it is less than 20% . This raTo this end, we are interested in the gender balance in computational biology and how it compares to other areas of biology, since computational biology is a relatively young field at the disciplinary intersection between biology and computer science. We examined authorship on papers from Pubmed published between 1997 and 2014 and compared computational biology to biology in general. We found that in computational biology, there is a smaller proportion of female authors overall, and a lower proportion of female authors in first and last authorship positions than in all biological fields combined. This is true across all years, though the gender gap has been narrowing, both in computational biology and in biology overall. A comparison to computer science papers shows that computational biology stands between biology and computer science in terms of gender equality.In order to determine if there is a difference in the gender of authors in computational biology compared to biology as a whole, we used data from Pubmed, a database of biology and biomedical publications administered by the US National Library of Medicine. Pubmed uses Medical Subject Heading (MeSH) terms to classify individual papers by subject. The MeSH term “Computational Biology” is a subset of “Biology” and was introduced in 1997, so we restricted our analysis to primary articles published after this date see .Pfemale) that an author in a particular dataset is female as well as a 95% confidence interval , we determined that the real Pfemale = 0.360 ± 0.018 . However, the discrepancy between biology and computational biology has been consistent over time  MeSH term  and those with a male last author . We found that women were substantially more likely to be authors at every other position if the paper had a female last author than if the last author was male  between impact factor and gender for the biology dataset. This is in contrast to previous studies from engineering that have found that women tend to publish in higher-impact journals [PZ > |z| = 0.568) between impact factor and Pfemale in computational biology publications. Further, for journals that have articles labeled with the computational biology MeSH term, the Pfemale for those articles is the same or lower than that for all biology publications in the same journal.One possible explanation might be that women are less likely to publish in high-impact journals, so we considered the possibility that the differences in the gender of authors that we observe could be the result of differences in impact factor between papers published in biology versus computational biology publications. We compared the t factor . There ijournals . By contPfemale, this difference is unlikely to explain the differences in Pfemale observed between our computational biology and biology datasets. Taken together, these data suggest that the authors of computational biology papers are less likely to be women than the authors of biology papers generally.We also examined whether computational biology or biology articles tend to have higher impact factors. Bootstrap analysis of authors in each dataset suggest that computational biology publications tend to be published in journals with a higher impact factor  section was added to arXiv in 2003. Quantitative biology is not necessarily equivalent to computational biology, and analysis of arXiv-qb papers that have been published and indexed on pubmed suggests that only a fraction of them are labeled with the “computational biology” MeSH term. However, this does allow us to make an apples-to-apples comparision between a field of biology and computer science. There are relatively few papers preprints prior to 2007, so we compared preprints in “quantitative biology” to those in “computer science” from 2007-2016.year, PZ > |z| < 0.005) increase in the proportion of female authors over time in quantitative biology, while there’s no significant increase in female representation in computer science preprints.Women were more likely to be authors in quantitative biology manuscripts than in computer science manuscripts in first, second, and middle author positions , Table 3Taken together, our results suggest that computational biology lies between biology in general and computer science when it comes to gender representation in publications. This is perhaps not surprising given the interdisciplinary nature of computational biology. Compared to biology in general, computational biology papers have fewer female authors, and this is consistent across all authorship positions. Importantly, this difference is not due to a difference in impact factor between computational biology and general biology papers.Articles with a female last author tend to have more female authors in other positions and this is true for both biology in general and computational biology. Since the last author position is most often occupied by the principal investigator of the study, this suggests that having a woman as principal investigator has a positive influence on the participation of women. This resonates with findings by Macaluso et al., who studied the nature of authorship contribution by gender in PLoS publications . They foSince biology attracts more women than computer science, we suspect that many women initially decide to study biology and later become interested in computational biology. If this is the case, understanding what factors influence the field of study will provide useful insight when designing interventions to help narrow the gender gap in computer science and computational biology.This dataset  containsSame as above , except Subset of author and gender data from Filardo et.al . This daThis dataset  containsThis dataset  containshttp://gender-api.com), which compares first names to a database compiled from government sources as well as from crawling social media profiles and returns a gender probability and a measure of confidence based on the number of times the name appears in the database. The API was queried with the 74,760 unique first names in the dataset .Genders were determined using Gender-API . The reported Pfemale represents the mean of means for 1000 samples. Error bars in figures represent 95% confidence intervals. Code and further explanation can be found on github [Mean gender probabilities were determined using bootstrap analysis. Briefly, for each dataset, authors were randomly sampled with replacement to generate a new dataset of the same size. The mean n github .Author positions were assigned based on the number of total authors. In papers with 5 or more authors, all authors besides first, second, last and penultimate were designated “other”. Papers with 3 authors were assigned only first, second and last, papers with two authors were assigned only first and last, and single-author papers were assigned only first.Pfemale using the the GLM.jl package for the julia programming language. Correlations were considered significant if PZ > |z| < 0.05.We used ordinary least squares regression analyses on IF and S1 FigA: Number of primary publications per year indexed under the “Biology” MeSH term. B: Number of primary publications per year indexed with “Computational Biology” as a major MeSH term. C: Comparison of computational gender inference (black) with known genders (white) for the dataset from Filardo et. al. . Grey re(TIF)Click here for additional data file.S2 Figbut not Computational biology (white). Error bars represent 95% confidence intervals. B: Mean probability that an author is female for articles in the “Bio” dataset (black) in the “Comp” dataset (white), or for articles in the Bio but not Comp (gray) for each journal that had at least 1000 authors plotted against the journals’ 2014 impact factor. Excluding computational publications from the biology dataset does not substantially alter the correlation between impact factor and Pfemale.A: Mean probability that an author in a given position is female for primary articles indexed in Pubmed with the MeSH term Biology (black), Computational Biology (gray) or for those articles with Biology (TIF)Click here for additional data file.S1 TableJournals identified as primarily computational are shaded grey.(TSV)Click here for additional data file.S1 Datacontact@gender-api.com.Permission to share these data was granted by Markus Perl. For additional information, e-mail (JSON)Click here for additional data file.