Millions of students follow online classes which are delivered in video format. Several studies examine the impact of these video formats on engagement and learning using explicit measures and outline the need to also investigate the implicit cognitive and emotional states of online learners. Our study compared two video formats in terms of engagement (over time) and learning in a between-subject experiment. Engagement was operationalized using explicit and implicit neurophysiological measures. Twenty-six (26) subjects participated in the study and were randomly assigned to one of two conditions based on the video shown: infographic video or lecture capture. The infographic video showed animated graphics, images, and text. The lecture capture showed a professor, providing a lecture, filmed in a classroom setting. Results suggest that lecture capture triggers greater emotional engagement over a shorter period, whereas the infographic video maintains higher emotional and cognitive engagement over longer periods of time. Regarding student learning, the infographic video contributes to significantly improved performance in matters of difficult questions. Additionally, our results suggest a significant relationship between engagement and student performance. In general, the higher the engagement, the better the student performance, although, in the case of cognitive engagement, the link is quadratic (inverted U shaped). Due to differences in practices and resources available to these content creators, videos are produced in a variety of ways and their production styles vary substantially within a platform [Even prior to COVID-19, millions of students attended online classes that can be followed asynchronously, without real-time interaction with an instructor ,3. The cplatform . Video pplatform . At the platform ,5.In traditional school settings, instructional material influences learner engagement . EngagemFurthermore, it has been demonstrated that neurophysiological measures allow us to better understand students’ experience in online learning environments ,14. EngaTherefore, there is a benefit to using these implicit measures to capture automatic and unconscious reactions of subjects ,16. EvenThus, this paper aims to advance current findings by comparing the lecture capture and infographic video formats to identify the format that engages the students more emotionally and cognitively over time (the duration of the video), and that provides better learning outcomes. In addition, we further examine the relationship between student engagement and learning outcomes.The current literature discusses video formats using the cognitive theory of multimedia learning (CTML)  and the Mayer  developeRegarding the first prediction, several authors in the multimedia learning field argue that active attention to the visual and verbal stimuli of the multimedia lecture is required for effective learning ,22. In vConsistent with the dual channel principle, it is suggested that video and audio input delivered at the same time provide better results in terms of engagement and learning performance in empirical studies . HoweverToo much load on the visuospatial sketchpad has been found to limit the ability of students to attend to all the content presented visually in multimedia learning, thus inhibiting the student’s acquisition of knowledge ,12,21, aAnother theoretical lens is the media richness theory. According to Trevino et al. , communiThe theoretical propositions for multimedia learning are that instructional material with higher levels of richness according to the four criteria will be better at transmitting information and conveying meaning. Empirical research suggests that rich media reduces the effort needed by parties to understand each other . In multHowever, when Chen et al.  comparedMany studies underline the importance of engagement in education. It improves student learning  and reteFredricks, Blumenfeld and Paris  propose Emotional engagement is broadly defined as positive or negative reactions that do not require cognitive effort and that are spontaneous . Such reA person is cognitively engaged when actively trying to understand new information . In partAs stated previously, our general research objective is to analyze two different video formats (lecture capture and infographic video) in terms of engagement (RQ1) and learning (RQ2). These formats are characterized by social cues, cognitive demand, and media richness. As supported by our literature review, these characteristics have an impact on the emotional and cognitive engagement of students. Additionally, the aim of this paper is to further examine the link between engagement and learning (RQ3).We argue that our two video production styles are characterized by a different level of media richness, and that this difference in media richness will produce a significant disparity in emotional engagement. In this regard, the infographic video provides more visual cues (second criteria) and richer vocabulary (third criteria) than the lecture capture, which has only simple video and audio. This will produce more excitement  and satisfaction  in the infographic condition. Furthermore, human presence in the lecture capture can activate substantial cognitive resources, increase the effort required to process the information and offset the benefits provided by social cueing ,12. In lHypothesis 1A.Emotional engagement will be higher for infographic video.As discussed for emotional engagement, we argue that the rich content of the infographic video sustains a student’s attentional engagement more effectively than a mere focus on the teacher for 15 min, similar to the results of Korving et al.’s  researchChen et al.’s  results Hypothesis 1B.Cognitive engagement will be higher for infographic video.Most research supports the hypothesis that higher media richness has a positive impact on learning performance, as in the case of infographic video compared to lecture capture. Interesting visual effects and graphics which convey further meaning and provide more context are likely to support more learning, especially in terms of difficult questions. In addition, according to the CTML, multimedia material which provides audio and video stimuli will promote better learning. An infographic video presents visual information in the form of graphics which are processed by the visual loop. At the same time, the narration provides additional explanations which are processed by the audio loop of the learner. By comparison, the lecture capture provides narration and an image of a teacher in the class. However, a simple image of the teacher does not necessarily convey or support additional information processing in the video loop . TherefoHypothesis 2.Learning performance will be higher for infographic video.As discussed, many studies argue that there is a significant relationship between the emotional engagement of students and their learning ,9,14,21.Hypothesis 3A.The higher the emotional engagement, the better the student performance.Cognitive engagement and, more specifically, attention, is considered key for effective learning in multimedia teaching ,28. ChenHypothesis 3B.There is a quadratic relationship between cognitive engagement and performance.To test these hypotheses, we conducted a between-subject lab experiment with two conditions. In both conditions the subjects passively watched an approximately 14-min-long pedagogical video on a segment of a course on Introduction to Organizational Behavior. During viewing, emotional and cognitive measures were recorded with neurophysiological instruments . Furthermore, pre- and post-test multiple-choice questionnaires were administered to measure student learning. Upon completion of the experiment, subjects were given a $30 gift card for the university bookstore. The research protocol was approved by the HEC Montréal-Comité d’éthique de la recherche (2017-2364-55) before the experiment took place. A total of 16 male and 10 female subjects participated in the study. They were recruited using an institutional panel of research participants. The subjects were on average 26.20 years old with a standard deviation of 6.93. Male participants had an average age of 25.56 (standard deviation (STD) 6.93), and female 27.33 (STD 6.98).Subjects were randomly assigned to one of the two conditions and were pre-screened before participation. In the infographic condition, the mean age was 24.33 (STD 6.93). There were nine males  and three females . In the lecture condition. the average age was 27.92 (STD 7.11), with seven males  and seven females . First, only subjects who had not previously attended any psychology classes at the university level were admitted. This was done to ensure that no one had prior knowledge of the content in the videos. Second, subjects who had a psychological diagnosis such as epilepsy were excluded as this could cause deviations in the electroencephalography (EEG) signal, similar to Gollan et al. [Two lecture videos with the assistance of the same professor were created specifically for the study. Both videos explained exactly the same content, namely, a chapter of the introductory psychology course at our institution, and had approximately the same length 14 min). This is within the length range suggested for videos by previous research  min. Thi,30. In tLike in Chen et al. , Table 1The students’ emotional and cognitive engagement over time was inferred on the basis of the related neurophysiological states  which we measured using implicit and explicit data. Implicit data was collected through neurophysiological sensors , electrodermal activity (EDA), and facial expression recognition). Explicit data was self-assessed . First, it needs to be noted that emotions are considered multi-componential, and they can cause different bodily responses  . These eIn particular, the SAM scale measures the self-reported affective state  using a non-verbal scale . BecauseIn addition, Keltner and Ekman  outlinedPhysiological reactivity in the form of EDA can be measured by looking at the skin’s electrical conductance levels (SCL) and changes in these levels due to sympathetic activity. For that purpose, two electrodes pass a small amount of electricity through the skin and SCL is measured in microsiemens or µS. EDA  is high when people are curious or anxious, and low then they are bored/relaxed . ElectroBased on the frequency and amplitude of the signal, and the spatial location of its origin, EEG can be used to infer cognitive states of the subjects. In particular, it allows us to assess attention ,41. ChenPope, Bogart and Bartolome  developeWe computed our cognitive engagement ratio according to a modified version of the cognitive engagement index as proposed by Mikulka et al.  using MeTo measure learning performance (achievement), subjects completed a multiple-choice questionnaire (25 questions), before (pre-test) and after (post-test) viewing either version of the learning video. Similar to previous research, there was no time limit to complete the questionnaire, and no feedback was given to avoid affecting the post-test ,16,21. PTo create the difficulty classification, the complexity of questions was rated by six experts using a 4-point Likert scale , as suggested by Cronan et al.  and simiThe median difficulty of all questions based on an expert rating was 2. Easy questions measure low complexity knowledge, whereas difficult questions measure high complexity knowledge. According to Bloom’s revised taxonomy, students who can answer actually difficult questions have acquired a deeper understanding of the material . They caThe experiment was conducted in a usability laboratory. Participants were seated in an experimental room with a computer to perform the task. In an adjacent room, the researchers monitored the experiment via a one-way mirror and computers to record the participants’ neurophysiological responses.There were no breaks during the experiment, and each session lasted on average 1 h and 30 min, including the setup and calibration of the equipment. Upon arrival, subjects had to first read and sign the Consent Agreement and their conformity to our selection criteria was verified. Next, they were informed that they would be taking part in a study which evaluates different lecture video designs, and they were given a summary of the experiment.Once the equipment was set up, and the EEG baseline was established, and all of the devices began their recordings, which continued until all of the tasks in the experiment were completed. The data was synchronized using markers which delineated each part of the experiment in all of the equipment . All tasks were performed on a 22-inch flat screen in front of the participants. Subjects were first asked to relax and close their eyes for 90 s to provide a baseline for the EEG signal. Then, for one minute, participants saw and counted randomly colored squares which appeared on the screen for 6 s each. In the next step, they completed a multiple-choice questionnaire (25 questions) to assess their pre-test knowledge of the video content. After completing the assessment, subjects started to watch one of the two videos.No note-taking or pausing was allowed during viewing to ensure comparability of the neurophysiological data over time between the two conditions and subjects. Once the video ended, a SAM questionnaire was administered. As a post-test, the same questions as in the pre-test were asked, to assess the learner’s performance after viewing the video.Once acquired, all of the data—i.e., EDA data from Biopac, FaceReader’s valence and arousal, cognitive engagement ratio from Mensia NeuroRT, SAM results, and pre- and post-test answers—was exported into csv files and imported to SAS . Data was then prepared, synchronized, and analyzed in SAS using statistical models appropriate for each research question/data type.For implicit granular-over-time data, we first computed a one second moving average for valence and arousal similar to previous research ,37. GiveFinally, we used multiple linear regression (“proc reg”) to model the relationship between the performance as dependent variable and a given implicit neurophysiological measure as independent variable. The overall implicit measures are averages calculated over the video session . We also included quadratic relationships and condition as a control variable in the model. The relationship between the SAM’s valence/arousal and performance was analyzed using Spearman correlation coefficients due to the small sample size.We compared lecture capture and infographic video formats by considering engagement over time (RQ1) and student performance (RQ2). An analysis of the link between student engagement and learning outcomes was also performed (RQ3). For RQ1, we hypothesized that emotional (H1A) and cognitive (H1B) engagement are higher for the infographic video. The latter should also allow for better learning outcomes (RQ2-H2). Finally, for RQ3, we proposed that the higher the emotional (H3A) and cognitive (H3B) engagement, the better the student performance. This relationship is, however, quadratic in the case of cognitive engagement.β = −0.19, Sig. = 0.15 > 0.05) according to facial expression analysis. However, there was a significant difference in the evolution of valence over time . Although valence of students who viewed the infographic video decreased over time, it increased over time for those who viewed the lecture capture.The results summarized in Explicit valence as reported with the SAM seems to confirm the above finding, as there was no significant difference (on average) in self-reported valence between the conditions (Sig. = 0.24 > 0.05). In general, subjects in both conditions were, however, pleased with the videos: infographic video  and lecture capture .β = −0.02, Sig. = 0.26 > 0.05) according to facial expression analysis. On average, the subjects in both infographic and lecture conditions had similar arousals. However, there was a significant difference in the evolution of arousal over time . Although arousal of students who viewed the lecture capture decreased over time, it increased over time for those who viewed the infographic video.Furthermore, there was no significant difference in arousal between the conditions  between the conditions (Sig. = 0.53 > 0.05). In general, subjects in both conditions were somewhat excited while watching the infographic video  and the lecture capture .β = 0.24, Sig. ≤ 0.0001 < 0.05). Subjects in the infographic condition had, on average, lower arousal than subjects in the lecture condition. However, over time, the infographic video was able to incite significantly more arousal than the lecture capture . At some point, around 7 min, the former managed to invoke more arousal than the latter.Regarding arousal operationalized through mean EDA, there was a significant difference between the two conditions  show that lecture capture induced less arousal over time than infographic video.β = −0.004, Sig. = 0.58 > 0.05). Subjects who watched the infographic video had, on average, a similar arousal variability to those who watched the lecture capture. However, over time, the infographic video was able to incite significantly more arousal deviation than the lecture capture . In addition, after 3 min, the former managed to produce more arousal variability than the latter. This result was supported by implicit arousal operationalized using facial expressions and mean EDA. It is interesting to note that variability of EDA in both conditions decreased. We can assume that students get tired over time and their EDA variability stabilizes as their arousal response to external stimuli diminishes.Regarding deviation in implicit arousal according to EDA recordings, our results suggest that there was no significant difference between the conditions on average  when seeing the professor at the beginning of the video and responded positively  over time to the social presence provided by the lecturer.From the theoretical standpoint, the CTML framework suggests that the lecturer in the video will have a positive effect on students’ emotional engagement by providing social cues. Regarding the media richness theory, the lecture capture shows the professors’ body movements, which could provide additional cues, increase personal meaning (fourth criteria), and affect arousal and valence positively. In empirical studies, it has been shown that social perception is related to arousal  and thatNevertheless, the infographic video is better at maintaining arousal. It provides more visual and vocal cues. In coherence with the media richness theory and related studies , this leβ = −0.06, Sig. = 0.81 > 0.05). On average, the subjects in both infographic and lecture conditions had similar attention levels. However, there was a significant difference in the evolution of attention over time . While the attention of subjects in infographic condition increased, it decreased for those in lecture condition. In other words, although attention in both conditions began similarly (significant intercept), over time the infographic video engaged the students much more. Furthermore, after 2 min the latter managed to evoke more attention than the lecture capture. Therefore, the results do support H1B: Cognitive engagement will be higher for the infographic video.Our results suggest that on average, there was no significant difference in attention between the conditions . In addition, there was no difference between the two groups in mastering the different types of questions . The post-test questionnaire revealed that students improved significantly after watching the video. Students performed significantly better after watching the videos, regardless of the condition and question type (Sig. = 0.04 < 0.05).In post-test questionnaires, participants in the infographic condition performed 3% better overall and their performance increased by 6% more. However, this overall difference between conditions was not significant (Sig. = 0.20 > 0.05). Examination of the results of the different question types separately shows that there was a significant difference in terms of mastering difficult questions (Sig. = 0.04 < 0.05). The number of correctly answered difficult questions increased by 38% in the infographic condition and only by 18% in the lecture condition. Four of six difficult questions were answered better by subjects in the infographic condition. The results support H2: Learning performance will be higher for infographic video. It is relevant to note that a significant difference for all question types would have been problematic. Both videos exhibit the same content. Therefore, there should not be a significant difference between the two conditions in terms of answering easy questions. The opposite could have indicated a design issue with our videos.The findings are supported by the media richness theory. Because the infographic video is a richer medium than the lecture capture, it is better at reducing uncertainty and equivocality. Thus, students are able to learn faster and better. Empirically, our results are supported by earlier research asserting that richer media improves student performance ,9,10. AsAccording to the CTML, enhanced integration of video and audio allows improved learning because information is processed by two separate channels . In thisR = 0.13, β = 20.27, Sig. = 0.03 < 0.05). However, self-reported valence seems to contradict the above finding, because there was no significant correlation between explicit valence and learning outcomes (Sig. = 0.53 > 0.05). Furthermore, there was a significant relationship between a subject’s overall performance and mean arousal , if we include deviation of arousal into the model. The higher the mean and variability of arousal, the higher the performance  and β = 110.22, Sig. = 0.04 < 0.05 (one-tailed)). This effect was true regardless of the condition, as can be seen in The results, summarized in β = 110.22, Sig. = 0.04 < 0.05). As deviation of arousal increased, performance decreased . This effect was true regardless of the condition. It is important to note that this significant relationship was only observable if we calculated mean EDA for the first 5 min of the viewing time. There was no significant relationship between mean EDA and performance if we took the average of the whole video session. This result can be explained by the evolution of arousal over time (H1A). Over the duration of the video, mean EDA decreased significantly for both conditions . As subjects became tired, mean EDA was lower at the end of the video viewing period for both conditions, regardless of the performance of the students. This masks the relationship between high mean EDA and high performance.According to EDA measurements, there was also a significant relationship between mean arousal and learning outcome, when we include deviation of arousal into the model. In particular, the higher the mean arousal, the higher the performance  with performance.In summary, our results suggest a significant positive relationship between emotional engagement and performance, in support of H3A: The higher the emotional engagement, the better the student performance. A decrease in negative emotions and a pleasant emotional state  allowed subjects to focus on the learning task and perform better. This result is widely supported by current literature ,9,14,21.The effect of variability of arousal warrants further investigation because results are inconsistent between EDA and facial expression analysis. Both measures could represent a different element of arousal and therefore show contrasting effects on performance . When HaR = 0.07). First, the higher the attention, the better the performance. However, at some point, attention has a negative effect on performance; the average attention varied between 4.29 and 7.18 and performance appeared to peak around 5.53. Too much attention can cause a decrease in performance as the students’ attentional resources get overloaded. This effect was found in both conditions. Previous research outlines that attention is a pre-requisite for processing information and learning [The results suggest a quadratic relationship between attention and performance . As subjects became tired, mean attention was lower at the end of the viewing period, regardless of the performance of the students. This masks the relationship between high attention and high performance. The same circumstances have been observed for arousal measured by EDA, as discussed in Hetland et al.’s [It is important to note that the significant relationship was only observable if we calculated the mean attention for the initial two-thirds of the viewing time (500 s). There was no significant relationship between attention and performance if we took the average of the whole video session. This can be explained by the evolution of attention (H1A). Over time attention significantly decreased in both conditions  and students’ achievement. This outcome is expected. According to our results, engagement influenced overall performance (H3A/B). At the same time, if we compare the conditions, a student’s performance was only different in regard to difficult questions (H2). There was no effect of the condition on students’ overall performance. Consequently, there can be no relationship between condition and overall performance that is mediated by engagement.As with any experiment, this research has limitations. Participants were only passively attending a single video. In a typical course setting, learners would be watching a sequence of online videos over a specific duration over time. Our experiment setup could not capture the effect of time, and other research methods will be needed to further investigate the longitudinal impact of video format. In addition, we only analyzed two different video production styles. There are obviously many video formats that could be compared using a similar methodology, including hybrid options using audio-visual aids in the lecture capture.Furthermore, we did not manipulate the quality of the rich media condition. Although we followed edX guidelines to develop the infographic condition, we acknowledge that various levels of richness quality might have a moderating effect on the learner’s engagement, and future research is needed to explore this effect.Additionally, the length of our videos was 14 min. It would be pertinent to explore if our results hold over shorter or longer duration of videos, and examine how performance and engagement vary throughout an entire online course.Finally, although our sample size is common for neurophysiological study, and our research provides a unique and rich perspective on the learner’s emotional and cognitive response, our findings should be replicated with a larger sample and a longitudinal research design to determine the extent to which this effect is observed over time.Researchers in the field suggest the most suitable video format should be selected based on how it affects the performance and engagement of students . To advaAs can be seen in Regarding cognitive engagement, our hypothesis (H1B) was supported. The infographic video retained students’ attention significantly better over time. In addition, students significantly improved in terms of correctly answered difficult questions in the infographic condition (H2). Finally, there was a noticeable relationship between learning outcomes and engagement (H3A/B). In general, the results suggest that the higher the engagement, the higher the performance. Too much attention, however, decreases performance at some point when the students become overwhelmed with information.Considering these results, it seems that students perform better in the infographic condition because the format is better at keeping students aroused and attentive over time. Even though subjects seem to be specifically aroused (EDA) at the beginning upon seeing the professor and are happier over time, the lecture capture is unable to maintain higher levels of attention and arousal. Furthermore, happiness does not seem to be enough to offset the decrease in arousal and attention over time and to encourage as much learning as the infographic video. Consequently, a combination of the two formats  could yield better engagement and learning. Rich content would help maintain arousal and attention over time, and timely social cues would bring in additional excitement and increase happiness of students.Future research could benefit from incorporating neuropsychological measures into the study design to gain better insight into students’ engagement, and to capture automatic, in addition to unconscious, reactions of subjects over time. Researchers could also perform a more granular analysis of neurophysiological data and try to understand how specific video design components affect engagement. Furthermore, although subjects in this study were mostly young students and we had a relatively small sample size, the online learning population is highly diverse and large. In the future, researchers could examine how different subpopulations engage with learning content using a larger number of subjects. Lastly, subjects were monitored during the whole experiment which could force engagement, and no breaks or note-taking were allowed. In real life, students mostly follow online courses at home, and subsequent studies could better mimic those conditions while capturing neurophysiological data.


Hypothermic circulatory arrest (HCA) has been considered to cause coagulopathy during cardiac surgery. However, coagulopathy associated with HCA has not been understood clearly in details. The objective of this study is to analyze the details of coagulopathy related to HCA in cardiac surgery by using rotational thromboelastometry (ROTEM).We retrospectively analyzed 38 patients who underwent elective cardiac surgery  in our hospital. Blood samples were collected before and after cardiopulmonary bypass (CPB). Standard laboratory tests (SLTs) and ROTEM were performed. We performed four ROTEM assays  and analyzed the following ROTEM parameters: clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF) and maximum clot elasticity (MCE). The amount of perioperative bleeding, intraoperative transfusion and perioperative data were compared between the HCA and non-HCA group.Operation time and hemostatic time were significantly longer in the HCA group, whereas CPB time had no difference between the groups. The amount of perioperative bleeding and intraoperative transfusion were much higher in the HCA group. SLTs showed no difference between the groups both after anesthesia induction and after protamine reversal. In ROTEM analysis, MCE contributed by platelet was reduced in the HCA group, whereas MCE contributed by fibrinogen had no difference.Our study confirmed that the amount of perioperative bleeding and intraoperative transfusion were significantly higher in the HCA group. ROTEM analysis would indicate that clot firmness contributed by platelet component is reduced by HCA in cardiac surgery. Hypothermic circulatory arrest (HCA) is an effective procedure to perform open anastomosis in bloodless fields while protecting whole organs in aortic surgery –3. It isRotational thromboelastometry (ROTEM) is one of a point-of-care coagulation tests with whole blood viscoelastic hemostatic assays (VHA), which can rapidly assess the detail of coagulation profiles including clot initiation, clot formation, clot stability and fibrinolysis . Thus, RThe objective of this observational study was to analyze coagulopathy in cardiac surgery with or without HCA, using not only SLTs but also ROTEM, and to compare the amount of perioperative bleeding and intraoperative transfusion of blood products.n = 16), acute aortic dissection (n = 1) and ventricular septal defect (n = 2) were excluded. Other exclusion criteria were incomplete ROTEM (n = 16), SLTs (n = 14), or both of them (n = 2). All 38 cases were electively performed with median sternotomy approach. Institutional Review Board of Asahikawa Medical University approved this retrospective observational study (no. 17068).We retrospectively investigated 89 patients who underwent cardiac surgery with cardiopulmonary bypass (CPB) in our hospital from January 2016 to December 2016. Among them, 38 patients who underwent cardiac surgery with or without HCA were reviewed. The cohorts were divided into two groups; 12 patients in the HCA group and 26 patients in the non-HCA group. ROTEM analysis was routinely performed in our institute in cardiac surgery. Both SLTs and ROTEM analysis were performed simultaneously before and after initiation of CPB. Minimally invasive cardiac surgery  was administrated to maintain activated clotting time (ACT) more than 480 s, and it was maintained during CPB. HCA was started when the rectal temperature reached mild to deep hypothermia (26 ± 0.4 °C). After weaning of CPB, heparin was reversed by protamine (3 mg/kg). After protamine administration, transfusion of blood products was started. Packed red blood cells were transfused when hemoglobin (Hb) level was less than 8 g/dL, and fresh frozen plasma was transfused when prolonged aPTT or PT-INR were detected. Platelet concentrate (PC) was transfused when platelet count (Plt) was less than 8 × 10Blood samples were taken at 4 time points: after anesthesia induction (S-1), during CPB, 5 min after protamine reversal (S-2) and 1 h after operation. SLTs were performed at all the 4 points, which included Hb, Plt, APTT, PT-INR, fibrinogen and antithrombin (AT3). ROTEM analysis was performed at the 2 points, after anesthesia induction (R-1) and protamine reversal (R-2).ROTEM analyses were performed with ROTEM delta  according to the manufacturer’s instruction. EXTEM, INTEM, FIBTEM and HEPTEM assays were performed. EXTEM assay shows clot strength with extrinsic pathway activation of whole blood with tissue factor. FIBTEM assay also indicates clot strength but platelet contribution to clot strength is prevented by addition of cytochalasin D. INTEM assay shows intrinsic pathway. HEPTEM assay is similar to INTEM assay but it contains heparinase to inhibit heparin. Figure U test was used to compare continuous variables between the two groups. Statistical significance was defined as P < 0.05.All statistical data were analyzed by using SPSS 26.0 for Windows . Values are described as mean ± SD for continuous data with a normal distribution and count or percent for categorical variables. Fischer’s exact test was used for categorical variables. Wilcoxon signed rank test was used to compare continuous variables between the two time points  in each group. Mann–Whitney All patient characteristics and surgical procedures are described in Table Perioperative data are shown in Table The results of SLTs after anesthesia induction (S-1) and after protamine reversal (S-2) are shown in Fig. To our knowledge, few studies have analyzed coagulopathy associated with HCA in detail by using ROTEM . The priThis study confirmed that the amount of bleeding and transfusion use in cardiac surgery with HCA were much higher than that without HCA. It is well-known fact that both CPB and HCA are one of the main causes of coagulopathy in cardiac surgery. During CPB, activation and dysfunction of intrinsic and extrinsic pathway, platelet dysfunction and excessive anticoagulation by heparin infusion could lead to excessive bleeding , 4, 9. IROTEM analysis in this study indicated that excessive bleeding in cardiac surgery with HCA might be mainly caused by decrease of clot firmness contributed by platelet components. ROTEM is one of VHA assays using whole blood sample and rapidly provides information about quality and dynamics of clotting cascade. ROTEM is often used in cardiac surgery, and its benefit in reducing perioperative transfusion use in cardiac surgery has been described , 11. FIBCorrelations between bleeding and platelet counts and aggregations are well-known . In our 9/L to 10 × 109/L in normal fibrinogen level (1.5–3.5 g/L), then clot strength tended to reach plateau in normal range of platelet count [9/L) and low platelet count causes inadequate clot strength in normal fibrinogen level. In our study, fibrinogen level in the HCA group was higher than the non-HCA group at both time points, but clot strength was not enough to prevent excessive bleeding. It might be because of low platelet counts (< 10 × 109/L) or platelet dysfunction. In such case, administration of much higher dose of fibrinogen concentrate or administration of PC might be needed. Our study indicated that not only adequate fibrinogen level but also platelet counts are important to make firmer clot formation. In ROTEM analysis, MCE platelets and FIBTEM MCE were useful parameters to see actual clot strength contributed by both platelet and fibrinogen. Further investigations are needed to detect the cut off value of the parameters to decide the need of PC and fibrinogen concentrate during surgery.Lang et al. reported that clot strength, expressed as MCE, increased when platelet count changed from 10et count . They alThis is a retrospective observational analysis of small sample size. Due to observational nature of the study, the sample size of the HCA group was smaller and the surgical procedures between the groups were different. The different sample size in the two groups limited the power of ROTEM analysis results between the groups. The number of aortic surgeries was smaller in the HCA group, which might affect the amount of intraoperative bleeding and transfusion use. We use fibrin sealant patches at anastomosis site more often in aortic surgery, so it might have some effects on hemostatic time or bleeding volume. MCE platelets, which calculated by EXTEM MCE and FIBTEM MCE, do not assess platelet aggregation, therefore platelet aggregometry is needed to assess further detail of platelet function. In our institute, the ROTEM analysis is not covered by the health care insurance, therefore, the study period and number of investigated patients were very limited due to our financial problem. Moreover, the ROTEM analysis was carried out by technicians in the clinical laboratory department and the tests could be done only in day time in the present study due to very limited man power in the clinical laboratory department in the night time. The heterogeneity of patient background, especially regarding operations was the strong limitation of the present study. Nevertheless, we think the present study has a certain meaning in this study field; relationship between coagulation disorder and HCA. Although definitive conclusion may be drawn only from experimental studies, the findings of the present study may contribute to such further studies.The volume of perioperative bleeding and intraoperative transfusion of blood products were significantly higher in patients who underwent cardiac surgery with HCA. In ROTEM analysis, MCE platelets were significantly lower in the group with HCA though FIBTEM MCE was similar. ROTEM analysis would indicate that clot firmness contributed by platelet component is reduced in cardiac surgery with HCA.


Compared to HBMVECs, AVMECs demonstrated increased invasion (p = 0.04) and migration (p = 0.08), impaired tube formation (p = 0.06) and increased EphrinB2:EphB4 ratios. Altering the EphrinB2:EphB4 ratio (by increasing EphrinB2 or blocking EphB4) in HBMVECs increased invasion . EphrinB2 expression was increased in AVM tissue, which correlated with increased urinary EphrinB2 levels in AVM patients. Using the optimal urinary cutoff value (EphrinB2 > 25.7 pg/μg), AVMs were detected with high accuracy (80% vs. controls) and were distinguished from other cerebrovascular disease (75% accuracy). Post-treatment urinary EphrinB2 levels normalized in an index patient. In summary, AVMECs have an EphrinB2:EphB4 ratio that is increased compared to that of normal HBMVECs. Changing this ratio in HBMVECs induces AVMEC-like behavior. EphrinB2 is clinically relevant, and its levels are increased in AVM tissue and patient urine. This work suggests that dysregulation of the EphrinB2:EphB4 signaling cascade and increases in EphrinB2 may play a role in AVM development, with potential utility as a diagnostic and therapeutic target.We investigated (1) EphrinB2 and EphB4 receptor expression in cerebral AVMs, (2) the impact of an altered EphrinB2:EphB4 ratio on brain endothelial cell function and (3) potential translational applications of these data. The following parameters were compared between AVM endothelial cells (AVMECs) and human brain microvascular endothelial cells (HBMVECs): quantified EphrinB2 and EphB4 expression, angiogenic potential, and responses to manipulation of the EphrinB2:EphB4 ratio via pharmacologic stimulation/inhibition. To investigate the clinical relevance of these in vitro data, Ephrin expression was assessed in AVM tissue (by immunohistochemistry) and urine (by ELISA) from pediatric patients with AVM , can be identified with a urine test, and the test protein may also help in treatment. AVMs often have no symptoms and can go undiagnosed, but when they rupture they can cause deadly brain hemorrhage. Better diagnostic tools and nonsurgical treatments are needed. Katie Fehnel and Edward Smith at Boston Children’s Hospital, USA, and co-workers identified an imbalance in a pair of signal/receptor proteins called ephrins in AVMs. Disturbing the balance of ephrin levels in blood vessel-forming cells disrupted growth, causing disorganized vessel formation with too many sprouts and insufficient junctions. Testing ephrin levels in patients’ urine reliably identified AVMs. These results offer a rapid and noninvasive new diagnostic tool and may help find new treatments for this mostly invisible and potentially fatal condition. The morbidity and mortality of AVMs is compounded by limitations in effective and cost-efficient detection methods and even further by existing high-risk interventional treatments without therapeutic options for the some of the highest risk lesions. There is a need for better diagnostic techniques and novel therapeutic targets to reduce risk and improve patient outcomes, particularly in the pediatric population.Cerebral arteriovenous malformations (AVMs) are vascular anomalies with an average hemorrhage risk of 2–4% per year cumulative over a patient’s lifetime, and a 25% fatality risk with each bleed4. Inhibition of EphrinB2 signaling can suppress endothelial cell migration and tube formation in vitro, and it can lead to aberrations in the actin cytoskeleton4. In addition to high affinity binding between the extracellular EphB4 binding domain to EphrinB2, there are additional lower affinity residues on the EphB4 receptors that allow for homodimerization as well as tetramer aggregation5. Aggregate binding can alter the strength of signaling with downstream effects influenced by the ratio of ligand to receptor. These data suggest that a balance of ligand to receptor is critical to influencing downstream vascular fate.We have identified axon guidance factors (AGFs), specifically EphrinB2 and EphB4, as promising biomarkers of pediatric AVM. Ephrins are cell surface proteins that are readily quantifiable and potentially amenable to manipulation; they play a critical role in normal cerebrovascular development, proliferation, migration, and adhesion, through complex bidirectional signaling . Arteriovenous cell fate determination occurs through canonical EphB4 signaling, and later angiogenesis, including vessel sprouting and remodeling, occurs through EphrinB28. Transgenic mice with loss of either Ephb4 or EphrinB2 are embryonically lethal (at E9.5), and they exhibit an AVM-like pathology with shunting of arterial blood via fusion of the dorsal aorta with the common cardinal vein12. Vascular endothelial-specific overexpression of EphrinB2 in mice results in intracranial hemorrhage13. Recent literature further suggests that AGFs play a critical role in pathological vascular development in the brain. EphrinB2 is expressed in hemorrhagic AVM endothelium14, and sophisticated genetic screens have identified EphB4 mutations in some Vein of Galen Malformations and a small percentage of patients with capillary malformation-arteriovenous malformation syndrome17.Ephrins have been implicated in AVM-like pathology in animal modelsWe hypothesized that there is a ratio of EphrinB2 to EphB4 that is critical for normal vasculogenesis and that this signaling ratio is abrogated in cerebral AVMs. In this study, we sought to understand the dynamic relationship between EphrinB2 and EphB4 in the normal and disease states to identify putative therapeutic targets and to investigate the potential utility of EphrinB2 as a quantifiable and therapy-responsive biomarker of pediatric AVM.18. Both the CD31− population and purified CD31+ cell populations were resuspended in growth media and plated. Under the microscope, CD31+ cells were visualized with magnetic beads still attached. Endothelial identity was confirmed by immunocytochemistry staining for CD31 .Human brain microvascular endothelial cells (HBMVECs) were supplied by Cell Systems . AVM-derived endothelial cells (AVMECs) were cultured primarily according to an Institutional Review Board-approved protocol. Fresh AVM tissue was dissociated in collagenase/dispase  at 37 °C, pelleted at 1200 RPM × 5 min, reconstituted in EGM2 media with 5% serum  and plated on attachment factor-coated plates . At near-confluence (~1 week of culture), sorting for CD31+ endothelial cells was performed using Invitrogen Dynabeads  according to the manufacturer’s protocolAVMECs and HBMVECs were fixed in 4% formaldehyde on glass slides, permeabilized in 0.1% Triton X-100 and blocked in 10% goat serum. Cells were stained with CD31  or VE-cadherin , and then they were incubated with an Alexa Fluor 594 goat anti-rabbit secondary antibody  and were counterstained with DAPI.Whole-cell protein extracts were prepared from cells at passage 8 that were lysed with cold RIPA buffer  that was supplemented with proteinase and phosphatase inhibitor . Protein concentration was determined with a Bradford assay . Then, 20 µg of total protein was loaded onto a NuPage 4–12% Bis-Tris Gel  with a Precision Plus Protein Kaleidoscope standard , which was followed by the transfer of proteins onto a nitrocellulose membrane . CD31 , αSMA , EphrinB2  and EphB4  were detected and compared to GAPDH, which was the housekeeping protein control .GAPDH, EphrinB2 and EphB4. All PCRs were performed using Applied Biosystems Universal PCR Master Mix and Taqman Gene Expression Assays primer/probe sets . Graphs represent the mean of duplicate experiments run in triplicate and normalized to GAPDH ± SE.RNA was harvested from AVMECs (passage 6) and HBMVECs (passage 11) ). cDNA was synthesized ) and amplified using gene-specific primers/probe sets for 19  were added to the lower chamber to act as a chemoattractant. After 24 h, cells remaining in the upper chamber were aspirated; cells in the bottom chamber were fixed, permeabilized, and stained using Diff-Quik Stain Set  as previously published21. Invasion assays were performed in a similar fashion as above using Matrigel invasion chambers . Experiments were performed in duplicate for each cell line, with a total of ten high-power fields analyzed per cell line. Representative images were captured at ×100, and the number of migrated (or invaded) cells was quantified using ImageJ software .Migration assays were performed using Costar 24-well Transwell plates . AVMECs (passages 6–10) and HBMVECs (passages 9–10) were plated at 100,000 cells/well in 100 μL of EGM2 media  with 0.1% serum in the upper chamber. In the lower chamber, either EGM2 with 0.1% serum (negative control), EGM2 full serum media (positive control), or EGM2 with 0.1% serum supplemented with EphrinB2-FC recombinant 250 ng/mL  or 100 nM EphB4 inhibitor22. Both AVMECs and HBMVECs were grown to ~80% confluence and then were split into a 48-well dish coated with Matrigel Matrix Basement Membrane . Cells were resuspended in EGM2 media with 0.5% serum alone or with either EphrinB2-FC 250 ng/mL or NVPBHG712 100 nM, and then they were plated at a density of 1 × 105 cells/well. After incubation at 37 °C for 18 h, the formation of tube-like structures was evaluated. Images of live cells were captured at ×100 magnification, and the number of completed junctions was quantified using ImageJ software .Tube formation was assessed as previously published23 in accordance with protocols approved by the Boston Children’s Hospital IRB (IRB#10-417); informed consent was obtained. Only patients with magnetic resonance imaging (MRI) of the central nervous system were included. In total, 73 patients were included, 30 of whom had a diagnosis of AVM of which half had hemorrhaged (n = 15) and the other half were nonhemorrhaged (n = 15). The date of urine collection from hemorrhagic patients ranged from 5 days to 4 months post-hemorrhage. Control urine samples were collected from healthy age- and sex-matched control patients (n = 29), all of whom had CNS imaging to confirm the absence of any neoplastic or vascular disease in the brain or spine. Additional patient cohorts for comparative vascular analysis included pediatric patients with a diagnosis of moyamoya (n = 14).Tissue and urine specimens were collected as previously publishedThe following clinical data were reviewed: AVM size, location, hemorrhage status, associated aneurysms, presence or absence of deep venous drainage as well as any additional comorbid disease or important clinical information. All angiographic imaging was reviewed, and AVM flow was graded by a pediatric neurointerventional radiologist (Orbach).25. No patient had evidence of other major physiologic stressors, such as stroke, recent surgery, chemotherapy or radiation, at the time of collection. Samples were collected, transported on ice and stored at −80 °C.As previously reported by our group and in accordance with an IRB-approved protocol, urine samples were collected preoperatively between 2006 and 2014Tissue specimens were obtained from the Division of Neuropathology at Children’s Hospital Boston through the Dana Farber Cancer Institute-Integrated Tissue and Clinical Data Bank for Patients with Neurological Disorders and in accordance with an Institutional Review Board-approved protocol. For immunohistochemistry staining, representative slides of AVMs were prepared from paraffin-embedded tissue. Normal human brain arterial sections were obtained commercially .. Assays were performed per company protocol after determining the urine protein concentration by Bradford assay, as previously reported25.Biomarker profiling was performed on all patient urine samples in duplicate. Commercially available ELISA assays were procured to test EphrinB2 23. Staining was quantified using ImageJ software. For each patient, ten separate fields were reviewed at ×200 magnification, and the percentage of positive cells was calculated. The values from these ten fields were then averaged to obtain the overall percentage of cells demonstrating immunoreactivity for each patient.Representative, paraffin-embedded 10-μm sections of AVMs were stained as previously described using antibodies to EphrinB2 U test with a two-sided α-level of 0.05 . Box-and-whisker plots were used to represent median and interquartile ranges for each biomarker comparison26. Receiver operating characteristic (ROC) curve analysis was applied to assess the predictive accuracy of the biomarkers with area under the curve (AUC) and 95% confidence intervals (CI) as indices of diagnostic performance in differentiating between AVM and controls, as well as AVM and moyamoya27. Multivariable stepwise logistic regression was used to confirm that EphrinB2 was a predictive noninvasive urinary biomarker for differentiating between AVM patients and controls as well as between AVM and moyamoya patients independent of age and sex as covariates; a likelihood ratio test was used to assess significance28. Youden J-index in ROC analysis was used to identify the optimal cutoff values (pg/μg) for EphrinB2, and based on these cutoff values, sensitivity, specificity and accuracy were calculated29. Statistical analysis was conducted using IBM/SPSS software . Paired sample t test analyses were performed for the in vitro assays. Two-tailed values of p < 0.05 were considered statistically significant.Statistical analysis was performed with the help of a biostatistician (D.Z.). Power analysis for the biomarker study indicated that the sample sizes of 30 AVM, 29 control and 14 moyamoya patients provided 80% power to detect twofold differences in median levels of the urinary biomarkers using a nonparametric Mann−Whitney 18  and, though not statistically significant, a trend towards increased migration (p = 0.086) and decreased tube formation (p = 0.06). The healthy control HBMVECs demonstrated a greater number of completed junctions, polygon formation and early capillary-like structures, while the AVMECs appeared to have stagnate sprouting, fewer completed junctions and no polygon or capillary-like formation.We next sought to understand the implications of the EphrinB2 to EphB4 ratio on angiogenesis in both AVMECs and HBMVECs Fig. . As a fi22.Of note, there is no universally agreed upon method for evaluating in vitro tube formation. Earliest methods focused on measuring tube length and absolute numbers of connected cells. More recent publications have focused on a combination of quantitative and qualitative measurements measuring branching/sprouting as well as assessing tube length and absolute connections. Here, healthy endothelial cells and AVMECs appear to peak at different points in the pathway to tube formation. Given this, angiogenesis was evaluated both by absolute connections and additionally by semi-quantitative evaluation of sprouting, connected cells, polygon formation, capillary-like structures and complex meshp = 0.06); however, subjectively, there appeared to be decreased invasion. On the other hand, HBMVECs behaved similarly to AVMECs and exhibited invasion that was increased from the baseline (p = 0.03) and was greater than that of the AVMECs (p = 0.056). Similarly, inhibition of EphB4 with 100 nM NVP BHG 712 induced increased HBMVEC invasion (p = 0.02), which was also increased with respect to AVMECs (p = 0.047). Neither stimulation with EphrinB2 nor inhibition of EphB4 with NVP BHG 712 altered AVMEC invasion. Here, we show that by increasing the ratio of ligand to receptor either by increasing the ligand or by blocking the receptor, we can stimulate normal HBMVECs to behave like and phenotypically imitate AVMECs.To understand the role of altered EphrinB2 to EphB4 ratios in the AVMEC phenotype, we sought to induce AVM-like behavior in HBMVECs by increasing EphrinB2 levels or decreasing EphB4 levels Fig. . Of the p = 0.01) in response to increased exogenous EphrinB2; however, the observed pattern remained simple, with redundant branching and sprouting and without demonstrable polygon or capillary-like formation. EphB4 inhibition had a minimal effect on AVMEC tube formation with respect to completed junctions, although there did appear to be a closer approximation to polygon formation than was observed in the untreated cells. Conversely, HBMVECs demonstrated a trend towards increased capillary-like tube formation and increased complexity in the presence of exogenous EphrinB2, and there was a statistically significant increase in tube formation in response to EphB4 inhibition (p = 0.02). Unlike what was observed in AVMECs, tube formation appeared organized in treated HBMVECs. This may reflect inherent differences between AVMECs and HBMVECs in addition to the Ephrin ratios that contribute to AVM pathology.As noted above, tube formation was determined by quantifying the number of completed junctions with high-power microscopy; an additional qualitative assessment was performed to assess the degree of organization with respect to sprouting, connected cells, polygon formation, capillary-like structures and complex mesh Fig. . Before We next sought to understand whether increased levels of EphrinB2 would be clinically significant and useful as a diagnostic test. To study this, we collected patient samples as previously described and specifically assessed urinary EphrinB2.A total of 73 patients were included in the study, of whom 30 had AVMs Tables  and 2. TUnivariate comparisons between AVM patients and control subjects did not show relevant differences by age or sex Table . No contLevels of EphrinB2 in urine were quantified using a commercially available ELISA assay. The results were normalized to overall urinary protein concentration, and levels from AVM patients were compared to levels from control patients. Subsequent analyses were performed to compare biomarker levels from AVM patients with biomarker levels in patients with other pediatric cerebrovascular disease (moyamoya). Statistically significant differences in biomarker expression were identified by multivariate analysis. Table .Multiple stepwise logistic regression analysis revealed that, independent of age and sex, urinary EphrinB2 was able to distinguish between AVM patients and controls Fig. . Median Optimal cutoff point for EphrinB2—determined by ROC analysis and regression modeling—demonstrated that a urinary EphrinB2 value of >25 pg/μg has a sevenfold increased likelihood of AVM presence when compared to controls, and this was independent of age and sex Fig. . The AUCTo establish that EphrinB2 is not just a ubiquitous marker of neurovascular pathology, we demonstrated that we could distinguish between AVM and ischemic vasculopathy (moyamoya) Fig. . Median n = 4) were obtained and subjected to IHC. EphrinB2 was detected in AVM tissue at higher rates than it was in normal artery control (n = 2). Staining for EphrinB2 in AVMs showed a tenfold increase over the normal control (p < 0.01). A heterogeneous population of cells, including nidal endothelial cells and perinidal cells, were noted to express varying levels of EphrinB2  analysis Fig. . IndividnB2 Fig. . EphrinBAs proof of concept, in one patient for whom pre- and postoperative urine was available, we investigated whether EphrinB2 levels normalized subsequent to treatment Fig. . Followiwith deep venous drainage. We hypothesize that the role of deep venous drainage is an important consideration for the utility of EphrinB2 as an AVM biomarker. Most consistent with the aforementioned findings, the lesion in the patient for whom urinary EphrinB2 levels were lowest, at 2.89 pg/µg, had both slow flow and evidence of deep venous drainage. The last patient below the cutoff value had a medium flow lesion, no deep venous drainage, and importantly had hereditary hemorrhagic telangiectasia—a pathology distinct from the other isolated AVMs included in this study.The most important angiographic correlations with urinary EphrinB2 levels were rate of flow and presence of deep venous drainage   Fig. . Importa01) Fig. . Further01) Fig. . Finally01) Fig. . These fTo further understand the clinical utility of these data, we cross-referenced our findings against an important clinical feature of cerebral AVM which is flow. We hypothesized that AVM flow patterns influenced EphrinB2 levels. AVMs were angiographically stratified by flow (high-flow and low-flow), size and drainage patterns  by a board-certified neuroradiologist. When correlated with urinary EphrinB2 levels, we found that high-flow lesions had higher levels of urinary EphrinB2, independent of size. In contrast, slow-flow lesions or AVMs with deep venous drainage had lower levels of EphrinB2.One explanation for this finding is that EphrinB2 is expressed as a cell surface protein, and turbulence in high-flow AVMs may lead to increased disruption of the endothelium and possible release of EphrinB2. Independent of nidus size, fast flow directly correlated with higher levels of urinary EphrinB2 > 100 pg/µg. Conversely, slow-flow AVMs had urinary EphrinB2 levels below 35 pg/µg. Deep venous drainage also correlated with low urinary EphrinB2 levels. As venous vasculature has been reported to have higher expression of the receptor EphB4, one potential explanation for lower ligand levels in AVMs with deep venous drainage could be proximity to a greater surface area of receptor to absorb AVM-generated ligand. This speculative hypothesis needs additional investigation; however, regardless what the underlying mechanism is, these data are clinically relevant.As with many clinical studies, there are limitations to what we can extrapolate from these data given the sample size and what we cannot control with respect to in vivo variables. While this work requires further validation with larger populations, we achieved statistical significance and provided substantial proof-of-principle data to encourage further investigation. A second issue that is true of any biomarker study of isolates is that we cannot control for in vivo variables, including inflammatory cells and other secreted cytokines, chemokines, and growth factors. In this vein, we recognize the heterogeneity of our AVMs, including both hemorrhage and nonhemorrhage patients. Ideally, subsets of each group could be analyzed independently; however, when we stratified the data based on prior hemorrhage, there was no significant difference between groups, suggesting that the underlying premise that EphrinB2 is a product of AVM cells (and not a byproduct of hemorrhage or trauma) is valid.In the future, further in vitro and in vivo validation will be important, given the potential clinical relevance of these findings. Our data provide a diagnostic biomarker and as a potential therapeutic target. We identified a relationship between EphrinB2:EphB4 expression in AVMs and correlated EphrinB2 overexpression with key mechanistic drivers of pediatric AVM pathology in both in vitro and clinical studies. Further validation is warranted, as these results may have significance in the development of noninvasive biomarkers and novel Ephrin-derived small molecule therapeutics to modify the disease course of this devastating disease.


In this study, we found virulence in both silkworms and mice was restored by introducing the yjbH gene but not the yjbI gene to both yjbI and yjbH genes-disrupted mutants, suggesting that yjbH, the gene downstream to the yjbI gene in a two-gene operon-yjbIH, is responsible for this phenomenon. We further observed a decrease in various surface-associated proteins and changes in cell envelope glycostructures in the mutants. RNA-seq analysis revealed that disruption of the yjbI and the yjbH genes resulted in differential expression of a broad range of genes, notably, significant downregulation of genes involved in virulence and oxidative stress. Administration of N-acetyl-L-cysteine, a free-radical scavenger, restored the virulence in both the mutants. Our findings suggested that YjbH plays a role in staphylococcal pathogenicity by regulating virulence gene expression, affecting the bacterial surface structure, and conferring resistance to oxidative stress in a host.We previously reported that disruption of the  Staphylococcus aureus expresses a plethora of virulence factors to invade, thrive, and multiply within the host S. aureus strains has challenged the treatments, thus warranting novel therapeutic options [S. aureus virulence factors and the genes responsible for pathogenicity, we performed screening using a silkworm infection model. Over the past years, we have identified several S. aureus virulence factors that are involved in both the silkworm and mice virulence [ options . It is cirulence .S. aureus USA300 of Nebraska Transposon Mutant Library, we found that disruption of the yjbI gene, lying upstream of the yjbH gene in a two-gene operon, resulted in the loss of pathogenicity in silkworm and mice. Pathogenicity was restored by the introduction of the yjbIH operon and not of the yjbI gene [yjbH gene might have a role in pathogenicity. Studies involving YjbH in Bacillus subtilis [S. aureus [S. aureus is obscure. In this study, we found that YjbH is essential to S. aureus virulence in a whole-body animal infection model and involved in the expression of the broad range of virulence genes. In addition, we found that YjbH affects the bacterial surface structure, and confers resistance to oxidative stress in the host.Recently, from the screening of 380 transposon insertion mutants of jbI gene , suggestsubtilis , S. aure. aureus , and oth. aureus  have rep. aureus ,24–26. HS. aureus strains were grown in tryptic-soy broth . Escherichia coli was grown in Luria-Bertani medium . Antibiotics were supplemented as required.Bacterial strains used in this study are summarized in Silkworms rearing until the fourth molt stage was performed on antibiotic-containing artificial diet Silkmate 2S  as previously described . The fif600) to 0.7 for microbial load and 4.0 for survival assay. The prepared suspensions (200 µl) of the strains were injected into the tail-vein of mice . The CFU of the injected suspensions was counted. For measuring the microbial load, kidney and heart of the infected mice were isolated after 24 h of injection, suspended in PBS, homogenized, and CFU was counted.Mice experiments were performed with approval from Teikyo University Animal Ethics Committee (18–024). Bacterial strains were cultured overnight in TSB with or without antibiotics. Erythromycin (5 µg/ml) alone or with chloramphenicol (12.5 µg/ml) was added to the medium for transposon mutants or gene complemented strains, respectively. The overnight cultures were 100-fold diluted with TSB and continued incubation for 16 h at 37°C with shaking. The cells were collected by centrifugation, washed with phosphate-buffered saline (PBS), and suspended in PBS to adjust the turbidity  sample buffer and centrifuged. The supernatant was then boiled for 5 minutes, and 20 µl was loaded to a 4–20% gradient Mini-PROTEAN®TGX™ precast gel . After electrophoresis, the gel was stained with Coomassie brilliant blue for visualization.Strains were grown overnight in TSB or TSB supplemented with antibiotic as necessary at 37°C with aeration, and the AyjbI::Tn, and yjbH::Tn strains were grown at TSA, 37°C for 24 h and were subsequently analyzed by whole-cell FTIR spectroscopy. Measurements were conducted on a Tensor 27/HTS-XT microplate adapter FTIR spectrometer . Unsupervised hierarchical cluster analysis  was performed on the second derivative, and vector normalized spectra using the highly discriminatory spectral region between 1200–800 cm−1, which is dominated by vibrations of various oligo- and poly-saccharides and their specific type of glycosidic linkages.Fourier-Transform Infrared (FTIR) spectroscopic measurements and spectral evaluation were performed as previously reported . BrieflyyjbI::Tn to have reduced pathogenicity, pigment, and protease productions [yjbI gene did not restore pathogenicity in yjbI::Tn, while the introduction of the operon consisting of the yjbI gene and the downstream gene yjbH  restore reduced , indicat ability ), and thyjbI::Tn .Figure 1yjbI::Tn was reduced in kidney and heart [yjbH::Tn was reduced only in the heart of infected mice . To furecreased ). Thus, yjbI and yjbH gene-disrupted mutants. We obtained consistent results in this study . Next, l region , upper).position , lower).. aureus .Figure 4yjbI and the yjbH genes in protease and pigment production, surface protein production and pathogenicity of S. aureus, we speculated YjbH might be involved in regulating the expression of various genes. Therefore, we performed RNA-seq analysis. We found that disruption of both the genes led to significant changes in the expression of the genes from diverse pathways. We found similar expression patterns among the yjbI and yjbH genes- disrupted mutants with a common upregulation and downregulation of 70 and 79 genes, respectively.  gene, encoding the immunoglobulin G binding protein A precursor; SAUSA300_2453, encoding ATP-binding protein of ABC transporters; SAUSA300_2454, encoding a putative membrane-spanning protein; SAUSA300_0114 (sarS), and SAUSA300_1890, encoding staphopain A protease. In addition, the alternative sigma factor sigB and the genes whose transcription exclusively depends on SigB, such as asp23, SAUSA300_2143, and SAUSA300_2144, were downregulated (Supplementary Table S1), suggesting that YjbH contributes to the control of SigB activity. Further, expression of the spxA was downregulated, which might be due to the autoregulation of spx transcription due to increased Spx level [aur and other proteases such as serine and cysteine proteases, which explains the reduced protease production in these mutants.To gain functional insight into the differentially expressed genes common in both the gene disrupted mutants, we used Gene Ontology term enrichment analysis. We found that the genes involved in pathogenesis and extracellular region were highly represented among the downregulated genes. Some of the most downregulated genes included the SAUSA300_0113 . Thus, YjbH might be necessary for pH homeostasis during acidic stress in S. aureus. These results suggested that YjbH is responsible for pleiotropic phenotypes in S. aureus, possibly by directly or indirectly regulating the expression of genes from diverse pathways and by the accumulation of Spx.Genes related to nickel cation binding were highly represented among the upregulated genes ). Along S. aureus [S. aureus against oxidative stress in the host. For this purpose, we determined the lethal dose fifty (LD50) values of the strains in the presence and absence of N-acetyl-L-cysteine (NAC). NAC is a free radical scavenger; it eliminates reactive oxygen species (ROS) such as OH•, HOX, NO2, and H2O2 [50 values were significantly decreased, rendering the strains more effective in killing the silkworms . We furilkworms ). These S. aureus in animal models, both silkworms and mice, and the first to show the function of YjbH regarding virulence is related to oxidative stress in the host. Previous studies had determined the viability of the yjbH and the yjbI genes-disrupted mutants in organs of infected mice [S. aureus surface and proteases.Our result is the first report showing the role of YjbH as a virulence factor of ted mice ,20; howeyjbH gene resulted in insufficient nitrogen supply. As S. aureus can utilize urea as a nitrogen source [yjbH::Tn enhanced urease production to compensate for the nitrogen supply in the absence of YjbH. This notion is further supported by the enhanced production of urease in the mutants with reduced proteolytic activities [Microbes acquire nitrogen through the degradation of proteins using proteases. Therefore, it is expected that the reduced protease production by the disruption of the n source , it is ltivities . In additivities , which itivities , YjbH miS. aureus against reactive oxygen species in the host [yjbH gene suggests that YjbH exerts oxidative stress resistance by regulating staphyloxanthin production. Based on the effect of YjbH on other oxidative stress-related genes, it is evident that YjbH plays a role in providing oxidative stress tolerance to S. aureus, which is an important defense mechanism against host immunity. This function was further verified by our in vivo oxidative stress model using silkworm, where an ROS scavenger was able to rescue S. aureus against host-immunity and increased the virulence of yjbH::Tn.Staphyloxanthin, the yellow pigment, has been shown to have roles in protecting the host . The losspxA and sigB, including SigB-specific genes, were downregulated. YjbH is involved in the proteolysis of Spx, a stress regulator required for oxidative stress tolerance [sigB gene-deficient mutant was found to be less virulent in mouse models of arthritis [S. aureus. Similarly, the absence of SigB causes reduced pigmentation irrespective of the presence of YjbH or Spx, and the absence of Spx was required for the enhanced proteolytic activity of sigB-yjbH double gene disruptant [In our RNA seq analysis, we found that the expression of stress regulators olerance ,19,21,36olerance . Despiterthritis  and intrrthritis ; whereasrthritis , and a rrthritis  found nosruptant . TherefoS. aureus and regulates virulence genes and surface proteins, and is necessary for oxidative stress tolerance inside the host. Moreover, the disruption of the yjbI and the yjbH genes led to reduced protease activity and reduced surface protein. Interestingly, most of the proteases are excreted into supernatant from the cell in general [S. aureus predominantly consist of proteins related to adhesion and evasion such as clumping factor, fibronectin, and collagen-binding proteins, staphylococcal protein A [sarA, agr, and other two-component systems such as srrAB, saeRS, and ArlRS [S. aureus virulence pathway and its utilization as a target for anti-virulence drug development.In our study, we have shown for the first time that YjbH is required for full virulence of  general , and surrotein A . In thisnd ArlRS , YjbH afClick here for additional data file.


The mechanism and rupture process of the giant 2011 Tohoku-oki earthquake (Mw 9.0) are still poorly understood due to lack of permanent near-field observations. Using seismic arrival times recorded by dense seismograph networks on land and at ocean floor, we determine a detailed seismic tomography model of the megathrust zone beneath the Tohoku forearc. Our results show that the coseismic slip of the 2011 Tohoku-oki earthquake initiated at a boundary between a down-dip high-velocity anomaly and an up-dip low-velocity anomaly. The slow anomaly at shallow depths near the Japan trench may reflect low-rigidity materials that are close to the free surface, resulting in large slip and weak high-frequency radiation. Our new tomographic model can account for not only large slip near the trench but also weak high-frequency radiation from the shallow rupture areas. Using data recorded by a new seafloor seismic network, the authors reveal the detailed 3D structure of the source zone of the great 2011 Tohoku-oki earthquake, which sheds new light on the mechanism of the great earthquake and tsunami. The mechanism of the large slip near the trench has been interpreted by different models, such as abundance of weak clay (smectite)12 and fault weakening13. A depth-variation frictional model was used to account for its energy radiation distribution16. However, the mechanism and rupture process of the great 2011 Tohoku-oki earthquake are still a matter of some controversy due to the lack of permanent near-field observations17.The great 2011 Tohoku-oki earthquake (Mw 9.0), one of the greatest earthquakes recorded by modern instruments, occurred in a megathrust zone formed by the subducting Pacific plate and the overriding Okhotsk plate  suggest that the huge shallow slip may be caused by weak clay (smectite) on the upper boundary of the subducting Pacific plate20. However, using residual topography and gravity anomalies, Bassett et al. 21 suggested that the structure and frictional properties of the overriding Okhotsk plate control the interplate coupling and seismogenic behavior in NE Japan. Nishikawa et al. 22 suggest that different slip behaviors exist in the Tohoku forearc, which may be caused by along-strike variations in pore fluids and lithology of the overriding and subducting plates. Previous tomographic studies25 of the Tohoku megathrust zone suggested that the 2011 Tohoku-oki earthquake hypocenter was located in a significant high-velocity (high-V) anomaly, which may represent an asperity in the megathrust zone. These studies have improved our understanding of the structural variation along the plate boundary. However, all the previous tomographic models have limited spatial resolution in the large-slip area near the Japan trench due to the lack of near-field seafloor observation.The mechanism of great megathrust earthquakes is influenced by the frictional properties, structure and composition of the plate boundary fault26. The deployment of the S-net provides us with an unprecedented opportunity to image the structure of the Tohoku megathrust zone and to clarify the mechanism of megathrust earthquakes. Using a large number of high-quality arrival-time measurements of local earthquakes recorded by the S-net27 and Hi-net28, here we investigate the detailed three-dimensional (3-D) seismic velocity structure beneath the Tohoku forearc, which sheds new light on the mechanism and rupture process of the great 2011 Tohoku-oki earthquake.Recently, the Seafloor Observation Network for Earthquakes and Tsunamis (S-net) Fig.  has been25, and the obtained results  earthquakes and historical great earthquakes occurred, which was designated as the Sanriku-oki low-seismicity region29. At shallow depths <20 km, our Vp model  may play a key role in the dynamic weakening processes. Recent numerical simulations32 suggest that the low shear strength of weak clay minerals is insufficient to arrest the inertial motion of rupture propagating along the megathrust, and so large slip could occur near the trench axis.One of the most striking characteristics of the great 2011 Tohoku-oki earthquake is its large coseismic slip at the shallow depth near the Japan trenchM ≥ 7.0) that occurred before 2011 are mostly located in the deeper high-V and high-Q  zones with strong ground motions and positive residual gravity35 , the up-dip low-V anomaly may have been able to arrest rupture propagation to the shallow part due to their relatively low slip velocities. However, for the great 2011 Tohoku-oki earthquake, the low-V anomaly at the shallow depth may not have arrested up-dip rupture due to the large strain rate changes resulting from the extraordinary large slip near the hypocenter. The low-V anomaly with low rigidity may cause not only large coseismic slips near the trench but also a deformed upheaval structure at the shallow depth5. The low-V anomaly near the Japan trench may be also responsible for a preceding slow slip event (M 7.0) in the source zone36  show distinct land-ward displacement rates in the large coseismic slip area and remarkable trench-ward displacement rates in the south of the FSB  that are located in a high-V anomaly and show small trench-ward movements , we can obtain more accurate ray paths and get a better tomographic modelExtensive checkerboard resolution tests (CRTs) are performed to evaluate the adequacy of ray coverage and the spatial resolution of our tomographic model (Supplementary Figs. Supplementary InformationPeer Review File