To determine whether radiomics features based on contrast-enhanced CT (CECT) can preoperatively predict lymphovascular invasion (LVI) and clinical outcome in gastric cancer (GC) patients.In total, 160 surgically resected patients were retrospectively analyzed, and seven predictive models were constructed. Three radiomics predictive models were built from radiomics features based on arterial (A), venous (V) and combination of two phase (A + V) images. Then, three Radscores  were obtained. Another four predictive models were constructed by the three Radscores and clinical risk factors through multivariate logistic regression. A nomogram was developed to predict LVI by incorporating A + V-Radscore and clinical risk factors. Kaplan-Meier curve and log-rank test were utilized to analyze the outcome of LVI.P < 0.01). Univariate logistic analysis identified three clinical features  and three Radscores as LVI predictive factors. The Clinical-Radscore  model that used all these factors showed a higher performance (AUC = 0.856) than the clinical  model (AUC = 0.810) and the A + V-Radscore model (AUC = 0.795) in the train cohort. For patients without LVI and with LVI, the median progression-free survival (PFS) was 11.5 and 8.0 months (P < 0.001),and the median OS was 20.2 and 17.0 months (P = 0.3), respectively. In the Clinical-Radscore-predicted LVI absent and LVI present groups, the median PFS was 11.0 and 8.0 months (P = 0.03), and the median OS was 20.0 and 18.0 months (P = 0.05), respectively. N stage, LVI status and Clinical-Radscore-predicted LVI status were associated with disease-specific recurrence or mortality.Radiomics related to tumor size and intratumoral inhomogeneity were the top-ranked LVI predicting features. The related Radscores showed significant differences according to LVI status (Radiomics features based on CECT may serve as potential markers to successfully predict LVI and PFS, but no evidence was found that these features were related to OS. Considering that it is a single central study, multi-center validation studies will be required in the future to verify its clinical feasibility. Gastric cancer (GC) is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide . In chinAccumulating evidence has confirmed that the recurrence rate is related to T, N and TNM staging , 9. In aTherefore, the purposes of this retrospective study were to investigate whether radiomics could be useful in predicting LVI and disease outcome in GC patients and in investigating the added value provided by integrating independent clinicopathological risk factors.n = 133); 2) any local or systemic treatment before surgery (n = 52); 3) poor CT image quality for postprocessing due to artifacts (n = 30); 4) a previous distal gastrectomy (n = 19); 5) undifferentiated tumor (n = 1) and well-differentiated tumor (n = 2). Figure This study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee of our hospital, with the requirement for informed consent being waived. From August 2015 to November 2018, 397 GC patients who had undergone radical gastrectomy were gathered and reviewed. The inclusion criteria were as follows: 1) histologically confirmed GC; 2) CECT performed before surgery. The exclusion criteria were as follows: 1) no definite information of postoperative pathological characteristics , cancer antigen 199 (CA199) and tumor node metastasis (TNM) stage, were retrospectively derived from electronic medical records. Laboratory analysis of CEA and CA199 was performed via routine blood tests within 1 week before surgery. According to the normal range used at our institution, the threshold value for CEA and CA199 level was 5 ng/mL and 37.0 U/mL, respectively. The TNM staging was reclassified according to the seventh edition of the Cancer Staging Manual of the American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) staging system.All patients underwent surgical treatment within 2 weeks of CECT examination. All surgical specimens were examined by two pathologists, especially in detecting the presence of LVI. The histological parameters ordinarily include tumor differentiation , number of LN metastases, surgical margin and LVI status of the resected tumor. LVI, which refers to as lymphatic and/or blood vessel invasion, was only visible under microscopy.Finally, a total of 160 patients  served as our study cohort. The patients’ clinicopathological characteristics are presented in Table According to the follow-up protocol of our institution, the patients were postoperatively followed up with abdomen CT every 6 months for the first 1 year and then annually. Follow-up data were collected from hospital records for patients who were lost during follow-up. The follow-up duration was measured from the time of surgery to the last follow-up date, and information regarding the survival status at the last follow-up was collected. The progression-free survival (PFS) was defined as the time to recurrence at any site, last follow-up, or all-cause death, whichever came first. The overall survival (OS) was defined from the date of surgery to the date of all-cause death, or on 1 March 2019, whichever came first.All patients signed informed consent forms for CECT examination. The patients were asked to fast from solid food for at least 8 h prior to CT examination and were encouraged to drink 800~1000 ml of water to achieve gastric distension. No anti-cholinergic agent was used. The patients were trained to hold their breath before CT examination. All patients underwent 64-slice multidetector spiral CT  prior to surgery. All patients were in the supine position and the scan covered the upper or the entire abdomen. The scanning parameters were as follows: tube voltage 120 kVp,180 reference mAs with automated tube current modulation system, slice thickness 5.0 mm, slice interval 5.0 mm, field of view 350 × 350 mm, matrix 512 × 512, rotation time 0.5 s, pitch 0.984 and reconstruction section thickness 1.25 mm. After an intravenous injection of contrast medium  via a syringe pump, the arterial phase and portal venous phase scans were acquired following delays of 30 s and 60 s, respectively.https://itk.org/). According to the literature, focal thickening of at least 6 mm or greater compared with the adjacent gastric wall was determined to be abnormal thickening and cancerous phase [Arterial and venous phase CT images (thickness: 1.25 mm) of all patients were downloaded from the picture archiving and communication system and uploaded into the open-source software ITK-SNAP  by drawing 2D ROIs layer-by-layer with a mouse. After the tumors were manually segmented, the arterial phase images, portal venous phase images and corresponding sketched VOIs were imported into AK software , 17  . The feaXi is the value of i th selected feature and Ci is the coefficient of the i th selected feature listed in Table After the ICC selected the repeatable features, Spearman correlation analysis (SPM) combined with the least absolute shrinkage and selection operator (LASSO) method  were utientclass1pt{minimaA univariate regression analyses model of clinical parameters was performed to determine LVI risk factors. The candidate clinical variables were gender, age, T stage, N stage, AJCC stage, tumor differentiation, CEA and CA199 level.To develop an optimal model, we evaluated 7 models by analyzing the Radscore of arterial (A) phase images, venous (V) phase images, the combination of two phases (A + V) images and then incorporated the independent clinical predictors to build the combination model by multivariable logistic regression analysis. The processes of features selection after ICC analysis and predictive models construction were shown in supplementary materials U test for continuous variables that were not normally distributed. The independent predictors for LVI status were identified by multivariate logistic regression analysis. In addition, receiver operating characteristic (ROC) curve analyses were performed. The area under the curve (AUC), accuracy, sensitivity and specificity were also calculated. Survival curves were generated with the Kaplan-Meier method and compared by log-rank tests.In this study, the patients were divided into groups with LVI and without LVI. The clinicopathological characteristics were compared using the chi-square test for categorical variables and the Mann-Whitney www.r-project.org/). The reported statistical significance levels were all two-sided, and the statistical significance level was set to 0.05. The multivariate logistic regression analysis was performed with the “stats” package. Nomogram construction was performed using the “rms” package.Statistical analysis was conducted with R software . The distribution of gender, age, tumor differentiation, CEA and CA199 levels were similar in the two groups (P ≥ 0.05). According to univariate analysis, T stage, N stage and AJCC stage were associated with LVI, whereas the age, gender, tumor differentiation, CEA and CA199 levels were not  patients had completed the PFS follow-up, and 155/160 (96.9%) had completed the OS follow-up. The overall recurrence rate was 21.3% (34/160), and the overall death rate was 8.75% (14/160).P < 0.001, Fig. P = 0.03, Fig. P = 0.024), N stage (P = 0.006), LVI status (P < 0.001) and Clinical-Radscore- predicted LVI status were associated with PFS. Multivariate Cox regression analysis showed that N stage , LVI status  and Clinical-Radscore-predicted LVI status  were independent predictors of disease specific recurrence.The median PFS of the patients was 9.0 (1.0~40.0) months, and 8.0 (1.5~26.5) months for those with LVI and 11.5 (1.0~40.0) months for those without LVI  and Clinical-Radscore predicted LVI status (P = 0.014) were associated with OS. Further, multivariate Cox regression analysis was performed, including N stage and Clinical-Radscore-predicted LVI status as inputs, which showed that N stage  and Clinical-Radscore-predicted LVI status  were independent predictors of disease specific mortality.The median OS for all patients was 19.0 (1.0~52.0) months, and specifically, the corresponding values were 17.0 (3.0~44.0) months for those with LVI and 20.2 (1.0~52.0) months for those without LVI  predicted model (AUC = 0.810). Because there is a lack of a single highly reliable factor to predict LVI, the radiomics signatures model combining clinical risk factors becomes a viable alternative . By incoP < 0.001). Similar results in PFS were observed in the Clinical-Radscore-predicted  LVI present and LVI absent groups . A potential explanation for the results may be as follows: patients with LVI showed more aggressive disease than those without LVI. However, this finding was not confirmed for OS . This result may be because OS is affected by many factors, such as late treatment, other diseases. The multivariate analysis indicated that LVI status, as well as N stage and Clinical-Radscore-predicted LVI status, is an independent prognostic factor in GC patients, which is in accordance with previous results of many studies [The current AJCC/UICC guidelines do not include LVI as an independent prognostic indicator of GC in the TNM staging system. However, many studies have shown that LVI is an independent risk factor for survival in GC patients , 27, 28. studies , 30.Our study has some limitations. First, the Clinical-Radscore-predicted LVI model overestimates the number of LVI+ cases . Although a predictive model with a higher rate of false-positives leads to a better prognosis than that with a higher rate of false-negatives. A high false-positive rate remains a major limitation of this work. More machine learning or deep learning methods will be utilized to further improve the accuracy of the predictions in future research. Second, our model only included traditional clinicopathological factors. The inclusion of additional variables, such as qualitative and quantitative features  that can be routinely extracted from multiphasic dynamic CT, may improve the prediction accuracy of LVI status and survival. This potential effect warrants future research. Finally, the study results were assessed in a single institution which has some inherent limitations. The generalization of the results in multi-center should be required in the future studies.Radiomics features based on CECT may serve as potential markers to successfully predict LVI and PFS, but no evidence was found that these features were related to OS. Considering that it is a single central study, multi-center validation studies will be required in the future to verify its clinical feasibility.Additional file 1.


This presentation will explore the development of AGHE’s major contemporary contribution: AGHE’s basic-competency guidelines and their role in program review and program enhancement. The presenter will describe in detail the steps in creating objective student learning outcomes and will explore how meeting these competencies improve instruction, self-reflection, program analysis and faculty discussion and ultimately gerontology programs. These guidelines and policies embody a living entity that is always evolving. Future iterations will be anticipated and discussed. Part of a symposium sponsored by the Geriatric Education Interest Group.


The relation between the size of an earthquake mainshock preparation zone and the magnitude of the forthcoming mainshock is different between nucleation and domino-like cascade models. The former model indicates that magnitude is predictable before an earthquake’s mainshock because the preparation zone is related to the rupture area. In contrast, the latter indicates that magnitude is substantially unpredictable because it is practically impossible to predict the size of final rupture, which likely consists of a sequence of smaller earthquakes. As this proposal is still controversial, we discuss both models statistically, comparing their spatial occurrence rates between foreshocks and aftershocks. Using earthquake catalogs from three regions, California, Japan, and Taiwan, we showed that the spatial occurrence rates of foreshocks and aftershocks displayed a similar behavior, although this feature did not vary between these regions. An interpretation of this result, which was based on statistical analyses, indicates that the nucleation model is dominant.  There is an intriguing discussion about the earthquake preparation process. Two major hypotheses proposed for this process are contradictory in the context of earthquake prediction. One of them is the most commonly cited cascade model ,2. In thm = 2 and 5, where m is mainshock magnitude. They employed the mainshock identification method proposed by Felzer and Brodsky  from the mainshock, divided by 0.2Δr and by their total number, i.e., the linear density [rmax = 3 km. We did not use Δrmax = 3 km because the range of mainshock magnitudes considered in our study was wider than that considered by Lippiello et al. [rmax = 100 × fL (km), where fL is the fault length obtained by substituting M into the equation between fL and M: log fL = 0.6M − 2.9 [rc values for different mainshock magnitudes were computed.Following the method used by Felzer and Brodsky  and Lippstance L . In addilsewhere . For mai density ,8 divide6M − 2.9 . By applrc, observed in other studies [rc, ρ(Δr) attenuated, following a clear power law. In addition, ρ(Δr) displayed a nearly constant fluctuation when the value of Δr was much larger. This feature, namely a nearly constant ρ(Δr), can be regarded as a region beyond the earthquake preparation area. The area of smaller than Δrc, possibly corresponding to slip areas, would be randomly generated seismicity, which yields increase or nearly constant of the linear density probability.The monomodal convex shape in  studies ,7, each rc and mainshock magnitude m. If we assume a circular asperity (Sa = πla2), where la is a characteristic asperity radius [al-M line correlates well with the Δrc-m correlation to be obtained from this study. y radius , the la-rc-m correlation and the la-M relation is valid, supporting our result. Skarlatoudis et al. (2005) used moment magnitude, and not local magnitudes used for Japan, California, and Taiwan, to obtain the scaling shown in rc-m correlation, which is similar to the la-M relation  earthquake hypothesis , are similar to each other, and this behavior is observable regardless of different earthquake catalogues (la. This indicates that the earthquake preparation and rupture areas grew as the magnitude of the mainshock increased, the latter being scaled with la [The spatial distributions of seismicity density for foreshocks and aftershocks, characterized by talogues . We focu with la . Our obs with la ,18,19. As described in the “Introduction” section, two major hypotheses of the preparation process have been proposed: the nucleation model ,4 and do


IntroductionPhysical distancing guidelines during the coronavirus disease 2019 (COVID-19) pandemic forced medical residency programs to move a large portion of required didactics to virtual settings. Toxicology, a core component of emergency medicine (EM) education, was forced to adapt to similar constraints. An in-person escape room style puzzle was modified to a virtual format for educational purposes, and shared with and evaluated by two different residency programs.Materials and methodsA virtual escape room, “Escape the Toxin: Online!” was created to test knowledge of toxicologic ingestion and antidote utilizing Google Forms and delivered using Zoom teleconference software to two EM residency programs in the Philadelphia region. After small groups completed the gamified activity, their scores were calculated and they completed an anonymous evaluation.ResultsResidents at the program where a Medical Toxicology fellowship is located found the virtual escape room to be more effective and enjoyable compared to the second program. Despite some differences in perceived effectiveness, the majority of participants were able to correctly solve the puzzle and get to the antidote.ConclusionThe majority of learners who participated from both residencies agreed that they would recommend this virtual program to other EM residents. Toxicology is a core component of emergency medicine (EM) resident education. In an effort to promote engagement with residents, the Division of Medical Toxicology at Albert Einstein Healthcare Network in Philadelphia, PA had previously created an immersive escape room experience titled “Escape the Toxin.” This team-based puzzle involved a geographical maze within departmental office space to find an antidote for a given toxidrome. As educators, we sought to share and replicate it within other regional residency programs. However, in an unprecedented era of physical distancing due to the coronavirus disease 2019 (COVID-19) pandemic, many residency programs found themselves planning for virtual educational content to replace in-person didactics. Adaptations to this new style of learning included live virtual conferences and lectures, in addition to increased asynchronous online learning . TherefoThe original “Escape the Toxin” innovation was created as a result of gamification becoming increasingly popular in medical education. Gamification in this sense is the incorporation of game elements into didactics to increase engagement among participants. Gamification has shown effectiveness in improving knowledge, skills, and satisfaction compared to traditional lectures -6. LearnIn an effort to continue resident education throughout the COVID-19 pandemic and add to the growing online medical education movement, we utilized Google Forms to develop a virtual version, “Escape the Toxin: Online!” Though the virtual version did not include physical locations or cryptex puzzle lockboxes containing a clue, it retained the same underlying theme: determine the antidote to the toxidrome presented. After an initial description of the patient, signs, symptoms, and vital signs, learners were then given multiple intermingled toxicology challenges. The ultimate goal of the activity was to pick the correct mechanism of action for the antidote to “escape the toxin.”The challenge was incorporated into weekly didactics at two regional EM residency programs that had successfully migrated onto a digital platform. The first program used the virtual escape room as either a live online activity during didactics or as an optional asynchronous activity, while the second program provided it as part of the required weekly didactics. During both sessions, the Zoom teleconferencing platform was utilized with breakout rooms. These rooms allowed for the task to be completed in small groups, providing an opportunity for a team-based approach to solve the challenges mimicking a live escape room.For each iteration, the entire group met in the main room before being divided into virtual breakout rooms of no more than six or seven participants. They received a brief introduction and the link to the Google Form containing the virtual escape room Figure . With thTo move forward through the Google Form, participants were required to answer all of the questions. In order to preserve flow, the answers did not have to be correct to move to the next puzzle, and participants were not notified in real time if their answer was incorrect. However, in order to accomplish the ultimate goal of picking the correct antidote , the participant needed to have identified the toxidrome described in the initial description of the case. The remainder of the questions and puzzles all had rhyming stems to apply gamification mechanics to mimic an in-person escape room experience. A variety of question types were utilized, including anagrams, fill-in-the-blank, multiple-choice or short answer Figure . The puzAt the end of the activity, a short three-question survey was embedded to obtain feedback about the learner’s attitude toward this modality for presenting toxicology content, the appropriateness of the time allotted, and whether the participant would recommend the activity to other EM residents. Responses to these Likert-scale questions were required to complete the game. After the live session, participants were invited to debrief about the puzzles and the case itself. The responses to the Google Form were reviewed. The top learners were identified, and considered to have “escaped the toxin” or “passed the case” if they met the following criteria: selected the correct mechanism of action of the antidote, had the most correct answers from the puzzles, and completed the activity within the allotted 45 minutes.“Escape the Toxin: Online!” is an innovative virtual activity that was successfully integrated into individual didactic sessions by two regional EM residency programs. With the gamification feel, it brought EM residents together in a fun, relaxed environment to use each other and their research skills to learn about a toxidrome and the associated antidote that they may encounter over their careers. Across the two iterations, 46 residents participated. The first program had 17 participants from a postgraduate year 1-4 (PGY1-4) residency with a Medical Toxicology fellowship at their institution. The second program had 29 participants from a PGY1-3 residency without an associated fellowship. Overall, 54% (25 out of 46) of the resident participants felt that this activity was helpful or very helpful towards learning toxicology. The participants at both programs all completed the activity in the time allotted. At the first program, 88% (15 out of 17) of participants completed the task by identifying the correct mechanism of action of the antidote and 95% (28 out of 29) of participants were correct at the second program.Data from the two programs was not only collated for total effectiveness but also compared, as shown in Table Overall, 58% of the total participants agreed or strongly agreed that they would recommend this activity to other EM residents as a way to learn toxicologic concepts. When comparing the two programs, the first iteration found 100% of participants to be in agreement, but only 41% (12 out of 29) at the second program. Seventeen percent (five out of 29) of participants felt disagreement or strong disagreement that they would recommend this activity to other colleagues. Interestingly, the majority of the residents who felt this activity was helpful came from the residency program with an associated Medical Toxicology fellowship. The duties of a medical toxicology fellow and faculty usually extend from bedside clinical consultations to a wide variety of lectures and on-the-job teaching . This reThe program without a Medical Toxicology fellowship had been previously exposed to gamification in their weekly didactics, but may have been fatigued of this concept. This could have contributed to less satisfaction with the activity at the time it was delivered. Adequate spacing of these innovative didactic sessions may improve engagement. The participants may not have known what to expect from this particular type of gamification. One of the free text comments from a participant at the second program alluded to the questions being somewhat vague. In an in-person escape room style puzzle, many of the clues are intentionally vague to lead the team to the correct answer with real-time brainstorming and interaction with teammates. After analyzing these results, we recommend an expanded explanation of the activity for programs that have not experienced an in-person escape room style puzzle for future activities.The presence of a Medical Toxicology fellowship is also a huge benefit for continuity and sustainability of the virtual program. The creation of this virtual learning modality may be more time-consuming and require more knowledge and effort than traditional learning methods . It woulThough there is a question as to the success of resident engagement among the two iterations, 88% of the first group and 95% of the second group ultimately answered the final question correctly. This indicated an overall understanding of the presented toxidrome, the diagnosis in the case and found the antidote in question. It also likely indicates decent engagement in the activity.For EM programs that have not participated in a similar activity or an in-person escape room, a thorough explanation of the concept is warranted to improve resident participation and experience. Lastly, an anonymous evaluation following a formal debriefing may be more reflective of participant engagement and thoughts on the success of the activity, and should be considered in future projects.Virtual medical education has been widely utilized due to physical distancing requirements during the COVID-19 pandemic and is likely to continue beyond this public health emergency. Constraints of time and availability of educators make virtual learning an appealing option to many EM programs. EM programs should familiarize their residents with different types of virtual learning, including gamification concepts such as “Escape the Toxin: Online!” Despite some differences in perceived effectiveness, the majority of participants in "Escape the Toxin: Online!" were able to correctly solve the puzzle and choose the correct antidote. Further study is needed to identify why an EM program with a Medical Toxicology fellowship may find increased satisfaction, though a plausible explanation is resident comfort with Medical Toxicology fellows and faculty. However, adequate explanation of the virtual escape room concept prior to implementation in a program’s curriculum may improve engagement, satisfaction, and ultimately, retention of information.


This study aims to evaluate a program promoting character strengths, positive psychological capital, learning flow, and sense of calling for nursing students. We conducted a concurrent embedded mixed methods study with 51 nursing students randomly classified into an intervention or a control group. The intervention group exhibited significantly higher scores than the control group for positive psychological capital, learning flow, and sense of calling. Program participation experiences were categorized as “change of views about oneself”, “change of views about the world”, “stress relief”, and “practice of positivity”. Among nursing students, this program demonstrated change toward a positive, committed, and meaningful life. Undergraduates are generally in young adulthood, a time of diverse changes and challenges ,2, it isPositive psychology is self-exploration that focuses on strengths, rather than weaknesses, and on health and happiness, rather than on illnesses and other problems: this perspective focuses on the best possible outcomes, instead of on previous failures . SeligmaPark and Huebner  reportedIn addition to a recent growing interest in positive psychology in Korea, there have been consistent attempts to develop strengths and increase happiness in undergraduates ,14,15. HThus, this study investigated the effects of a positive psychology-based character strength exploration program that encouraged living a happy life, which encompasses having a positive, engaged, and meaningful life. We anticipate that this program will contribute to developing extracurricular programs that promote happiness and stimulate personal growth in nursing students. We developed three hypotheses for this study:Hypothesis 1 (H1).The intervention group will show increased positive psychological capital, compared with the control group.Hypothesis 2 (H2).The intervention group will show increased learning flow, compared with the control group.Hypothesis 3 (H3).The intervention group will show a heightened sense of calling, compared with the control group.We used a concurrent embedded mixed method design, and qualitative data and quantitative data were concurrently collected and separately analyzed. The results were then integrated into the discussion stage of the study, according to the appropriate research standards .n = 26) or control group (n = 26) using a computer program. After excluding one participant who wished to withdraw from the study prior to the beginning of the program for personal reasons, 25 participants in the intervention group and 26 participants in the control group were included in the final study.Participants were first- and second-year nursing students at a single university, all of whom provided informed consent to participate. Third-year and fourth-year students were excluded due to their clinical training during the semester and the high academic burden of the upcoming national licensure examinations. The sample size was computed using the G*Power 3.1 program. With reference to the effect size of 0.91 for promotion of positivity in a meta-analysis of the effects of positive psychology-based group programs by Eom, Jeon, and Goh , we perfPositive psychological capital was assessed using the Psychological Capital Questionnaire (PPC) , in its Learning flow refers to the degree of immersion during learning , which rLearning flow was measured using the Learning Flow Scale for Adults developed and validated by Kim, Tack, and Lee . This 29Sense of calling was measured using the Korean version of the Calling and Vocational Questionnaire developed by Dik, Eldridge, Steger, and Duffy , as valiWhat did you like about this program?What are some of the regrets that you have after participating in this program?What has changed in you or in your environment after participating in the program?The participation experience survey was an open-ended questionnaire consisting of the following items:http://www.strengthgarden.co.kr/) using Seligman et al.’s [This program was structured by modifying the Strength 5 Program . Baseline surveys for both groups were conducted prior to the program, and the post-program survey was conducted one week after completion of the six-session program for the intervention group and four weeks after the baseline survey for the control group. To prevent a diffusion effect, the intervention group began after the control group completed the post-program survey. To supplement data that could not be obtained using a quantitative instrument, and to thoroughly understand participants’ experiences of the intervention, a qualitative questionnaire was administered during the post-program survey. To minimize bias, the research assistant collected the data, and was blinded to group allocation. The baseline and post-program questionnaires were given individualized IDs, and collected data were coded to prevent identification of personal information before the assistant entered the data into an Excel sheet.® SPSS® Statistics 24.0 software . The demographic characteristics of the intervention and control groups were summarized using means and standard deviations or frequencies and percentages. Normality of the dependent variables was tested using Shapiro–Wilk’s test. Homogeneity of the demographic characteristics and dependent variables between the intervention and control group was analyzed using χ2-tests, Fisher’s exact test, and Mann–Whitney U-tests. The effects of the program were analyzed using paired t-tests and independent t-tests.Quantitative data were analyzed using IBMQualitative data collected through the participation experience questionnaire were analyzed using the content analysis method developed by Elo and Kyngäs . We readThe mean age of the participants was 19.8 years; among the participants, 38 (74.5%) were women and 13 (25.5%) were men. Twenty-nine (56.8%) had a family income of less than 5 million KRW, while 22 (43.2%) had a family income of 5 million KRW or higher. Twenty-nine (56.8%) had no religion, and twenty-two (43.2%) were religious. There were no significant differences in demographics between the intervention and control groups at the start of the study .t = 1.56, p = 0.253), learning flow , and sense of calling . The Shapiro–Wilk’s test confirmed that all variables were normally distributed , learning flow , as well as calling and vocation , compared to the control group  and learning flow . For calling and vocation, the mean for the intervention group increased from 2.85 to 3.04; however, it decreased from 3.05 pre-intervention to 3.00 post-intervention in the control group. The intervention group had a statistically significant increase in positive psychological capital  represented participants’ determination to continue expressing their gratitude to others, help others, and view life positively, even after the program’s conclusion. These changes confirm that the program helped increase participants’ sense of calling. However, further studies should re-examine the relationship between positivity and sense of calling using instruments that are more objective. Park  identifiOur participants stated that they felt happy while striving to discover and use their key strengths during the program. According to Seligman , using oThe major topics of interest in positive psychology include positive mental states, positive traits, and positive institutions, all of which help individuals attain happiness and self-realization . It is cNevertheless, this study has the following limitations. First, there is a limit to generalizing only nursing students enrolled in one university to participate. In addition, it is difficult to ascertain the persistence of the post-test measured one week after the end of the program. Therefore, it is necessary to perform repeated studies including expanded subjects in more schools in the future and repeated measurement studies to confirm the persistence of effects.This study developed, implemented, and examined the effects of a character-strength exploration program tailored for nursing students in Korea. The program effectively promoted happiness in nursing students, thereby helping them to enjoy, engage in, and pursue meaningful college lives. Use of this program may benefit other nursing students and ultimately positively affect their competencies as healthcare providers. The results provide significant foundational data for use by universities and medical institutions to develop and implement interventions to increase positivity in nursing education. Further research could explore the duration of the benefits of this program among the participants.


Go/no-go decisions after phase II and sample size chosen for phase III are usually based on phase II results . Due to the decision rule (only promising phase II results lead to phase III), treatment effect estimates from phase II that initiate a phase III trial commonly overestimate the true treatment effect. Underpowered phase III trials are the consequence. Optimistic findings may then not be reproduced, leading to the failure of potentially expensive drug development programs. For some disease areas these failure rates are described to be quite high: 62.5%.We integrate the ideas of multiplicative and additive adjustment of treatment effect estimates after go decisions in a utility-based framework for optimizing drug development programs. The design of a phase II/III program, i.e., the “right amount of adjustment”, the allocation of the resources to phase II and III in terms of sample size, and the rule applied to decide whether to stop or to proceed with phase III influences its success considerably. Given specific drug development program characteristics , optimal designs with respect to the maximal expected utility can be identified by the proposed Bayesian-frequentist approach. The method will be illustrated by application to practical examples characteristic for oncological studies.In general, our results show that the program set-ups with adjusted treatment effect estimate used for phase III planning are superior to the “naïve” program set-ups with respect to the maximal expected utility. Therefore, we recommend considering an adjusted phase II treatment effect estimate for the phase III sample size calculation. However, there is no one-fits-all design.Individual drug development planning for a specific program is necessary to find the optimal design. The optimal choice of the design parameters for a specific drug development program at hand can be found by our user friendly R Shiny application and package (both assessable open-source via [1]). Exploratory studies are usually carried out to provide a basis for deciding whether or not to proceed with a confirmatory trial and, if necessary, to provide information for planning purposes. In drug development programs, this strong link between exploratory  and confirmatory  studies favors integrated planning. In particular, the costs of phase III studies have increased remarkably in recent years , 3, whilAlready over 30 years ago, Hughes and Pocock  pointed Ellenberg et al.  as well In our framework, we follow the concept of “assurance” , 17, whiKirby et al.  and WangDe Martini , 25 repoIn this article, we integrate the general concepts of using a multiplicative or additive adjustment method to correct for overestimation of the treatment effect in a framework of utility-based optimization of phase II/III development programs . That isIn the second section of this paper, we will introduce the basic setting and notation, explain the adjustment methods and show how they are incorporated in our optimization framework. After introducing the utility function and explaining the optimization procedure, we present optimal designs for exemplary settings of drug development programs in Section 3. We finish with a discussion in Section 4 and a conclusion in Section 5.θ =  − log(HR)), which is the ratio of the hazard functions of the treatment and the control group. In order to reflect the uncertainty in the true treatment effect, θ can be modelled by a prior distribution f(θ). In phase II, the total number of events is denoted by d2 and the maximum likelihood estimate of θ is given by κ is a predefined threshold value. In case of a go decision, the number of events for the phase III trial is calculated based on the observed treatment effect of phase II. If the confirmatory analysis in phase III reveals a significant result, program success is declared , performed independently, investigating the same time-to-event primary endpoint and the same population. The true treatment effect is given by the negative logarithm of the true hazard ratio . The inequation holds as κ in the unconditional expectation κ in Due to the decision rule after phase II, the treatment effect estimate of phase II is biased. The bias is positive with Note that the representation of the bias cannot be further simplified, neither by calculating Therefore, in the following, multiplicative and additive adjustment methods for the treatment effect estimate obtained in phase II will be investigated. Afterwards, dependent on the respective adjustment method, launch criteria and approaches to calculate the number of events for phase III will be presented.In this section, we introduce two methods (an additive and a multiplicative adjustment method) to adjust for the overestimation of the phase II treatment effect estimate. It should be mentioned that the terms “multiplicative” and “additive” relate to the specific type of scale and endpoint considered here.αCI ∈  and define the additive adjusted treatment effect estimate by zγ1 −  = Φ−1(1 − γ), where Φ(.) denotes the distribution function of the standard normal distribution. Note that our version of the additive adjusted treatment effect estimate is a generalization of that of Wang et al., as they use the lower limit of the one and two standard deviation two-sided CI  and we allow αCI ranging from 0.025 to 0.5. For αCI = 0.5, the additive adjusted treatment effect estimate is not discounted as Wang et al.  advise tλ, which can be understood as a retention factor, that is, the fraction of the treatment effect retained. Integrated in our setting, we define λ ∈  can be viewed as the result of discounting the observed treatment effect of phase II by 1 − λ. Note that for λ = 1 the multiplicative adjusted treatment effect estimate is not discounted.Kirby et al.  propose s1); 2. when calculating the phase III sample size , exceeds a predefined threshold value κ, it is decided to go to phase III and otherwise to stop the program. Hence, the expected probability to go to phase III can be determined by1.: If the treatment effect estimate s1 = λ, αCI or u  in order to calculate the bias (compare Table A0). The expectation of the estimate (of phase II) used for the sample size calculation can be calculated by Martini , 25, theH0 : θ ≤ 0 is rejected in favour of H1 : θ > 0 at a one-sided significance level α. This is the case if T3 > zα1 − , where T3 is the normalized log-rank test statistic in phase III, which is assumed to be asymptotically normally distributed, i.e., t3 is a realization of T3 > zα1 −  under H0) as program success. We discuss this aspect in detail in Section A1 of Additional file α that it is a false positive decision. Thus, we keep the commonly used term “success” and PsP which should be regarded as probability of market access and not a probability of a correct decision.The program is considered to be successful, if the one-sided null hypothesis t result , 27, cans1) and, in case of a go decision, by the choice of the treatment effect estimate used for the calculation of the number of events for phase III (selection s2).We investigate the impact of using adjusted treatment effect estimates , with two “multiplicatively adjusted” set-ups s1 ∈ {u, λ}, s2 = λ), and two “additively adjusted” set-ups s1 ∈ {u, αCI}, s2 = αCI). Note that if s1 ≠ u, we define s2 = s1, which means that if an adjustment parameter is used for the decision rule, the same adjustment parameter is used for the calculation of the expected number of events for phase III (for reasons which will be given later).Table λ or αCI, the number of events in phase II d2, and the go/no-go decision threshold value κ. Therefore, we set up a utility function, which utilizes the difference between program costs and potential gains after successful market launch  and variable per-patient  costs are included for the phase II and III trial, respectively. By dividing the number of events by the event rate ξi, the total number of patients can be calculated for the respective phase i = 2, 3. Obviously, only in case of a go decision the costs of the phase III trial apply. In case of program success, a benefit b is obtained, and we assume that the level of benefit depends on the observed treatment effect in the phase III trial as suggested by a report of the German Institute for Quality and Efficiency in Health Care  = E[g] − E[c], where the expected costs and gain with respect to θ, T3 are given byThe utility is defined as the difference between costs and gain and expressed as a function of δ =  that maximizes the expected utility E[u] for programs with program set-up d2, κ, and λ in the multiplicatively or αCI in the additively adjusted set-ups, respectively. The optimal design δ∗ for each program set-up D = {δ = } is the optimization set.The aim is to find a design The optimization is solved by using numerical integration procedures written in the programming language R . In ordeIn this paper, the parameters in the oncology trial example are chosen as in Kirchner et al.  to allowξi = 0.7 for i = 2, 3. Therefore, the total sample size can be calculated by di/0.7, i = 2, 3. In practice, estimates on the event rates could be obtained by taking recruitment rates and duration as well as drop-out rates and treatment group specific hazards into account. However, using those parameters often leads to event rates around ξi = 0.7 as it is a compromise between data maturity and avoidance of long follow-up times if drop-out rates are higher than expected. If ξi < 0.5 the median event time might not be observed while if ξi is too high, the planned number of events might not be reached at all with substantial drop-out rates.The event rates for phase II and III are set to $ are reported  ≤ K ,optimal total number of events for phase II κ ∈ D in “HR-scale”, i.e., optimal go/no-go decision rule threshold value s2 ∈ D) for the multiplicative and additive adjustment method, respectively, optimal adjustment parameter The optimization results are presented in Table u∗ = E[u(δ∗)],maximal expected utility β = 0.9 and a one-sided significance level α = 0.025,expected number of events for phase III total number of expected events in the program expected probability to go to phase III expected probability of a successful program expected estimate of phase II used for sample size calculation with corresponding program characteristics for the optimal design:s1 = u or bs 1-7) and weights for the prior distribution of the true underlying effect , where .for program set-ups b1, b2, b3)) lead to more liberal optimal decision rules .Dividing the optimal number of events in phase II by the expected number of events in phase III  compared between the two multiplicatively and the two additively adjusted program set-ups do not vary (much) for each benefit scenario bs and choice of weight for the prior distribution w, respectively. However, there are differences in the optimal choice of the threshold value for the decision rule The operating characteristics for the optimal designs , the assumed benefit, and the development risk  are taken into account. By maximizing the expected utility with respect to the design parameters , optimal phase II/III drug development program designs can be found. Therefore, it enables quantitative reasoning for the design  for specific drug development programs at hand.We investigated two adjustment methods (additive and multiplicative adjustment), several benefit scenarios , different distributions for the true treatment effect (with the same and different distributions in phase II and III), scenarios with a real life budget constraint, scenarios with a predefined clinically relevant effect, and scenarios where phase II could be skipped, hence presented a method for the implementation of a variety of possible oncology drug development program scenarios, and an opportunity for assessing associated changes of the optimal design parameters. Of course, the implementation of alternative  or more complex planning situations and broader application to other research areas are possible by choosing relevant  parameters appropriately –39. As tIn general, our results show that the adjusted program set-ups are superior to the unadjusted program set-up with respect to the maximal expected utility. This is associated with higher investments in terms of number of events and lower expected probabilities to go to phase III in the adjusted program set-ups compared to the unadjusted approach. Thus, in the adjusted program set-ups it is less often decided to go to phase III, but in case of a go decision, the investment in terms of sample size is higher. These aspects are particularly true for the multiplicatively adjusted program set-ups, which have also higher expected probabilities of a successful program compared to the additively adjusted and unadjusted program set-ups. Simply said, the money is spent more wisely when adjustment methods are used.αCI = 0.5 and λ = 1 in the additively and multiplicatively adjusted program set-ups, respectively) were included but never selected in the optimization. Thus, the results suggest that adjustment should always be considered, which is in line with Chuang-Stein and Kirby [Values for the adjustment parameters that do not lead to an adjustment  ranBoth multiplicatively adjusted  adjusted phase II treatment effect estimates for calculation of the phase III number of events in a phase II/III drug development program with go/no-go decision rule  and investigation of an alternative definition of program success is given (A1). Furthermore, more details and results of the application example when modelling different population structures in phase II and III (A2), when using a predefined minimal clinically relevant effect for phase III planning (A3), when using a budget constraint (A4), when skipping phase II (A5) and when using a linear function for modelling the gain (A6) are presented. The file Code.R includes the main function calls for generating the datasets and tables, using the R package drugdevelopR.


Pleurotus ostreatus strain yang1 with high laccase activity. The enzymatic properties of LAC-Yang1 and its ability to degrade and detoxify chlorophenols such as 2,6-dichlorophenol and 2,3,6-trichlorophenol were systematically studied. LAC-Yang1 showed a strong tolerance to extremely acidic conditions and strong stability under strong alkaline conditions (pH 9–12). LAC-Yang1 also exhibited a strong tolerance to different inhibitors , metal ions , and organic solvents . LAC-Yang1 showed good stability in the presence of Mg2+, Mn2+, glycerol, and ethylene glycol. Our results reveal the strong degradation ability of this laccase for high concentrations of chlorophenols  and chlorophenol mixtures . LAC-Yang1 displayed a strong tolerance toward a variety of metal ions  and organic solvents  in its degradation of 2,6-dichlorophenol and 2,3,6-trichlorophenol. The phytotoxicity of 2,6-dichlorophenol treated by LAC-Yang1 was significantly reduced or eliminated. LAC-Yang1 demonstrated a good detoxification effect on 2,6-dichlorophenol while degrading this compound. In conclusion, LAC-Yang1 purified from Pleurotus ostreatus has great application value and potential in environmental biotechnology, especially the efficient degradation and detoxification of chlorophenols.In this study, a laccase LAC-Yang1 was successfully purified from a white-rot fungus strain  Pleurotus ostreatus is a type of edible fungus with great edible and nutritional value. Recently, this fungus has also been studied as source of bioactive proteins, such as a specific ribonuclease (ribotoxin-like protein) able to inhibit protein synthesis in vitro. A novel ribotoxin-like protein named Ostreatin was purified and characterized from P. ostreatus [P. ostreatus is also an important member of white-rot fungus. P. ostreatus and its ligninolytic enzymes also have important application values in the field of the treatment of environmental pollutants and bioremediation [White-rot fungus is a class of filamentous fungi that forms white decay on wood. With the strong ability of lignin decomposition, white-rot fungus has good potential in numerous energy and environmental biotechnology applications, including the development and utilization of renewable resources, and the biodegradation of different xenobiotic compounds ,2. Pleurstreatus . P. ostrediation .Laccase EC 1.10.3.2) is a type of polyphenol oxidase with four copper atoms in its catalytic site. Laccase catalyzes the oxidation of phenols and aromatic compounds, and by transferring four electrons, reduces molecular oxygen to water. The four copper atoms in the active site of laccase combine with oxygen to form a copper superoxide complex and an electron transport chain for redox reactions ,6,7. In .3.2 is aChlorophenols are chlorine-containing aromatic compounds and are widely used in present-day industry as important raw chemical materials. Because of their toxicity, potential carcinogenic and mutagenic effects, and low degradability, which make them persistent organic pollutants, the widespread application of chlorophenol compounds in industrial production and the discharge of industrial wastewater containing these compounds have brought serious environmental pollution problems. Study on the degradation of chlorophenols is thus of importance and value to the management of the ecological environment and human health ,18,19. LAlthough some researchers have previously studied the degradation of different chlorophenols by laccase, some key problems remain to be explored and solved to achieve better application of this enzyme and more efficient degradation of chlorophenols: (1) in an actual polluted environment, chlorophenols are largely found in the industrial waste and industrial wastewater discharge, and other metal ions and organic solvents are usually present at high concentration in the chlorophenol pollutants encountered in real life . What arP. ostreatus strain yang1 with high laccase activity. The enzymatic properties of this laccase and its ability to degrade chlorophenols with different chemical structures  were systematically studied. The tolerance of LAC-Yang1 toward different organic solvents and metal ions in the degradation of 2,6-dichlorophenol  and 2,3,6-trichlorophenol  was emphasized. Detoxification of chlorophenol by LAC-Yang1 was also investigated. LAC-Yang1 has great application value and potential in the degradation and detoxification of chlorophenols.In this study, a laccase LAC-Yang1 was isolated and purified from a white-rot fungus strain 2+ and syringic acid were used as inducers to induce laccase production by the fungal strain in tomato juice medium. As shown in P. ostreatus strain yang1. LAC-Yang1 was purified by precipitation with ammonium sulfate, anion exchange chromatography and hydrophobic interaction chromatography. The summary of the purification process is shown in CuThe purified LAC-Yang1 showed only one band in SDS-PAGE and Native-PAGE D,E. The max = 4.41 × 10−7 mM/s), and the lowest reaction rate was observed on guaiacol substrate. The affinity of LAC-Yang1 for the three substrates was 2,6-DMP > ABTS > guaiacol. The conversion efficiency of LAC-Yang1 was higher on ABTS than on the other two substrates  (2+ inhibited LAC-Yang1 activity. The enzyme activity was reduced to 16.54% by 10 mM concentration of Fe2+ (2+ + Na+ + K+ + Cu2+ + Mg2+)  A–C. Amon of Fe2+ I. In add + Mg2+) M. The abThe effect of 11 organic solvents with different concentrations on the activity of LAC-Yang1 was also studied. As shown in 1/2 of Mg2+ (2 mM) and Mg2+ (5 mM) was 24 h and 12 h, respectively. The values of t1/2 suggest that LAC-Yang1 was more stable in the presence of Mg2+ and Mn2+.The effect of metal ions with different concentrations on the stability of LAC-Yang1 was studied . As show1/2 suggest that LAC-Yang1 showed relatively strong stability in the presence of glycerol and ethylene glycol. t1/2 of glycerol  and glycerol  was 72 h and 24 h, respectively. Among the 11 organic solvents studied, LAC-Yang1 was most stable in the presence of glycerol  by LAC-Yang1 was shown in The above results show the good degradation effect of LAC-Yang1 on 2,6-DCP and 2,3,6-TCP. On this basis, metal ions of various concentrations were added to the degradation mixture to investigate their effect on 2,6-DCP and 2,3,6-TCP degradation by LAC-Yang1.+, Zn2+, Mn2+, Mg2+, K+ in the degradation of 2,6-DCP. Various metal ions such as Na+, Zn2+, Mn2+, Mg2+, K+ in the concentration range of 10–200 mM had no obvious effect on the degradation of 2,6-DCP by LAC-Yang1 . As shown in 2+, Al3+, Li+, Co2+, Cd2+ and Ni2+ had a strong inhibitory effect on the degradation of 2,6-DCP by LAC-Yang1. Fe2+ exerted even stronger inhibition on the degradation .The effects of different concentrations of metal ions on the degradation of 2,6-DCP by LAC-Yang1 were shown in AC-Yang1 A–E. LAC-2+, Mn2+, Na+, Zn2+, K+ in its degradation of 2,3,6-TCP, though at a significantly weaker level as compared to 2,6-DCP degradation. Mg2+, Mn2+, Na+, Zn2+, K+ impacted the LAC-Yang1 degradation of 2,3,6-TCP to varying extent.The effects of different concentrations of metal ions on the degradation of 2,3,6-TCP by LAC-Yang1 were shown in The effect of metal ion mixtures on the degradation of 2,6-DCP and 2,3,6-TCP by LAC-Yang1 was further studied. As shown in Following the addition of different concentrations of organic solvents to the degradation mixture, the effects of 11 organic solvents on the degradation of 2,6-DCP and 2,3,6-TCP by LAC-Yang1 were studied.v/v), the percent degradation of 2,6-DCP could still reach 34.90% and 29.44%, respectively  were measured   E,F.Triticum aestivum seed and Mung bean seed were shown in Triticum aestivum seed, there was no significant difference in the length of shoot and root between the group treated with degraded 2,6-DCP (200 mg/L) and the control group . LAC-Yang1 degradation could completely eliminate the toxic effect of 2,6-DCP (200 mg/L) on wheat seed  on Mung bean seed  effect of temperature on the activity of laccase, (2) effect of pH on the activity and stability of laccase, (3) effect of inhibitors on the activity of laccase, (4) effect of different metal ions on the activity of laccase, (5) effect of different organic solvents on the activity of laccase, and (6) effect of metal ions and organic solvents on the stability of laccase.In this study, the laccase LAC-Yang1 was purified from P. ostreatus strain 10969, P. ostreatus IBL-02, P. ostreatus UAMH7992, P. ostreatus UAMH7988, P. ostreatus IE-8, P. ostreatus strain Florida were reduced to a very low level or were completely inhibited when the temperature exceeded 60 °C [Pleurotus nebrodensis [Pleurotus [A comparison of the effect of temperature, pH, EDTA and metal ions on the activity of LAC-Yang1 and other reported laccases is summarized in ed 60 °C ,32,33,34ed 60 °C ,32,33,34rodensis . In summleurotus , LAC-YanPleurotus. As shown in P. ostreatus strain 10969 [P. ostreatus 7992 [P. ostreatus 7972 [P. ostreatus 7980 [P. ostreatus strain 10969, P. ostreatus 7992, P. ostreatus 7972, and P. ostreatus 7980 reported previously [In terms of the influence of pH on laccase activity, differences were observed between LAC-Yang1 and other reported laccases derived from the genus in 10969 , P. ostrtus 7992 , P. ostrtus 7972 , and P. tus 7980  were vereviously ,33.Pleurotus, most of the previous research was performed at pH 1–7 [P. ostreatus var florida was very stable at alkaline pH. t1/2 of POXA1b at pH 9.0 was 30 days [P. ostreatus K16-2 under the alkaline condition of pH 9.0–11.0. The residual activity of the laccase from P. ostreatus K16-2 was about 90% after incubation at pH 9.0 for 20 h. However, the previous study only detected residual laccase activity after 20 h of incubation at pH 9.0, 10.0, and 11.0 and no stability after a longer duration of incubation (such as 24 h or more) under strong alkaline conditions. The stability of the laccase from P. ostreatus K16-2 after incubation at pH 9.0–11.0 for a longer time (such as 72 h and 7 days) was not studied in the previous research [The stability of LAC-Yang1 at different pH values was compared with other laccases reported previously. Although there have been some reports on the influence of pH on the stability of laccase from the genus t pH 1–7 ,34,35, w 30 days . Okamotoresearch . Our resresearch , the staTrametes polyzona [P. ostreatus K16-2 [P. ostreatus strain 10969 [In terms of the effect of inhibitors on laccase activity, LAC-Yang1 showed strong resistance to some enzyme inhibitors such as EDTA and SDS, which was the significant advantage of this laccase. Comparison of the effect of EDTA on the activity of LAC-Yang1 and other reported laccases was summarized in polyzona , P. ostrus K16-2 , and P. in 10969 . The resin 10969 ,37,38.2+, Mn2+, Zn2+, and Cu2+ than the laccase from P. ostreatus strain 10969 [Trametes polyzona KU-RNW027 [+ and Co2+. Taken together, compared with other laccases reported previously [2+, Mn2+, Zn2+, Cu2+, Na+, and Co2+. LAC-Yang1 had good broad-spectrum tolerance to various metal ions.In terms of the influence of different metal ions on laccase activity, we also compared the metal-ion tolerance of LAC-Yang1 with that of other laccases reported previously. As shown in in 10969 . CompareU-RNW027 , LAC-Yaneviously ,38, LAC-While many studies have examined the effect of metal ions on laccase activity, fewer studies have examined the effect of organic solvents on laccase activity. However, previous research did not examine the effects of glycerol, propylene glycol, ethylene glycol, and butanediol on laccase activity ,33,38,39P. ostreatus strain 10969, Trametes polyzona KU-RNW027, Pycnoporus sp. SYBC-L10, Trametes maxima IIPLC-32, P. ostreatus 7988, and P. ostreatus 7980 was not investigated in previous research [2+, Mn2+, glycerol and ethylene glycol. Such findings were not reported in previous research [To our knowledge, there are few reports on laccase stability in the presence of different metal ions and organic solvents. The effect of different metal ions and organic solvents on the stability of laccase from research ,37,38,39research ,37,38,39research ,41. Prevresearch ,43. Glycresearch . Magsumoresearch . Therefo2+, Mn2+, Zn2+, Cu2+, Na+, and Co2+ than some other laccases reported previously. (5) LAC-Yang1 had strong tolerance toward some organic solvents such as glycerol and propylene glycol. LAC-Yang1 was most resistant to glycerol. (6) LAC-Yang1 showed good stability in the presence of Mg2+, Mn2+, glycerol and ethylene glycol. The above characteristics and advantages of LAC-Yang1 give this enzyme great value and potential in industrial applications.In summary, compared to other laccases reported previously, LAC-Yang1 has the following novelty and advantages in terms of enzymatic properties: (1) LAC-Yang1 displayed greater tolerance to high-temperature conditions than some other laccases. LAC-Yang1 also remained highly active at low temperature. The high activity level of LAC-Yang1 could be maintained across a wide range of temperatures (10–85 °C). (2) LAC-Yang1 showed a strong tolerance to extremely acidic conditions and strong stability under strong alkaline conditions. (3) LAC-Yang1 displayed stronger tolerance and resistance to different inhibitors such as EDTA and SDS. The resistance of LAC-Yang1 to EDTA was significantly stronger than that of some other laccases reported previously. (4) LAC-Yang1 exhibited stronger tolerance to different metal ions such as MgChlorophenols are chlorine-containing aromatic compounds which can be directly used in modern industry. Chlorophenols are widely used as insecticides, herbicides, wood preservatives and acaricides. In addition, chlorophenols are widely used in the production of various chemical products as important raw chemical materials. 2,6-dichlorophenol is one of the important raw materials for the synthesis of non-steroidal antipyretic analgesics and herbicides. Chlorophenol is also a key intermediate compound in the industrial production of pesticides, dyes, phenolic resins and paper . The extIn this study, LAC-Yang1 had a strong ability to degrade and detoxify chlorophenols. Compared to the previous studies on the degradation of chlorophenol by other laccases ,26,27,28Because of its low substrate specificity, a wide range of substrates and unique degradation function, laccase is widely used in the fields of industrial and environmental biotechnology. Our study suggests that LAC-Yang1 laccase showed strong ability to degrade and detoxify chlorophenols. Compared to other laccases reported previously, LAC-Yang1 had unique and novel characteristics in terms of enzymatic properties. Therefore, LAC-Yang1 has a good application prospect and value in the field of environmental biotechnology and bioremediation, such as the efficient degradation of persistent organic pollutants that are difficult to degrade. The perspectives of use of LAC-Yang1 laccase are described as follows. In the area of environmental pollutant degradation, LAC-Yang1 laccase can be used in the following aspects: (1) Wastewater polluted by chlorophenols usually contains a large number of metal ions and organic solvents besides chlorophenol. In the treatment of chlorophenol wastewater from different sources and with complex composition, these metal ions and organic solvents may affect the ability of laccase to degrade chlorophenols. Our study indicates that LAC-Yang1 had strong ability to degrade high concentrations of chlorophenols and chlorophenol mixtures. LAC-Yang1 also showed a strong tolerance toward a variety of metal ions and organic solvents in its degradation of chlorophenols. Therefore, LAC-Yang1 can be used in the treatment of actual industrial wastewater containing high concentrations of chlorophenols in the future. LAC-Yang1 can be practically applied to the effective treatment and degradation of chlorophenol-polluted wastewater containing different metal salts and organic solvents. LAC-Yang1 laccase has great potential to achieve efficient degradation and detoxification of chlorophenol pollutants in the actual polluted environment . (2) LAC-Yang1 laccase may be used for the degradation and detoxification of other environmental organic pollutants except chlorophenols, such as polycyclic aromatic hydrocarbons , industrial dyes , dyeing wastewater, environmental endocrine disruptors, and pesticides. Our other study suggested that LAC-Yang1 had a strong ability to degrade polycyclic aromatic hydrocarbons such as fluorene. (3) In order to better improve the reusability and stability of LAC-Yang1 in the degradation of chlorophenol pollutants, laccase needs to be immobilized. Immobilized laccase can often achieve good reusability. Therefore, we will further study the degradation and detoxification of different chlorophenols and industrial wastewater containing chlorophenols by immobilized laccase. LAC-Yang1 laccase can be immobilized on different media, and the reusability of immobilized LAC-Yang1 in the degradation of chlorophenols may be greatly improved.P. ostreatus strain yang1 was preserved in Central China Normal University, Wuhan, China. The tomato juice medium was used for the production of laccase. After incubation at 28 °C in a shaking incubator (180 rpm) for 3 days, syringic acid and Cu2+ were added to the culture. After addition of the inducers, the fungal cultures were then grown at 28 °C with continuous shaking at 180 rpm. Laccase activity was measured daily. The fungal cultures were harvested at the peak of laccase activity. The laccase activity was measured as described previously using 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate [ubstrate .g for 20 min. The culture supernatant was precipitated by (NH4)2SO4 (80% saturation) at 4 °C overnight. The precipitates were dissolved in 20 mmol/L citric acid-disodium hydrogen phosphate dodecahydrate buffer  and dialyzed in a dialysis bag  for 48 h to remove ammonium sulfate. The dialyzed crude enzyme was then concentrated using PEG20,000, filtered, and applied to the DEAE Sepharose anion-exchange chromatography column equilibrated with 20 mmol/L CPBS (pH 6.5). The laccase protein was eluted with a linear gradient of 0–1 mol/L NaCl in the 20 mmol/L CPBS (pH 6.5) at a 1 mL/min flow rate. Proteins in the eluted fractions were monitored by measuring the absorbance at 280 nm. The activity of laccase was measured. The active fractions were collected and dialyzed by 20 mM CPBS (pH 6.5) for removing NaCl. The dialyzed enzyme fractions were subsequently applied to the Phenyl-6FF (HS) hydrophobic chromatography column equilibrated with 20 mM CPBS (pH 6.5) containing 1 mol/L (NH4)2SO4. The laccase protein was eluted with a linear gradient of 1–0 mol/L (NH4)2SO4 in the same buffer at a 1 mL/min flow rate. Fractions possessing laccase activity were collected, dialyzed, concentrated and stored at 4 °C. The purity of the purified laccase was detected by SDS-PAGE which contained a 5% polyacrylamide-stacking gel and a 12% polyacrylamide-separating gel. The purified laccase was named as LAC-Yang1. Native-PAGE (non-denaturing polyacrylamide gel electrophoresis) was also used to detect LAC-Yang1 laccase. The main step of Native-PAGE is described as follows. (1) Preparation of protein electrophoresis gel (gel preparation): The gel for Native-PAGE contained a 5% polyacrylamide-stacking gel and a 12% polyacrylamide-separating gel. Different from SDS-PAGE, 10% SDS was not added in the preparation of gel for Native-PAGE. (2) Sample loading and electrophoresis: The original activity of the protein needs to be maintained in Native-PAGE. Therefore, the protein sample was not subjected to denaturation treatment such as boiling, and the sample was directly mixed with the non-denatured protein loading buffer and loaded. (3) Stain of gel (colouration): The laccase substrate 2,6-Dimethylphenol  was used as the staining agent to detect the active laccase. After electrophoresis, the protein gel was peeled off and washed twice with distilled water. Then, the gel was stained with the acetic acid-sodium acetic buffer (pH 5.0) containing 1.0 mM 2,6-DMP. The laccase protein with laccase activity could oxidize 2,6-DMP and show a brownish-yellow band on the protein band. The active laccase protein could react specifically with 2,6-DMP and show a brownish-yellow band on the protein band.Fungal cultures at the peak of laccase activity were collected and centrifuged at 5000× Kinetic studies were performed in 50 mM acetic acid-sodium acetic buffer (pH 5.0) at 30 °C. ABTS and 2,6-DMP  and guaiacol  were used as substrates. The absorbance at 420 nm, 470 nm and 465 nm was measured respectively. The amounts of laccase used for each substrate were the same (0.01 U/mL). The kinetic parameters of LAC-Yang1 were calculated by non-linear regression model based on the Michaelis–Menten equation.The effect of different pH on the activity of LAC-Yang1 was determined in citric acid-disodium hydrogen phosphate dodecahydrate buffer (50 mM) within a pH range of 1.0–8.0 at 30 °C using ABTS (1 mM) as the substrate. The amount of the laccase was 0.01 U/mL. The laccase activity measured at the optimal pH was set as 100%.The effect of different temperatures on the activity of LAC-Yang1 was determined in acetic acid-sodium acetic buffer  within a temperature range of 10–85 °C using ABTS (1 mM) as the substrate. The amount of the laccase was 0.01 U/mL. The laccase activity measured at the optimal temperature was set as 100%.To evaluate the pH stability, the purified LAC-Yang1 was incubated at different pH (1–12) at 30 °C for 3, 6, 12, 72 and 168 h, respectively. The buffer used was citric acid-disodium hydrogen phosphate dodecahydrate buffer (50 mM). The amount of the laccase was 0.1 U/mL. The laccase activity was measured at different time points using ABTS (1 mM) as the substrate. Then, the residual laccase activity was calculated based on the original activity before incubation. The initial activity of LAC-Yang1 was set as 100%.To evaluate the thermal stability, the purified LAC-Yang1 was incubated at 30–70 °C for 1, 2, 3, 6, 9, 12 and 24 h, respectively. The buffer used was acetic acid–sodium acetic buffer (50 mM). The amount of the laccase was 0.1 U/mL. The laccase activity was measured at different time points using ABTS (1 mM) as the substrate. Then, the residual laccase activity was calculated based on the original activity before incubation. The initial activity of LAC-Yang1 was set as 100%.The effects of different concentrations of EDTA-2Na , SDS , mercaptoethanol , sodium azide , and DTT  on the laccase activity were measured in 50 mM acetic acid–sodium acetic buffer (pH 5.0) containing different inhibitors at 30 °C. The amount of the laccase was 0.01 U/mL. An amount of 1 mM ABTS was used as the substrate. The residual activity was calculated based on the control in the absence of any inhibitor (set as 100%).+, Na+, Mg2+, Mn2+, Cu2+, Zn2+, Al3+, Fe2+, Cd2+, Co2+, Li+, Ni2+  on the laccase activity of LAC-Yang1 were determined by measuring the residual activity relative to the control without adding any metal compound. The laccase activity of the control was set as 100%. The buffer used to evaluate the effect of metal ions on the activity of laccase was 50 mM acetic acid–sodium acetic buffer (pH 5.0). The amount of the laccase was 0.01 U/mL. 1 mM ABTS was used as the substrate for measuring the laccase activity.The effects of different metal ions including KTo evaluate the effect of different metal ions on the stability of LAC-Yang1, the purified LAC-Yang1 was incubated with different concentrations of metal ions  at 30 °C for 7 days. Then the laccase activity was measured at different time points . The residual activity was calculated based on the original activity before incubation. The initial activity of LAC-Yang1 was set as 100%. The buffer used to evaluate the effect of metal ions on the stability of laccase was 50 mM acetic acid–sodium acetic buffer (pH 5.0). The amount of the laccase was 0.1 U/mL. 1 mM ABTS was used as the substrate for measuring the laccase activity.v/v) on the laccase activity of LAC-Yang1 were determined by measuring the residual activity relative to the control without adding any organic solvent. The laccase activity of the control was set as 100%. The buffer used to evaluate the effect of organic solvents on the activity of laccase was 50 mM acetic acid–sodium acetic buffer (pH 5.0). The amount of the laccase was 0.01 U/mL. 1 mM ABTS was used as the substrate for measuring the laccase activity.The effects of different organic solvents including methanol, ethanol, ethylene glycol, propylene glycol, glycerol, isopropanol, butanediol, acetonitrile, acetone, DMSO, DMF  at 30 °C for 7 days. Then, the laccase activity was measured at different time points . The residual activity was calculated based on the original activity before incubation. The initial activity of LAC-Yang1 was set as 100%. The buffer used to evaluate the effect of organic solvents on the stability of laccase was 50 mM acetic acid-sodium acetic buffer (pH 5.0). The amount of the laccase was 0.1 U/mL. 1 mM ABTS was used as the substrate for measuring the laccase activity.To evaluate the effect of different organic solvents on the stability of LAC-Yang1, the purified LAC-Yang1 was incubated with different concentrations of organic solvents : 25–2000 mg/L chlorophenol , 50 mM acetic acid-sodium acetic buffer (pH 5.0), purified LAC-Yang1 . The total volume of the reaction mixture was 2 mL. Degradation was performed at 30 °C for 12 h. The reaction mixture was then extracted with an equal volume of ethyl acetate. A Shimadzu LC-20A HPLC  equipped with a diode array detector was used to measure the concentration of 2,6-dichlorophenol and 2,3,6-trichlorophenol. The chromatographic grade methanol was used as the mobile phase. An amount of 20 µL of each sample was injected into the HPLC. Chromatographic separation was performed using a WondaSil C18 column (250 mm × 4.6 mm × 5 µm) at a flow rate of 1.0 mL/min at room temperature. The method used for elution was linear gradient elution (60–90% methanol). 2,6-dichlorophenol and 2,3,6-trichlorophenol were detected at 215 nm.0 − Ct)/C0 × 100, where C0 was the initial concentration of chlorophenol before degradation and Ct was the residual concentration at time t.The degradation efficiency of chlorophenol was expressed as a degradation percentage (%) which was calculated by the following formula: Degradation efficiency (%) =  by LAC-Yang1 was also performed as described above. 2,6-dichlorophenol and 2,3,6-trichlorophenol were added into the reaction mixture together .v/v). Degradation was performed at 30 °C for 12 h. The reaction mixture without adding metal ions and organic solvents was set as the control. Degradation was monitored and calculated as described above.To evaluate the effects of metal ions and organic solvents on the degradation of chlorophenol by LAC-Yang1, different concentrations of metal ions and organic solvents were added into the reaction mixture, respectively. The reaction mixture contained : 50 mg/L 2,6-dichlorophenol or 2,3,6-trichlorophenol, 50 mM acetic acid-sodium acetic buffer (pH 5.0), purified LAC-Yang1 (the amount of the laccase was 1 U/mL), different metal ions (10 mM–800 mM) or different organic solvents  were measured according to the method described before [Oryza sativa, Triticum aestivum and Mung bean (20 seeds of each) by watering separately 10 mL of original 2,6-dichlorophenol  and 2,6-dichlorophenol treated by LAC-Yang1. Plant seeds were also treated with distilled water at the same time as the control. The shoot and root lengths of Oryza sativa, Triticum aestivum, Mung bean treated under different conditions were measured after 5 days, 3 days, 2 days, respectively.The phytotoxicity of 2,6-dichlorophenol before and after its degradation was evaluated by plant seed germination and growth. The toxic effects of different concentrations of 2,6-dichlorophenol (not treated or treated by LAC-Yang1) on the germination of three types of plant seeds . p-value < 0.05 indicated significant difference (presented as *).The data were presented as mean ± standard error. Statistical analyses were performed using GraphPad Prism 5 software . A P. ostreatus strain yang1 with high laccase activity. The enzymatic properties of LAC-Yang1 and its ability to degrade and detoxify chlorophenols were systematically studied. LAC-Yang1 possesses unique and novel characteristics in terms of enzymatic properties as well as chlorophenol degradation and detoxification compared to other laccases from other fungi reported previously. LAC-Yang1 showed a strong tolerance to extremely acidic conditions and strong stability under strong alkaline conditions. LAC-Yang1 exhibited strong tolerance to different inhibitors, metal ions and organic solvents. LAC-Yang1 also had a good stability in the presence of some metal ions and organic solvents. Our results show the strong degradation ability of LAC-Yang1 for 2,6-dichlorophenol, 2,3,6-trichlorophenol and chlorophenol mixtures . Our study provides new laccase resources for the efficient degradation and detoxification of chlorophenol pollutants.A laccase LAC-Yang1 was successfully purified from a white-rot fungus strain


Pklr and Acyl expression. It has recently been demonstrated that ChREBP plays a role in the conversion of gut microbiota-derived acetate to acetyl CoA by activating its target gene, Acss2, in the liver. ChREBP regulates fatty acid synthesis, elongation, and desaturation by inducing Acc1 and Fasn, elongation of long-chain fatty acids family member 6 (encoded by Elovl6), and Scd1 expression, respectively. ChREBP also regulates the formation of very low-density lipoprotein by inducing the expression of Mtp. Furthermore, it plays a crucial role in peripheral lipid metabolism by inducing Fgf21 expression, as well as that of Angptl3 and Angptl8, which are known to reduce peripheral lipoprotein lipase activity. In addition, ChREBP is involved in the production of palmitic-acid-5-hydroxystearic-acid, which increases insulin sensitivity in adipose tissue. Curiously, ChREBP is indirectly involved in fatty acid β-oxidation and subsequent ketogenesis. Thus, ChREBP regulates whole-body lipid metabolism by controlling the transcription of lipogenic enzymes and liver-derived cytokines.Carbohydrate response element-binding protein (ChREBP) plays an important role in the development of type 2 diabetes, dyslipidemia, and non-alcoholic fatty liver disease, as well as tumorigenesis. ChREBP is highly expressed in lipogenic organs, such as liver, intestine, and adipose tissue, in which it regulates the production of acetyl CoA from glucose by inducing  Dietary carbohydrate is metabolized to acetyl CoA, which is a key intermediate in lipid metabolism (Pklr), which is activated by ChREBP and converts phosphoenolpyruvate to pyruvate (Acly) (Acc1) and fatty acid synthase (encoded by Fasn).Excess carbohydrate intake causes hepatic triglyceride accumulation through the activation of carbohydrate response element binding protein (ChREBP) and tabolism . Acetyl tabolism . In the tabolism , 2. In ctabolism , 2. Acettabolism Figure 1Pklr  plays a pivotal role in the pathogenesis of metabolic diseases and tumors –6. ChREBand Fasn , 8, and ic genes –12. Studtabolism –16. Intetl genes , 17–23. tl genes –28. In amulation . ChREBP mulation –32. ThisAcly is expressed mainly in the liver and white adipose tissue and its expression is upregulated at the transcriptional level by glucose-activated ChREBP and the insulin-induced transcription factor sterol response element-binding protein-1c (SREBP-1c)  , 33, 34.REBP-1c) , 36. In REBP-1c) –39  and Elovl6, both of which are required for the synthesis of monounsaturated fatty acids (MUFAs), while SREBP-1c induces Elovl2, fatty acid desaturase 1, and fatty acid desaturase 2, which are required for the synthesis of polyunsaturated fatty acids (PUFAs), as well as Scd-1 and Elovl6 (Chrebp overexpression reduces the concentrations of saturated fatty acids (SFAs), such as palmitic acid (C16:0) and stearic acid (C18:0); and increases that of the MUFA oleic acid (C18:1 n-9)  particles to be delivered to the periphery . These petyl CoA , 34, 51.d Elovl6 Figure 2d Elovl6 –56. Cons8:1 n-9) , 57. Fur8:1 n-9) . Importa8:1 n-9) , 60. Mor8:1 n-9) . Thus, bvia regulation of its nucleocytoplasmic translocation  inhibitor -mediated ChREBP O-GlcNacylation, which enhances ChREBP DNA-binding and protein stability by reducing ubiquitin-mediated degradation –68. Recehylation . H3K4 mehylation Figure 1Chrebp knockout mice -triglyceride, as well as the number of VLDL particles, are lower in out mice , 58, 76.out mice . Furtherinvolved . Hepatictrations . The heprol mice . Becauseycerides , it has ntration , 32, 57.tabolism Figure 3Chrebp knockout mice have lower PAHSA concentrations in their adipose depots  are a recently discovered class of endogenous lipids that have anti-diabetic and anti-inflammatory properties –83. FAHFe depots . RecentlChrebp overexpression reduces plasma OHBA concentration and increases free fatty acid concentrations  or acetoacetyl CoA through ketogenesis. Although ChREBP is activated by feeding, it is also involved in β-oxidation and ketogenesis in the fasting state , 32, 76,ferase-1 . Hepatic oxidase . Therefoogenesis , 58, 76.f21 gene . Althougf21 gene , these rvia acetyl-CoA produced by the conventional pathway (glycolysis and fructolysis) and a novel pathway (conversion of gut microbiota-derived acetate). It also regulates lipoprotein metabolism in the liver and PAHSA production in adipose tissue. Furthermore, ChREBP regulates β-oxidation and ketogenesis in concert with PPARα. However, despite remarkable progress in understanding of the roles of ChREBP in these processes, a number of questions remain regarding its relationship with lipid metabolism. First, to what extent does gut microbiota-derived acetate induce hepatic triglyceride accumulation? Although ChREBP regulates Acss2 mRNA, does acetate itself affect ChREBP transcriptional activity? Second, the role of ChREBP in cholesterol metabolism remains to be determined. Chrebp knockout mice have lower plasma concentrations of cholesterol, despite normal cholesterol synthesis in their livers, and the low plasma cholesterol concentrations may affect physiological processes, such as steroidogenesis in the adrenal gland. Third, the physiological and pathophysiological roles of ChREBP in the fasting state require more in-depth investigation. Because ChREBP is normally activated by glucose, it is conceivable that it regulates post-prandial lipid metabolism. However, it remains unclear why the ability of ChREBP to regulate β-oxidation and ketogenesis evolved. Finally, it remains unclear whether ChREBP suppression modifies the microbiota and lipid metabolism in humans. As discussed above, Chrebp knockout mice have an altered gut microbiota due to impaired fructose absorption from the gut  have made a substantial, direct intellectual contribution to the work, and approved it for publication.This work was supported by grants from Japan Society for the Promotion of Sciences (JSPS) [KAKENHI Grant 17K00850 and 20K11645 (to K. I.) and 17K09825 (to D.Y.)].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.