Performance management systems have been introduced in health and social services institutions to improve organizational performance, supporting the emergence of new management behaviors that are more rooted in collaborative management practices. This study aims to understand how different leadership styles emerge through the implementation of a performance management system and its related tools, and how these can foster distributed leadership.Over two years, the implementation of an integrated performance management system supporting the integration of social services for children, youth, and families was studied at a recently merged Canadian healthcare organization. Qualitative analysis of data collected from 15 interviews, 3 focus groups, and over 350 h of non-participant observation was conducted.The results show that leadership evolved to adapt to the context of organizational integration and was no longer confined to a single manager. Transformational leadership was needed to encourage the emergence of a new integrated performance management system and new behaviors among middle managers and team members. Transactional leadership was legitimized through the use of a status sheet when the integration project did not deliver the expected results. Both transformational and transactional leadership paved the way to distributed leadership, which in turn promoted collaborative practices associated with activities in control rooms and dialogue stemming from the status sheets. Distributed leadership among team members made a difference in the outcome of the integration project, which became a driver of collaboration.The integrated performance management system and the use of its tools can help renew leadership in health and social service organizations. The results lend credence to the importance of distributed leadership in promoting collaborative practices to improve services for children, youth, and families. The results also highlight how various leadership styles can contribute to the emergence of distributed leadership over time. In recent decades, health and social service organizations have attempted to implement various initiatives to improve performance , particuA condition for successful service integration is collaboration, which is often presented as a tool to find new solutions to the complex problems of the fragmentation of care and services  and to rOn April 1, 2015, the province of Quebec, Canada, restructured its health and social services network by reducing its number of institutions from 182 to 34. These new institutions now group the facilities and services for the population of its region. The initial goal of the reform was to reduce bureaucracy by flattening organizational structures from three to two levels and centralizing powers with the Minister of health and social services. This flattening of the management hierarchy resulted in the elimination of 1300 managerial positions.The transformation of the health and social services network is supported by the deployment of a new performance management system that encourages stakeholders to reflect on the evolving role of managers. An IPMS encourages traditional models, in which leadership resides in a single individual, to evolve into leadership development efforts that extend to all levels of the organization. Transformational leadership and transactional leadership are powerful concepts for improving organizational performance , 18. HowThis paper studies how different leadership styles evolve during the deployment of an IPMS. Through a longitudinal case study, it aims to demonstrate how the adaptation of different leadership styles in conjunction with IPMS tools can support the integration of social services for children, youth, and families. The following section draws from a literature review to summarize the main leadership typologies and presents the integrated performance management system and the various tools used by the organization. This is followed by a presentation of the organizational context and the research methodology. The results reveal a multi-dimensional experience of how leadership styles evolved over more than two years. The paper concludes with a discussion of the results, the implications, and future avenues of research.Over the last forty years, the literature on leadership has evolved from the “Great Person Theory” into how leaders behave and develop relationships with followers . AccordiLaissez-faire leadership is a passive form of leadership, where leaders provide little to no feedback which can often leave employees in the dark. This is usually detrimental to employee engagement. This type of leader is nowhere to be seen when problems arise and is not very engaged with his or her employees. These leaders seek a hands-off approach to management .Transactional leaders focus on the resources they manage. They also employ management by exception either in an active way (trying to prevent problems) or a passive way (only intervening when a problem occurs) . Transac and care  about each employee, who can articulate a vision that inspires employees, and foster the acceptance of group goals  when the managers signed the collaboration contract at the beginning of the project  that’s when people really became engaged.”To reinforce collaboration between directors and middle managers, a strategic A3From the strategic A3 report, four other A3 projects were developed using rapid improvement events and were named Charlotte, Henriette, Rosalie, and Juliette. These activities required more tactical and operational approaches, and the goal was to develop an action plan to help integrate services in the desired trajectory. To monitor these A3s, operational control rooms were quickly deployed in the integrated youth teams, where employees, directors, and middle managers were brought together every week to set targets for the service integration project.“During the summer  I had a team member take over to get us to the point that the room was part of our team […] and all employees had taken ownership of it.”This reality was further unpacked by the following statement from a staff member:“Since we became an integrated team, it has been much easier for us to work together and in proximity to each other. It’s been really positive.”The operational control rooms of the new integrated youth teams provided part of the foundation for the emergence of distributed leadership, by democratizing management activities, i.e. team members take turns leading the activity, and this task is no longer under the purview of the manager. As one middle manager explained about the team’s ownership and accountability:“We dramatically reduced the wait time between assessment and the start of case management.”From the start of activities in the operational control rooms, the participants submitted many improvement initiatives , which dInitial staff engagement in the integration project appeared good, especially following efforts made to urgently remove daily irritants that had persisted for many years. However, once these irritants had been dealt with, improvement initiatives became increasingly rare or addressed routine issues, such as poorly soundproofed consultation rooms. One middle manager said, “These are minor annoyances.” After one year, no improvements had been made on the key performance indicators, which indicated that problem-solving to find solutions to trajectory-related challenges was unfortunately not taking place.“I think there are challenges in making the numbers reflect the living reality of the service offer.”The project encountered major difficulties during the first year not so much due to the project managers’ commitment and belief in the project’s rationale but because of how performance indicators were monitored. According to one middle manager, performance measurement was one of the main reasons for low levels of engagement from staff, who felt that quantitative evaluation did not properly represent the fundamentally human dimension of their jobs:“Take away the statistics, performance, and everything. They are completely useless. It just puts pressure on staff.”Observations from the research showed a laissez-faire leadership style on the part of some directors who experienced difficulties engaging their teams in measuring indicators. Comments from one staff member speak to this problem:The action plans for the A3 projects, therefore, remained conceptual and were not conducive to quick action. An informal status quo bogged down the project in a kind of non-performance, and the service trajectory was not monitored at all. At this point, it was observed that some leaders with formal power over the team were negatively influencing the project.“The meeting was key for the project. It gave a glimmer of hope [for us to] work more as a team. We had to look at what wasn’t working.”After observing the directors’ laissez-faire style, the CEO and her associate (COO) organized a project review day in June of the following year. They started the meeting by sharing their disappointments and communicating their reservations about any real progress having been made with the service integration project with the various middle managers involved since the project started in June 2017. The integration project was not bearing the expected fruits. The directors and middle managers found it difficult to be confronted and questioned about why collaborative spaces were being hindered. However, as one middle manager expressed:Aware that the philosophy of the IPMS reinforces a bottom-up approach to problem-solving to help staff identify problems and find the right solutions, the CEO set up a status sheet so that everyone could become better aware of the trajectory and could more effectively document it. Every week, using the status sheet, a performance review was conducted by monitoring the effectiveness of the interfaces of the trajectory through accessibility and continuity indicators. This action by the CEO embodied a more directive transactional leadership approach aimed at getting the project back on track. At first, middle managers were reluctant to adopt this practice with their teams because they felt obligated to apply top-down decisions. However, it proved to be a turning point toward the success of the integration project. As one middle manager explained:“I feel like the status sheet is what changed things.”The inevitable centralization of leadership toward the executives led to a genuine renewal in the deployment of the IPMS so that the trajectory targets could be achieved. Furthermore, the difficulties encountered during the first year of the integration project and the subsequent changes implemented by the CEO contributed to the departure of two key opinion leaders (directors) who did not agree with the organizational priorities expressed by the executives.Status sheet sessions were held weekly during a Friday morning conference call attended by all middle managers. The observations suggest that transactional leadership was an excellent vehicle for leading the status sheet, as this tool is task and results-oriented. The sessions began with a review of indicators related to accessibility, continuity, and quality so that everyone could quickly detect any deviations from the standard. Leaders could focus on generating possible solutions from an employee in order to have corrective action taken. However, the leader (director) could also help collaboration emerge during the second part of the status sheet, which focused on qualitative aspects, particularly the perceptions of the team climate, risk management, and comments received by users.“The other thing I really like is how good performance is recognized. This is very positive because we never used to take the time to do this every week. It happened maybe once or twice a year. We definitely point it out and we have peers recognize each other. Peer recognition increases team spirit.”Without calling it a recognition program as such, team members’ positive performance was highlighted by their peers every week both during the control room activities and during the status sheet sessions. The purpose of this recognition was to improve the working climate so that each team member felt valued, which was appreciated by both middle managers and employees:After six months of using the status sheet in conjunction with the control rooms, significant improvements to accessibility and continuity had been achieved. Initially, the average wait time in child protection was 16.96 days, compared to the government target of 12 days. The achieved result was 8.35 days. Service intensity at the start of the project was 0.96 interventions per day per worker and improved to 3.6 interventions per day per worker. Significant gains were also achieved regarding the implementation of intervention plans, which rose from 70% to the government target of 85% and from 10 to 43% in statutory and voluntary contexts respectively. Furthermore, the disability insurance rate decreased to 8.70% from its initial level of 14.30%. The target was 6.03% at the time.“We’ve got a director of youth protection and a director of youth programs working side by side, and that’s a big win.”As mentioned previously, two directors left and were replaced during the first year of the integration project. This change in top management for the youth programs led to a new dynamic that supported greater interprofessional collaboration, particularly in the control room. After this change, leadership started to be distributed based on trust and based on the managers’ mutual knowledge of each other. As one middle manager pointed out:“You have to be completely in sync. Since the fall, we haven’t felt that the two departments were working as well together, [nor did we have anything] facilitating a strong feeling of cohesion in our control room.”When the new directors arrived, they began sharing leadership and moved toward better collaboration that left no room for interdepartmental competition, which confirms that service integration is more dependent on human factors and added value for the client experience rather than on organizational and structural issues . The twoThe two new directors supported the clear and integrated vision of Jimmy’s trajectory articulated by the CEO and upheld the need for measurement to achieve the performance targets. Meetings in the tactical control room provided opportunities for both departments together to evaluate team performance by sharing essential information to raise important issues about Jimmy’s trajectory and devise collective solutions through consensus, thus supporting Barnas’ view  that the“Implementing the tactical room jointly between the youth program and the youth protection directorate has advantages, such as continuity  there are projects that, even though they are more the responsibility of the other department, considering that I am involved, I take responsibility for them.”Given that no formal activities were held in the tactical control room during the first year of the project’s deployment, the new directors had to take ownership of how the tactical control room functioned. The decision that the two departments with the same clientele should share the same control room led to a change in leadership and gave the teams the legitimacy to commit to the service integration project. The goals that the directors shared with the teams to help everyone understand the reasoning behind the changes created a climate of mutual trust, which was a key factor that allowed distributed leadership to emerge after two years of experimentation. Democratizing control room activities also fostered the emergence of distributed leadership. Both directors made this a collective responsibility. Other departments were also involved in Project Jimmy. Managers or staff were invited to attend control room activities to find collective solutions to the problems of accessibility, continuity, and quality throughout Jimmy’s trajectory. Distributed leadership made it possible to develop a culture of integration between the first and second service lines. For example, as a director pointed out:Distributed leadership is considered an effective way to foster team performance in complex situations, such as those found in programs for children and youth in difficulty. This paper tracks the evolution of the different leadership styles adopted during the implementation of a new management model Fig. . The resAt the onset of the integration project, the CEO exhibited transformational leadership by articulating a clear vision, by challenging directors and managers to actively participate in the integration project, and by fostering their participation by signing the Jimmy A3. Transformational leadership exhibited by top administrators is frequently cited as behavior that allows institutions to evolve through continuous improvement . The traThe matrix organizational structure implemented because of the new reform seemed to have negatively impacted the development of collaboration, as the leader with formal power had a purview that was too broad. This partially explains why distributed leadership at the strategic level still had not fully emerged by the end of our study.This case study demonstrates that transformational leadership from executives did not initially influence the type of leadership adopted at the tactical level, as highlighted by the dotted arrows in Fig. Laissez-faire leadership led to the emergence of transactional leadership from the CEO, through the implementation of the status sheet, as a response to address the absence of progress made in the project. This course of action is typically more conducive in crises . TransacThe executives used the reform as an opportunity to create territorial teams dedicated to children, youth, and families. The pooling of professional and multidisciplinary expertise and the initial familiarity of members contributed to the emergence of distributed leadership. From the start of the project and the set-up of the control rooms, each member’s contribution to providing accessible services was an important mechanism for achieving the required interdependence to develop distributed leadership . Our obsDespite good proximity, a certain degree of familiarity, and high task complexity, our findings show that the teams still seemed to wait for a leader to provide solutions to their problems. The control room lets staff identify problems and propose appropriate solutions. The control rooms, particularly the operational control rooms, therefore became more like information rooms. According to Pearce and Sims , the basThe implementation of an IPMS can support a new way of managing organizations where leadership and decision-making are shared in teams at all levels of the organization. Notwithstanding this willingness to share and adopt distributed leadership through collaborative practices, a more traditional top-down management style is a fundamental source of decision-making for organizations to run smoothly.In the end, some leadership responsibilities have been retained by the department head while other responsibilities are now distributed among the integrated team through the control rooms and status sheet. Distributed leadership is more in keeping with collaborative profiles or competencies that are strongly ingrained in individuals. The complementarity between transformational leadership and distributed leadership at all hierarchical levels is a promising avenue to reinforce the organization’s capacity to change. The more that this leadership combination evolves, the more the organizational capacity to change will improve as new top and middle managers with a collaborative style arrive and real improvements are made in terms of care and service accessibility, and continuity.recognizing individuals). Through transformational leadership, she asked top and middle managers to contribute to this project, based on their knowledge and expertise in the field of services for children. This would ultimately legitimize the emergence of distributed leadership further down the line, even though the short-term effects were not conclusive.Overall, this case study shows that a service integration project does not need to be guided by a single leadership style but rather by distributed leadership that emerges over time. This study also highlights the interdependence of leadership styles, where one is usually the result of another. While distributed leadership did not emerge quickly, its foundations were laid at the onset of the integration project. The CEO recognized the complexity of the project to integrate psychosocial services and that she needed to work with people who had skills and expertise that were complementary to her own  in the deployment of an IPMS. This study was carried out in the child, youth, and family social services sector, where different hierarchical levels, i.e. both managers and staff, were required to adopt new behaviors so that distributed leadership could emerge. This qualitative study makes a useful contribution regarding the complementarity of the foundations of distributed leadership, the tools of the IPMS, and integration processes applied over time.This case study raises questions about its transferability, along with certain limitations. The scope of this study was limited to the youth sector. A comparison with other sectors could be useful. Future research could be done in similar contexts  to explore the conditions and barriers for success, and for organizations to learn collective lessons to improve the performance of health and social services networks. Our results also show that recognition of team performance may have helped trigger the emergence of shared leadership. Hence, future research could explore this phenomenon through a positive psychological lens , to stud


In this work, we did our best to develop a novel and interesting analytical method based on coupling of spectrofluorimetry with first-order multivariate calibration techniques for simultaneous determination of lead (Pd), zinc (Zn) and cadmium (Cd) in HeLa cells. To achieve this goal, quenching of the emission of graphene (GR) was individually investigated in the presence of Pb, Zn and Cd and then, according to the linear ranges obtained from individual calibration graphs, a multivariate calibration model was developed based on modeling of the quenching of the emission of GR in the presence of the mixtures of Pb, Zn and Cd. First-order multivariate calibration models were constructed by partial least squares (PLS), principal component regression (PCR), orthogonal signal correction-PLS (OSC-PLS), continuum power regression (CPR), robust continuum regression (RCR) and partial robust M-regression (PRM) and their performances were evaluated and statistically compared. Finally, the OSC-PLS was chosen as the best model with the best practical performance for analytical purposes.  •The GR was uptaken by the HeLa cells and then, the cells uptook Pb, Cd and Zn.•Individual and multivariate calibration models were developed by the use of several first-order algorithms.•Comparing the performance of the models was performed and the OSC-PLS showed the best performance.•Performance of the OSC-PLS was compared with a reference method.•Our records showed that the OSC-PLS had a comparable performance with the reference method. Sometimes, the heavy metals are considered as contaminants which can be hazardous for human health therefore, monitoring of them is important. Lead (Pb) and cadmium (Cd) are heavy metals which are widely and naturally distributed toxic metals. There are some reports on determination of these metals with zinc (Zn) HeLa is an immortal cell line which is the most commonly used human cell line in scientific research. The HeLa cell line is durable and prolific which make it to be extremely suitable for scientific research. Therefore in this study, we have used the HeLa cells as a very interesting case for developing a novel analytical method for simultaneous determination of the Pb, Cd and Zn.Chemometrics combines chemical data with mathematical and statistical methods to extract useful information which can help the chemists to better justify their observations. Chemometricians have performed different projects by the use of instrumental data 22.13)2 were purchased from Sigma. Commercial Pb, Cd and Zn standards (1 g l−1) were prepared from Merck. Graphene quantum dots (blue luminescent) were purchased from Sigma-Aldrich. The other chemicals which were needed for doing this project were available in archive of our laboratory which had been purchased from Sigma or Merck. Doubly distilled water was used wherever water was needed. A phosphate buffer solution  was prepared from Na2HPO4 and its pH was adjusted at 7.4 by the use of H3PO4 and NaOH.Trypsin-EDTA, Dulbecco’s modified Eagle’s medium (DMEM/F-12 (1:1)), fetal bovine serum , penicillin-streptomycin (PEN-STREP), zinc nitrate hexahydrate, cadmium nitrate tetrahydrate and Pb(NO2.2Spectrofluorimetric data were recorded by a Cary Varian spectrofluorimeter equipped with a quartz cell (1 cm length path). First-order multivariate calibration algorithms including PLS, PCR, OSC-PLS, CPR, RCR, PRM, smoothing of the data and elliptical joint confidence region (EJCR) were run in MATLAB (Version 7.5) by the use of a series of m-files. The first-order multivariate calibration algorithms have been run in MATLAB with the help of PLS-toolbox or TOMCAT. The HeLa cells were prepared from the cell bonk of Kermanshah University of Medical Sciences. Then, the flask was transferred into a culture room where a deep-freezer (−80 °C), a memmert incubator, a JTLV CZS hood and a Motic microscope were existed for cell culturing. pH adjustments were performed by a Jenway pH meter 3510. Performance of the developed methodology was compared with the results of an Agilent atomic absorption spectrometer as reference method (AAS). Operating conditions for the AAS were: PMT voltage (450 V), slit width (0.40 nm), lamp current (9.0 mA), sample volume (20 µl), purging gas (argon), sample injection replicates (2) and measurement (peak height). All the calculations which were needed for data processing were performed on a Dell XPS laptop.2.32 +95% air) at 37 °C during a day (24 h). For uptaking the GR, 100 ng mL−1 GR was added to different culture dishes and incubated at different times and then, the cells were washed with PBS  and left to be in the PBS.Dispersion of the HeLa cells were performed in DMEM + FBS (10%) + PEN-STREP (1%) and seeded on five confocal dishes and then, they were incubated at an humidified atmosphere  were added to the dishes. The cells were further incubated for 2 h and washed with the PBS for three times and kept in the PBS. Spectrofluorimetric monitoring of the Pb, Cd and Zn was performed by excitation at 405 nm. For performing background correction on the data, the control cells which had not been incubated with GR (didn’t have any GR) was prepared. The procedures described above were continued by digestion of the treated and control cells with trypsin and then, the cells were kept in the PBS. Afterwards, the cells were counted, broken by ultrasonic and centrifuged. Finally, the supernatant of cells were measured spectrofluorimetrically.For simultaneous determination of Pb, Cd and Zn in HeLa cells, the seeded cells were allowed to grow during a day (24 h) and 1 mL DMEM having 1300 ng mL2.4In this work, we are going to develop a novel spectrofluorimetric method assisted by chemometric methods which will enable us to simultaneous determine Pb, Cd and Zn in living cells. Data treatment and development of multivariate calibration models must be very carefully performed to achieve the final goal. Prior to data modeling, all the spectrofluorimetric data were treated according to the following equation RMSEP: root mean square error of prediction and REP: relative error of prediction):yact and ypred are nominal and predicted concentrations, respectively, and ymean is the mean of the nominal concentrations. n are the number of samples in the validation set. Precision and accuracy of the developed calibration models will be compared according to the ellipses of the EJCR as well. Univariate calibrations and multivariate calibration and validation sets were performed in internal medium of the cells and by digestion of the cells with trypsin, the medium was extracted. This is a very important advantage which causes having a same medium for calibration and validation of the method which can help us for exploiting first-order advantage.All the data used in this work after passing this correction step was used for the next steps. Emission of the control cells was subtracted from the emission of the all of the cells and the corrected emissions were used for developing multivariate calibration models. Background correction was performed on the whole of data by subtracting emission of the control cells from emission of the whole of sets. Performance of the calibration models will be compared by the use of the following equations  and PRM: number of LVs = 3 and PDC = 0.12. After application of the algorithms and optimization of their parameters and constructing multivariate calibration models, their performance was verified by their application to a validation set having cells with different concentrations of Pb, Cd and Zn whose composition is shown by In order to multivariate calibrate the emission of the GR with concentration of Pb, Cd and Zn, a central composite design was developed based on linear ranges obtained from individual calibration graphs. Composition of the calibration set is shown in In order to further verification of the performance of the spectrofluorimetric method assisted by OSC-PLS, the AAS was applied to the prediction of the concentrations of the validation set as reference method and the results are shown in RSD) for the analysis of 800 ng mL−1 Pb, Zn and Cd in six replicates which gave us RSDs of 2.08%, 2.15% and 2.11% for Pb, Zn and Cd, respectively. Inter-day precision was determined by the analysis of six replicates 800 ng mL−1 Pb, Zn and Cd on three consecutive days which gave us RSDs of 2.34%, 2.28% and 2.21% for Pb, Zn and Cd, respectively. The results obtained for examination of intra-day and inter-day precision confirmed acceptable precisions for the developed methodology.The intra-day precision of the assay was estimated by calculating the relative standard deviation (4In this work, a novel and interesting analytical methodology based on coupling of spectrofluorimetry and chemometrics was developed for simultaneous determination of Pb, Cd and Zn in Hela cells. Among the tested chemometric algorithms, the OSC-PLS showed the best performance for simultaneous monitoring of Pb, Cd and Zn whose performance was comparable with AAS as reference method. The results of this work showed that chemometrics has a great potential for assisting instrumental techniques to develop accurate novel methods which have very better performance than those instrumental alone. As a new research field for our research group, we are going to continue coupling of chemometric method with instrumental techniques for bioanalytical purposes and definitely, this work will be a bridge to connect the world of chemometricians with the world of bioanalysts.The main idea of this project belongs to Dr. Ali R. Jalalvand and the other authors contributed equally in this project.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


We thank Dr. Landry and colleagues  for theiAdherence to dietary prescriptions is of pivotal importance in studies comparing different nutritional interventions. In our randomized clinical trial, self-reported dietary compliance was assessed by trained dietitians at 2 and 4 weeks of diet using semiquantitative food frequency questionnaires, according to previously described methods ,4. NutriAs already pointed out and discussed , a low-cLastly, participants’ satisfaction with their assigned diet was not formally assessed and quantified in our study. However, none withdrew the diet or complained about its composition during the 4-week intervention, suggesting the feasibility of these dietary prescriptions, at least in the short term. In conclusion, our study emphasizes important differences in the effectiveness of two calorie-restricted eating patterns on weight loss and glucose homeostatic mechanisms in morbidly obese individuals. A great effort has been made to maximize participants’ engagement to nutritional interventions and reduce heterogeneity, with the final aim to minimize the influence of dietary adherence on study findings.


The authors are listed out of order. The last author should be Paula Garcia Chiarello. Please view the correct author order, affiliations, and citation here:1, Gabriel Ruiz Sizoto2, Lorena Vega-Piris3, Guillermina Barril Cuadrado4, Paula Garcia Chiarello5Natália Tomborelli Bellafronte1 Post-graduate Program in Health Sciences, Ribeirão Preto Faculty of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil, 2 Nutrition and Metabolism Undergraduate Course, Ribeirão Preto Faculty of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil, 3 Methodology Unit, Instituto de Investigación Sanitaria del Hospital Universitario de la Princesa, Madrid, Spain, 4 Nephrology Department, Hospital Universitario La Princesa, Madrid, Spain, 5 Department of Health Sciences, Ribeirão Preto Faculty of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazilhttps://doi.org/10.1371/journal.pone.0242671Bellafronte NT, Sizoto GR, Vega-Piris L, Cuadrado GB, Chiarello PG (2020) Bed-side measures for diagnosis of low muscle mass, sarcopenia, obesity, and sarcopenic obesity in patients with chronic kidney disease under non-dialysis-dependent, dialysis dependent and kidney transplant therapy. PLoS ONE 15(11): e0242671.


Since reproductive interference in plants may occur through interspecific pollination, the effective range of reproductive interference may reflects the spatial range of interspecific pollination. Therefore, we hypothesized that the coexistence of congeners on a small spatial scale would be less likely to occur by chance but that such coexistence would be likely to occur on a scale larger than interspecific pollination frequently occur. In the present study, we tested this hypothesis using spatially explicit woody plant survey data. Contrary to our prediction, congeneric tree species often coexisted at the finest spatial scale and significant exclusive distribution was not detected. Our results suggest that cooccurrence of congeneric tree species is not structured by reproductive interference, and they indicate the need for further research to explore the factors that mitigate the effects of reproductive interference.In plants, negative reproductive interaction among closely related species ( However, the CRH has also been widely discussed  limits the coexistence of closely related species  are less likely to coexist by chance on a local scale  congeneric species do not coexist on a fine spatial scale where reproductive interference frequently occurs but coexist on a large spatial scale; and (2) on large scales where reproductive interference does not occur, congeneric species coexist while avoiding each other, resulting in an exclusive or checkerboard distribution .2) was located in the Kasugayama primary forest, Nara prefecture, western Japan (34′41′N135′51′E) (Sapium sebiferum and Nagia nagi (The study area (∼1 km35′51′E) . Because35′51′E) . In the 35′51′E) ; howevergia nagi . The fieField studies were conducted from June to September 2015. In the study area, 30 transect plots (∼0.1 ha in size) at 200 m intervals were established . Tree spIn this study, we defined congeners as closely related species based on the definition by a priori-defined spatial scales . In this study we established 30 plots at 200 m intervals. A priori-defined spatial scales corresponded to the area of each plot extended horizontally.We employed the species-to-genus ratio (S/G ratio) as an indicator of intragenic interactions for the categorized tree, shrub, and liana species. The S/G ratio has long been used to describe community patterns and to infer levels of competitive interactions among species within genera . Note that there are differing opinions among researchers on whether the genus Prunus should be considered as a single genus or not .where N species, there are P = N(N − 1)/2 species pairs; thus, the C-score is calculated as follows: For The C-score becomes larger as the two species occur more commonly across different plots. We simulated null models to compare the observed C-score with stochastic distributions. The null models, which were run 999 times for each species pair, randomly shuffles the number of species (α-diversity) among sampling locations while preserving the species occurrence totals (the plant density). All statistical analyses were performed using R software version 3.6.1 . EcoSimRAt the study site, we recorded 42 tree species  from 31 genera, 20 shrub species  from 19 genera, and seven liana species from six genera . The resultant S/G ratios for trees, shrubs, and lianas were 1.350, 1.021, and 1.200, respectively. Only the S/G ratio for tree species significantly deviated from the 1:1 ratio, whereas those for shrub and liana species did not .The average S/G for trees increased as spatial scale increased. Even at the smallest spatial scale, however, the average S/G ratio for trees exceeded the 1:1 ratio , indicatQuercus, Carpinus, and Prunus. It indicates that statistically significant exclusive distribution of species from the same genus did not occur . Since a low S/G ratio is generally a product of strong intrageneric competition  is within a few tens of meters (Quercus, shows considerable variation in annual seed production (In plants, the spatial extent of reproductive interference corresponds to pollen transfer distance ; consequf meters . This suf meters . One of oduction , which moduction . Eaton ePrevious studies reveal that specialist natural enemies, such as herbivores and pathogens, maintain tree species diversity by the reducing survival rates of conspecific seeds and seedlings located close to reproductive adults or in high conspecific density areas (Janzen–Connell effect; In future research, investigation on a larger spatial scale with a more complex analysis will be required to determine the relationship between plant life history and the spatial scale of exclusive distribution. Previous studies on herbaceous plants suggest that reproductive interference plays an important role in community assembly ; however10.7717/peerj.12150/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12150/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12150/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.12150/supp-4Supplemental Information 4Click here for additional data file.


Hartmann’s procedure involves resecting the rectosigmoid colon, closure of the distal rectal stump, and forming an end colostomy for complicated left colon diverticulitis or malignancy. Recovery from the initial operation can, in a second stage, be followed by a reversal stage with the restoration of bowel continuity. This study aimed to assess the reversal rate and its correlation with demographic data, ASA grade, and length of hospital stay. All patients who underwent Hartmann’s emergency procedure from 2014 to 2018 at Lewisham and Greenwich hospital were enrolled in this retrospective study. Data was collected from the inpatient electronic files and NELA . 118 patients were included in the study, with 57.6% females and a median age of patients of 69 years (range 35–91). Findings of the study indicate that the most common indications for Hartmann’s procedure were diverticular complications 60% (n=71) and benign perforated sigmoid or rectosigmoid cancer 16% (n=19). The average length of hospital stay was 24 days (range n=2 – 212 days). The reversal rate was 34.9% (41/118 cases). No significant difference was observed between gender and length of hospital stay in relation to the reversal rate while there was a significant correlation between age and ASA grade in relation to reversal rate; the calculated P values were recorded as (<0.000) and (<0.009) respectively. Our results show that the highest reversal rate was observed in younger and fitter (I–II) ASA grade patients. The most common medical complication from reversal of Hartmann’s procedure was an anastomotic leak . Reversal rate of Hartmann’s procedure was 34.9%. The average timeframe for reversal was within 18–20 months. There was a significant correlation between age and ASA grade in relation to reversal rate. In the early twenties of the past century, the French surgeon Henri Albert Hartmann introduced and described a surgical procedure for obstructive rectosigmoid cancer in 1921, with postoperative sequel superordinate for other surgical techniques. Hartmann’s procedure is defined as a surgical procedure that mainly includes rectosigmoid resection and closure of rectal stump plus establishment of an end colostomy . MoreoveAmerican Society of Anesthesiologists Physical Status (ASA PS) classification was first implemented in clinical practice more than seventy years ago to classify patients according to tolerance and fitness for specific surgical procedures. The ASA class is a subjective evaluation of a patient’s overall health, and it only measures the amount of physiological allowance that a patient has at the time they are evaluated for a surgical procedure. Also, it should not be used as the only indicator of an operative risk to the patient . ASA scoOur study aims to identify the factors that affect the reversal rate, mainly the age, gender and ASA grade, and length of hospital stay . In addiThis is a retrospective observational study. All patients who underwent emergency Hartmann’s procedure between 2014 and 2018 at Lewisham and Greenwich hospitals were identified and included. We excluded patients who have their Hartmann’s procedure on an elective basis. The cases were retrieved from a computerized medical records database and NELA  bowel database. Analysis was completed by examining the inpatient files in selected patients where appropriate. Collected data included the following parameters: demographics, indications for Hartmann’s procedure, duration of the surgery, hospital stay, ASA grade, and reversal rate. In addition, the type of surgery (open or laparoscopic) covering ileostomy and postoperative complications was collected for patients who underwent a reversal. All the reversal procedures were performed by qualified colorectal surgeons, while the original Hartmann’s procedure was performed by a mixture of emergency surgeons, general surgeons, and colorectal surgeons. Data analysis was performed using the SPSS software. A chi-squared test was used to assess categorical variables. A p-value of less than <0.05 was considered to be statistically significant.A total of 118 patients were identified during the study period from 2014 to 2018 at Lewisham and Greenwich hospital. 68 (57.6%) of the 118 patients were female. The median age was 69 years (range from 35 to 91). A substantial proportion of patients were ASA grade III–V , as summarized in Sigmoidoscopy to assess the rectal stump length and colonoscopy through the stoma to rule out residual proximal pathology were routinely performed prior to reversal. The reversal rate of Hartmann’s procedure was 34.7% (41/118 cases). Seventy-seven patients (65%) did not proceed to reversal . The reaForty-one patients underwent successful reversal (34.9%). Defunctioning ileostomy was used in 10 patients (24%), an anastomotic leak occurred in 6 patients (14.6%). The operative time mean was 3 hrs.15 min, and the average length of hospital stay following reversal was 11 days (range 2–159 days). The Chi-square test was used to determine the significance of differences between the reversal rate of Hartmann’s procedure and the demographic data of the patients, ASA grade, and length of hospital stay . No signet al. [et al. [et al. [This study involved the selection of 118 consecutive patients who underwent Hartmann’s procedure to measure the reversal rate and mortality rate and assess the correlation between demographic data, ASA grade, length of hospital stay, and the reversal rate of Hartmann’s procedure. The most common indication for Hartmann’s procedure in our study was diverticular complications (60%), similar to the studies conducted by Hallam et al.  and Chri [et al. , while t [et al.  was coloet al. [et al. [In this study, the reversal rate of Hartmann’s procedure was assessed. 34.9% of the patients underwent a successful reversal, which is higher to previously reported data by Christou et al. , where o [et al. , who repet al. (16.1%) [et al. observed a low rate of laparoscopic surgery for a colostomy reversal. However, good results have been published with its use, and a laparoscopic reversal is comparable or superior to open reversal [et al. [Thirty-day mortality following Hartmann’s procedure was 16.9%; it was higher in those aged above 80 years. This was in accordance with previously reported results by Zarnescu  (16.1%) . It woulreversal . Post re [et al.  was diaret al. [Our results revealed that there was no significant difference between gender and reversal rate (p<0.737), while a significant relation was observed between age (p<0.001) and ASA grade (p<0.009) in relation to reversal rate, the highest reversal rate was observed in younger and (I–II) ASA grade patients. In addition, there was a higher rate of reversal in benign conditions than malignant conditions. These results were in accordance with previous results reported by Hallam et al. , where tThere are several other surgical trends to approach the management of diverticular complications or obstructed rectosigmoid tumors, such as colonic stent insertion and primary anastomosis with or without a stoma. The colonic stent can convert the emergency surgery to a semi-elective surgery which allows more time for patient optimization and a procedure performed by a colorectal surgeon. Primary anastomosis with or without diverting ileostomy is another option that can save the patient from undergoing another major and morbid surgery. This latter approach was not adopted in our current study as most of Hartmann’s procedures were performed by emergency consultants who are not specialized in colorectal surgery and only occasionally perform colon resections. On the other hand, colorectal surgeons performed all reversal operations, who conducted primary anastomosis and diverting ileostomy in one-quarter of the reversed patients. Moving towards centralizing services with 24 hours access to colorectal surgeons may reduce the number of Hartmann’s procedures performed overall. In addition, providing up-to-date information on the management of those common problems in the surgical department is crucial.One limitation of our study is the retrospective data collection from a dual centers group, involving many surgeons not necessarily specialized in colorectal surgery. Second, our patient group was rather heterogeneous. However, this variety in the patient mix was a caveat to assess possible risk factors for non-reversal. The value of our study is that it demonstrates that Hartmann’s procedure is still a very prevalent surgery that carries high mortality and morbidity, but it can still save lives in high-risk comorbid patients, and by implementing new evolving surgical options, we can improve the quality of patient care.In conclusion, Hartmann’s procedure is still a commonly performed emergency colorectal operation. Reversal rate of Hartmann’s procedure was 34.9%. The predicted mortality on NELA v/s actual mortality is an important figure to compare to identify areas needing a particular focus. The number of patients turned down for reversal may well reflect the poor general health of the cohort and may have a relation to the low socioeconomic status of the population in this area. As expected, there is a significant correlation between age and ASA grade in relation to reversal rate. Our high comorbidity rate and high ASA grade may have contributed to the high leakage rate.The authors declare that there is no conflict of interest.The study was approved by the Audit Committee of the Lewisham and Greenwich Trust .Written informed consent was obtained from the participants in the study.MF contributed to data collection and writing the article. PS, RK, and OO designed the methodology and PN reviewed the article.


Food is an integral part of human culture. Aside from its nutritional role, it connects us on a daily basis through the act of sitting and eating together, it contributes to feelings of shared identity in populations, it is an essential component of many formative events in our lives, and is a gateway to experiencing new cultures.While the influence of food in defining human culture is well-understood, there is a growing appreciation of the role of microbial cultures in defining the properties of food. Archaeological evidence points to the use of fermentation over many thousands of years for the production of a range of foods and beverages. Fermentation serves to make foods more digestible, longer-lasting, safer, and more palatable. For our species, the role of fermentation in food production has arguably been as influential as the role of fire for cooking food.For much of our history, these fermentation processes have been carried out by complex assemblages of organisms, with the use of single strains for fermentation of bread, beer, wine, etc. being a relatively recent development. We have however been seeing a growing interest in the use of microbial communities in food production. In the western world, this revival has been driven in part by the popularity of craft beer, natural wine, fermented non-alcoholic beverages , and sourdough bread. In other parts of the world, complex cultures in fermentation represent continuation of tradition rather than revival.This Research Topic from Frontiers in Microbiology aims to highlight the beneficial role of microbial complexity in driving fermentation processes and influencing food quality. The issue features ten articles describing investigations into complex microbial fermentation for the production of food, feed, and beverages. Contributions were received from Africa, Asia, Australia, Europe, and South America—highlighting the global importance of fermentation. Demonstrated benefits of particular communities during fermentation include improved flavor and color, greater safety, increased nutritional value, faster fermentation rates, reduced alcohol content, and better industrial applicability.Elhalis et al., who show improved sensory properties of coffee after fermentation with the yeast Pichia Kudriavzevii. Likewise, Köhler et al. note how specific yeast and bacteria combinations can improve the flavor of water kefir. Liu et al. describe the relationship between bacterial composition and flavor profile of Xifeng liquor (a spirit prepared from fermented sorghum).One of the most direct impacts of fermentation is the creation of specific flavor profiles. We see this in the work of Su et al. for example, note how fermentation of corn by-products with Saccharomyces yeast and lactic acid bacteria can improve their suitability for use as feed. Dahunsi et al. provide a comprehensive account of how the presence of lactic acid bacteria within fermentative microbial consortia have a critical role in ensuring the safety and shelf-life of many fermented African foods. Likewise, Huang et al. describe the metabolites produced by the yeast Debaryomyces hansenii can prevent the growth of contaminant molds during Danish cheese production.Palatability of foods is not the only factor influenced by fermentation. Benefits extend also to improved digestibility and safety. Martinez et al. who describe how coffee fermentation is influenced by altitude, and how this is associated with changes in the natural microbiota. Substrate also influences the development of populations, as illustrated by Liu et al. who show how different microbial communities develop depending on the type of sorghum used in Xifeng liquour production. Su et al. likewise show how communities develop differently in different by-products of the corn starch industry. These, and other studies described in this Research Topic, demonstrate the power of high-throughput sequencing in studying microbial communities. This is particularly important for dynamic, complex or under-researched communities. This is exemplified in the studies on dark tea fermentation by Yan et al. or the work of Atter et al. on Ghanian cereal fermentation.The composition of species in a fermenting culture is strongly impacted by the environment, a fact that must be considered when trying to control or direct fermentation processes. The effect of environment is clearly seen in the study of Huang et al. coffee fermentation by Elhalis et al. and water kefir production by Köhler et al.Despite the advantages of complex fermentations with respect to product quality, it should be noted that industrial application of such cultures is not without complications. More complexity may lead to less consistency in fermentation processes. The controlled use of cultures, e.g., supplementing natural microbial consortia with specific species, or rationalizing consortia so that they contain only the keystone species, represents a compromise in this respect. Examples are seen here in the work on cheese production by Complex fermentations demonstrate great potential for improving food process efficiency, enhancing food quality, and increasing diversity of available foods and beverages. It is clear however that our ability to fully exploit such fermentations is limited by insufficient knowledge of how individuals in mixed populations interact to influence each other and their environment. It is our hope that this Research Topic represents a step forward in our understanding of these complex systems, and will to some extent facilitate the efficient production of a range of high quality food products in the future.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A comprehensive review of relevant clinical literature on evidence-based recommendations and existing prediction models specific to lung cancer surgery was undertaken. Preoperative risk assessment parameters such as pulmonary function tests (PFT), cardiopulmonary exercise testing (CPET), Brunelli models, Thoracoscore and frailty were analyzed for predicting postoperative risk of complications. (DLCO ) showed conflicting evidence in determining a positive correlation with postoperative mortality. CPET variables predicted higher complication risk when VO2peak < 10ml/kg/min, AT < 11ml/kg/min and ventilation/carbon dioxide production (VE/VCO2) was in range of 34-40. While a cardiac risk index like the Thoracic Revised Cardiac Risk Index (ThRCRI) predicted major cardiovascular compromise, a thoracic risk index like Thoracoscore proved imprecise. Lastly, frailty is used to risk stratify patients in clinical practice but a recognized validated model specific to thoracic surgery is non-existent.When assessing fitness for surgery, the primarily used PFT parameters such as predictive postoperative forced expiratory volume in one second (FEV1) and diffusion capacity for carbon monoxideWhen considering patients for lung cancer surgery, some dilemma exists regarding the accuracy of clinical prediction models and their external validation. There is a pressing need for the development of a consolidated clinically robust risk stratification model to predict complications after thoracic resections. Lung cancer is the leading cause of oncological deaths worldwide . It is aTreatment of early-stage lung cancer primarily involves surgical resection as it delivers a strong probability of cure . DespiteThis highlights the need to risk stratify to predict mortality, morbidity and long-term shortness of breath in lung cancer patients. In the last decade, an increasing amount of literature has addressed the role of various parameters used in preoperative risk assessment. This often involves cardiorespiratory evaluation in conjunction with assessment of operative mortality, lung function and exercise testing. More recently, there has been an emphasis on frailty as a significant surgical risk factor and practical screening tool. Individual preoperative investigations such as pulmonary function tests (PFT), cardiopulmonary exercise testing (CPET), Brunelli models, Thoracoscore and frailty can be useful in clinical practice and the accuracy of these in predicting postoperative risk has been analyzed in this article. Pulmonary function testsppoFEV1Assessment of pulmonary function parameters such as forced expiratory volume in one second (FEV1) and predicted postoperative FEV1 (ppoFEV1) is important in-patient selection for lung cancer surgery. This is to evaluate the possibility of postoperative pulmonary complications (PPC) and operative mortality, defined as death within 30 days of surgery. Patients with an estimated ppoFEV1>40% are considered as having an average risk, and high risk is determined by an estimated ppoFEV1 <40% [Various small studies have been carried out at local or regional units to establish the risk indicators for developing PPC. A four-year prospective observational study of 285 patients undergoing video-assisted thoracoscopic surgery (VATS) for non-small cell lung cancer (NSCLC) was carried out. Data on gender, age, predicted %FEV1, ASA score, perioperative activity level, BMI, smoking status, and NSCLC staging were recorded. Of the 21 participants who went on to develop postoperative pulmonary complications, the percentage of predicted FEV1 was 88.8% compared with 87.5% in patients who did not develop complications. Predicted %FEV1 was not found to be a significant independent risk factor in determining PPC such as pneumonia, atelectasis, longer hospital stays in high-dependency units or admission to intensive care following VATS   and %FEV1 was collected. A cut-off value of 70% for FEV1 was defined as obstructive ventilatory impairment. In Japan, FEV1 of less than 70% is defined as obstructive ventilatory impairment which is in contrast to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification. Postoperative complications were present in 35% of patients with a %FEV1 <70% compared to 23% of patients with an FEV1 ≥70%. Univariate analysis showed that FEV1 <70% was associated with a greater risk of postoperative atelectasis, pneumonia, and air leakage. These findings oppose the aforementioned studies likely due to the larger population size and demonstrate %FEV1 as an important predictor and significant risk factor of postoperative complications [Moreover, a study using whole lung CT to determine preoperative pulmonary function was carried out in 390 patients who underwent resection for lung cancer. The development cohort had 290 patients and 100 patients formed the validation cohort. Whole lung CT along with patient demographics was used to develop a regression equation for FEV1 to determine preoperative pulmonary function and ppoFEV1. The ppoFEV1 was also calculated based on the anatomic equation: measured %FEV1 x (42 - number of subsegments resected)/42. Both regression analysis and actual measured FEV1/FVC, %FEV1 and %ppoFEV1 were shown to be significant variables associated with predicting postoperative cardiopulmonary complications in both cohorts. Both calculated ppoFEV1 and actual ppoFEV1 were positively correlated with the occurrence of PPC . The useTaking these studies into account, pulmonary function parameters continue to provide conflicting evidence for their ability to predict postoperative complications. The smaller population size of these studies is a limiting factor and we need stronger studies collaborated at national or international units to improve reliabilityDiffusion capacity for carbon monoxide (DLCO)In addition to spirometric parameters, the diffusion capacity for carbon monoxide (DLCO), also known as the transfer factor for carbon monoxide (TLCO), is considered in most preoperative assessments in addition to ppoFEV1. DLCO is based on the ability of carbon monoxide to bind to haemoglobin, therefore DLCO represents the efficiency of gas transfer from inspired air into red blood cells. A low DLCO predicts (a) a higher likelihood of pulmonary complications requiring either supplemental oxygen or hospitalization and (b) a worse dyspnea score. DLCO is also strongly associated with the risk of higher operative mortality . DLCO isA prospective study involving 351 patients with NSCLC was conducted to determine the risk factors associated with PPC after lung cancer surgery. The preoperative evaluation demonstrated a lower predicted DLCO in the COPD group compared to non-COPD counterparts. Univariate analysis found that patients with COPD who went on to develop a PPC had a lower percentage of predicted DLCO of 86% compared to 93.1% in patients who did not develop PPC. Moreover, in the non-COPD group, predicted DLCO was 87.7% in patients who developed a PPC compared with 96.7% in patients who did not develop a PPC. This study signifies low DLCO as a vital risk factor for postoperative complications in both COPD and non-COPD groups thus highlighting the importance of measuring DLCO in patients with normal spirometry prior to resection for NSCLC [DLCO was also found to be a significant prognostic factor following surgical resection for stage I NSCLC. In a study, 426 patients who underwent resection for stage I NSCLC were recruited to investigate the short and long-term outcomes in those with a 'marginal-risk' versus 'normal-risk'. Marginal-risk was defined as a ppoFEV1 of 30-60% and/or ppoDLCO of 30-60%; while, normal-risk was defined as ppoFEV1 ≥ 60% and ppoDLCO ≥ 60%. After applying exclusion criteria, 391 patients were included with 73 classified as marginal-risk and 318 as normal-risk. Overall, marginal-risk patients compared to normal-risk had higher postoperative morbidity (48% vs 21% respectively), pulmonary complications (34% vs 11%) and prolonged hospital stay (23% vs 6%). Among six patients with a ppoFEV1 and/or ppoDLCO of <40%, postoperative morbidity was found to be 83%. Furthermore, in the 27 patients with a ppoFEV1 and/or ppoDLCO of <50%, postoperative morbidity occurred in 44%, PPC occurred in 33% and 22% had longer hospital stays . Cardiopulmonary exercise testing (CPET) Cardiopulmonary exercise testing (CPET) is a complex, non-invasive approach to cardiorespiratory evaluation during exercise and at rest under controlled physiological conditions . Of lateVO2 peak is the maximum value of oxygen uptake measured during CPET at the end of the exercise phase . It is tAnaerobic/ lactate threshold (AT) is a measure of submaximal exercise capacity, i.e. oxygen consumption achieved almost exclusively under aerobic conditions . Since iVE/VCO2 is not only a ratio of the minute ventilation to CO2 output but also an expression of gas exchange efficiency . ElevateCardiac and thoracic risk indicesAs cardiovascular complications are potential risks following lung cancer resection, a cardiovascular risk assessment must be performed preoperatively . The NatThoracic Revised Cardiac Risk IndexThe Revised Cardiac Risk Index (RCRI) was proposed in 1999 by Lee et al. for evaluation of stable non-urgent major non-cardiac surgery patients . This waThe sum of these scores then divides patients into different risk classes that estimate their risk of cardiac complications, thus determining those who require further cardiac assessments: (1) Class A - 0 points (1.5%), (2) Class B - 1-1.5 points (5.8%), (3) Class C - 2-2.5 points (19%) (4) Class D - >2.5 points (23%).The validity of the ThRCRI in predicting cardiovascular risk after major lung resection has been supported in multiple studies -20 , which specifically looks at in-hospital mortality following general thoracic surgery. The nine variables it uses include age , sex, American Society of Anesthesiologists (ASA) score , Zubrod score , dyspnea score , the priority of surgery  and procedure class  . NICE reThoracoscore and the modified Thoracoscore (dyspnea score excluded) have previously been externally validated, including a retrospective study in which 1675 patients who underwent thoracic surgery (n=626 for lung resections) were analyzed from 2002 to 2006. The modified Thoracoscore proved to be an effective predictor of in-hospital mortality and midterm mortality (mean follow-up of 25 months), with in-hospital mortality risk increasing by 20% for every 1% increase of the modified Thoracoscore, and midterm mortality increasing by 12% for every 1% increase  Table 33.However, studies in recent years have shown the inaccuracy of Thoracoscore, particularly when compared to other models. One such study aimed to externally validate Thoracoscore, as well as other multivariable prediction models , looking at their effectiveness in estimating perioperative mortality. The survival time (number of days from surgery to date of death) was measured in 6600 patients who underwent lung resection between 2012 and 2018. Each model was produced to predict either in-hospital or perioperative mortality; 5/6 of the models had an inaccurate estimation of the type of mortality they were made for, with the modified Eurolung model producing the only acceptable prediction (observed:expected ratio of 0.92). However, this model has limited variables and does not factor in patient comorbidities, thereby bringing its clinical validity into question .A brief summary of some of the available risk models in thoracic surgery along with their disadvantages is mentioned in the table below -28 Tabl. Their uFrailty Frailty assessment is defined as an age-related decline in multiple physiological systems and can be used to flag vulnerable patients who may benefit from pre-operative rehab or non-surgical therapies . The curmFI was formed by matching the Canadian Study of Health and Aging Frailty index (CSHA-FI) to the 11 variables, for example, MI history and diabetes, compiled by the American College of Surgeons NSQIP . RCRI usA study on frailty assessment was conducted at a single-site thoracic surgical clinic - out of the 180 eligible patients, 125 completed screening. Results showed that 71 (57%) were prefrail and 15 (12%) were frail . This isPreoperative risk assessments for lung cancer resection play a vital role in determining whether it is appropriate to operate, deciding if further assessments or treatments are required, and predicting a patient’s prognosis. Some of these assessments, such as a frailty assessment, CPET and ThRCRI, have been externally validated by studies, proving their accuracy in predicting complications. Their use in combination, depending on the patient, would provide a detailed picture of what a patient’s outcome following surgery would be. However, other assessments, such as pulmonary function tests and Thoracoscore, still have conflicting evidence regarding their efficacy. This is possibly skewed by small underpowered studies, hence need for larger studies using acceptable cut off values. These assessments require further external validation, and should not, at this time, replace the use of the assessments that have proven to be effective.


The Phosphohistone H3 (PHH3) antibody is recognized as a biomarker of cell proliferation, specific for cells in mitosis, of prognostic value in different malignant neoplasms, however it has been poorly studied in oral squamous cell carcinoma (OSCC). The main objective of this study was to evaluate the immunoexpression of the PHH3 in the OSCC, through the correlation with the immunoexpression of Ki-67, the mitotic activity index (MAI), histological grading, clinical-morphological parameters and the rate of survival. The study sample consisted of 62 cases of OSCC diagnosed in the Pathological Anatomy Laboratory of the Faculty of Dentistry, University of the Republic (Uruguay). In each of them, an immunohistochemical technique was performed for Ki-67 and PHH3 (serine 10) antibodies. Image J software was used for the MAI and biomarker quantification, defining the percentage of positivity and mitotic Figures per 1000 tumor cells.p 0.016) and MAI (p 0.031) with survival time. However, no similar relationship was found with Ki-67 (p 0.295). Although it was confirmed a statistical association between histological grade and Ki-67 immunoexpression (p 0.004), PHH3 did not show a similar relationship (p 0.564). a significant association was obtained between the expression of PHH3 ( It was confirmed the role of the PHH3 antibody as a biomarker of mitotic Figures in OSCC and as a potential marker of cell proliferation. It is noteworthy that this is one of the first works that evaluates a possible relationship between the expression of this antibody and survival in OSCC. Key words:Oral squamous cell carcinoma, phosphohistone H3, Ki-67, cell proliferation. Oral squamous cell carcinoma (OSCC) represents the most common cancer of head and neck region ,2. It isCell proliferation is an essential biological process, key in the growth and maintenance of tissue homeostasis, whose loss of control plays a fundamental role in the development of malignant neoplasms ,6. In faMAI represents the oldest method for determining the proliferative capacity of malignant neoplasms ,11. It iThe PHH3 antibody is recognized as a biomarker of cell proliferation, specific for cells in mitosis, by identifying phosphorylated histone H3 by IHC ,11,16. HThe main objective of this study was to evaluate the immunoexpression of PHH3 in OSCC, through the correlation with the immunoexpression of Ki-67, MAI, histological grading, clinical-morphological parameters and the rate of survival.The study sample consisted of 62 cases of OSCC, corresponding mostly to incisional biopsies diagnosed in the Pathological Anatomy Laboratory of the University of the Republic (UdelaR) School of Dentistry, in the period 2007-2015. The clinical‑pathological records of each of the cases were reviewed, recording the variables corresponding to gender, age, topography, date of pathological diagnosis and histopathological diagnosis. Survival was defined as the time elapsed between the initial histopathological diagnosis and death from cancer. The histopathological diagnosis of each of the cases was made according to the WHO classification (2017) for OSCC, in well, moderately and poorly differentiated  and IHC histological slides were digitized with the Motic VM 3.0 Digital Slide Scanning System for image acquisition and processing. Motic VM 3.0 Motic Digital Slide Assistant software  was used for its analysis. Cell quantification was performed with the Image J software manual counting tool . For the calculation of the MAI, in the H-E slides, normal and atypical MF present in 1000 tumor cells were counted. The identification of the MF was carried out according to the morphological characteristics described by Van Diest anaphase .Immunohistochemical processing: from the blocks of tissue fixed in formalin and embedded in paraffin, two sections of 4 μm thickness were obtained. To unmask the antigenic epitopes, recovery was performed with sodium citrate solution (pH 6.2)  in a microwave pressure cooker at maximum power for about 5 minutes. Endogenous peroxidases were blocked with 0.9 % hydrogen peroxide for 5 minutes. Tissue sections were incubated with the primary antibodies Ki‑67  and PHH3 (Serine 10)  for about 45 minutes and then with a biotinylated anti-mouse/anti-rabbit secondary antibody for 30 minutes. To visualize the products of the antigen-antibody reaction, 3,3' diaminobenzidine H2O2  was used, followed by counterstaining with Harris hematoxylin . Breast carcinoma samples were used as a positive control for IHC processing. Likewise, as a negative control, IHC processing was performed omitting the incubation step with the primary antibodies.IHC evaluation: positive cells for the Ki-67 antibody were considered to be those neoplastic epithelial cells that presented a brown nucleus, regardless of the intensity and pattern of staining ,9,22. FoPrior to the IHC evaluation, interobserver calibration was performed between two pathologists, who previously agreed on the morphological criteria necessary for the identification of MF and positive cells for biomarkers. The observers carried out their quantifications independently, without knowing the Figures established by each other. The interclass correlation coefficient (ICC) was used to calculate the degree of interobserver agreement .Statistical analysis: in the considered markers comparation, descriptive statistics (mean and standard deviation) were used for categorical variables, and frequency distribution for continuous variables. To compare the expression of the markers according to sex, age, location and histological grade, when it came to two groups, the comparison of means based on the student's t-test for independent samples was used. Likewise, in cases where three or more groups were compared, the analysis of variance (ANOVA) model was used. In cases where a significant association was identified, multiple comparisons were carried out considering the Tukey test. Additionally, scatter diagrams were made to investigate the degree of linear correlation between the three markers, calculating in each case, the Pearson linear correlation coefficient. Survival analysis was carried out using the Kaplan-Meier survival curves. The association of patient survival was analyzed according to the three markers considered, in a multivariate analysis through the Cox proportional hazards model, adjusting the results by sex, age, grouped location and histological grade. While their comparisons were made using the Cox model. All tests were carried out with a significance level of 5%.- Descriptive analysis of the clinical-pathological variables of OSCC: of the 62 cases, the 64.6% corresponded to men and the 59.7% were older than 65 years, the 43.5% were located in others , 25.8 % in tongue, 21.0 % in palate and 9.7 % in the mouth´s floor. Regarding histological grading, 61.3 % corresponded to moderately differentiated OSCC (Grade 2), 27.4 % to well-differentiated OSCC (Grade 1), and only 1.3 % were classified as poorly differentiated OSCC (Grade 3) .p 0.004) (p 0.564). Neither could a statistically significant correlation be demonstrated between the biomarkers studied and the independent variables, sex, age and location  . Thus, ilocation .p 0.041)     and the survival rate at five years was 27 %  Fig. . Likewis16) Fig. . A signip 0.194)  Fig. . In contThe evaluation of cell proliferation in cancer is considered an important histological parameter for defining the biological behavior of the tumor and for determining the individualized prognosis for each patient . Howeverp 0.295). Along the same lines, the results obtained by Brockton et al. and Gonzales Moles et al. . Although it was previously established in invasive breast and urogenital cancer, it wasn´t found any work in the literature that had previously studied this relationship in OSCC ; expected since both are biomarkers of cell proliferation expressed by the fraction of cells that are actively passing through the cell cycle. This association was described in breast cancer by Kim et al. and in follicular lymphoma by Bedekovics et al.(p 0.004), which supports the potential usefulness of this biomarker in the histopathological classification of OSCC , expected because the PHH3 antibody is a specific IHC biomarker of MF and because it was used the same quantification criteria, both in the H-E slides for MAI and in those for IHC (When compared the immunostaining patterns of the studied biomarkers, in the Ki-67 slides, nuclear immunostaining was shown with a wide range of intensities, a factor that contributes to low reproducibility -11,29. O for IHC ,19,21.The main limitations of the study are the sample size, not including the tumor invasion front as an evaluation parameter because most of the biopsies were incisional, and not having the TNM stage of the cases analyzed.The PHH3, is a biomarker of cell proliferation specific to cells in mitosis, with respect to Ki-67, has been underinvestigated in the literature. In this work, a significant relationship was demonstrated between the immunoexpression of the phh3 and the survival of patients with OSCC. A significant association of MAI with survival time was also observed. Regarding the Ki-67, as was previously described in the literature, a positive association was confirmed between the degree of histological differentiation of the OSCC and the marker´s positive immunoexpression. Based on the results obtained, it is important to continue investigating the PHH3 proliferation biomarker in OSCC, with a uniformly standardized IHC protocol and in a large cohort of cases.


Although neoadjuvant therapy (Nac) is recommended for high-risk resectable pancreatic cancer (R-PDAC), evidence regarding specific regimes is scarce. This report aimed to investigate the efficacy of S-1 Nac for R-PDAC. In a multicenter phase II trial, we investigated the efficacy of Nac S-1  in R-PDAC patients. The protocol involved two cycles of preoperative S-1 chemotherapy, followed by surgery, and four cycles of postoperative S-1 chemotherapy. Two-year progression-free survival (PFS) rates were the primary endpoint. Overall survival (OS) rates and median survival time (MST) were secondary endpoints. Forty-nine patients were eligible, and 31 patients underwent resection following Nac, as per protocol . Per-protocol analysis included data from 31 patients, yielding the 2-year PFS rate of 58.1%, and 2-, 3-, and 5-year OS rates of 96.8%, 54.8%, and 44.0%, respectively. MST was 49.2 months. Intention-to-treat analysis involved 49 patients, yielding the 2-year PFS rate of 40.8%, and the 2-, 3-, and 5-year OS rates of 87.8%, 46.9%, and 33.9%, respectively. MST was 35.5 months. S-1 single regimen might be an option for Nac in R-PDAC; however, the high drop-out rate (36.7%) was a limitation of this study. However, evidence regarding specific regimens for Nac in R-PDAC is scarce, and participation in clinical trials is encouraged. A recent meta-analysis reported that the median overall survival (OS) of R-PDAC ranged from 12 to 25.3 months in upfront surgery, while the median OS associated with Nac group ranged from 10 to 50.2 months2. A separate meta-analysis based on intention-to-treat analyses has shown that patients with PDAC who received Nac had better long-term survival outcomes than patients who received upfront surgery 3. In a large-scale propensity-score matched analysis, Nac with upfront surgery was associated with improved survival 4. At present, at least eight randomized trials have investigated the use of Nac for R-PDAC. However, only two trials have reported long-term outcomes associated with preoperative treatment6. One of the trials was PREOPANC trial that showed survival benefit of preoperative chemoradiotherapy using gemcitabine with radiation 5. The other trial was Prep-02/JSAP-05 trial that showed survival benefit of Nac using gemcitabine with S-1 6. Thus, there is sparse evidence with respect to the administration of Nac for R-PDAC and the optimal protocol.The National Comprehensive Cancer Network (NCCN) guidelines recommend upfront surgery for patients with resectable pancreatic ductal adenocarcinoma (R-PDAC) , or neoadjuvant therapy (Nac) is also recommended for high-risk R-PDAC cases, such as those involving high levels of tumor marker, large primary tumors, weight loss, and so on9. As such, the main purpose of Nac in the treatment of R-PDAC is the prevention of postoperative recurrence, which is consistent with postoperative adjuvant therapy. Preoperative treatment of R-PDAC does not need to reduce tumor size, as even if the local effect is weak, R0 resection is possible in cases of stable disease.The theoretical benefits of neoadjuvant therapy in R-PDAC are (a) early treatment of potentially metastatic disease, (b) identification of patients diagnosed with metastatic disease during treatment who can be spared surgical procedures that are unlikely to have survival benefit, and (c) delivery of chemotherapy and radiation to the primary tumor while it is in an intact, well-vascularized condition. In cases of R-PDAC, downsizing strategies to improve R0 resectability are not as important as they are in cases of borderline resectable PDAC or locally advanced PDAC. It should be noted that early treatment of potentially metastatic disease involves the same strategy as postoperative adjuvant therapy; however, only approximately 60% of patients with PDAC receive postoperative adjuvant therapy in the real world setting due to perioperative morbidity or early disease recurrenceP < 0.0001)10. In the same trial, disease recurrence in the liver was observed in 29% of patients in the Gem group and in 19% of patients in the S-1 group (P = 0.0016). Based on these findings, we hypothesized that S-1 might decrease the risk of micrometastasis, such as occult liver metastasis, in cases of R-PDAC. We conducted a multicenter single-arm phase II clinical trial to investigate the efficacy of S-1 Nac in patients with R-PDAC. This study is the first trial to focus on the prevention of R-PDAC recurrence using only a single oral agent for preoperative treatment.Given the advantages of Nac, S-1  was selected for use in the present study; this regimen has been associated with relatively good outcomes as postoperative adjuvant therapy. The JASPAC01 trial has shown that the S-1 regimen is a superior adjuvant therapy to gemcitabine (Gem) in patients with R-PDAC  findings, under the informed consent. We enrolled 49 patients in this trial between January 2014 and October 2015. The CONSORT study flow summary is presented in Fig. Treatment-related adverse events (AEs) are shown in Table After completing Nac, 33 patients proceeded to surgery Fig. . ConcurrThe postoperative complications after pancreatectomy are shown in Table Of 31 patients who completed Nac followed by R0/R1 resection, 28 (90.3%) started S-1 adjuvant therapy and three patients did not (2 patients had poor performance status (PS) and one patient had bone metastasis before adjuvant therapy). Twenty-two patients completed all pre- and postoperative therapies as per study protocol (45% of 49 eligible patients and 71% of completed Nac patients). Meanwhile, among 12 patients who discontinued Nac, but received R0/R1 resection, 8 (66.7%) patients started adjuvant therapy and six patients completed it. Another four patients refused any chemotherapy.The 2-year progression-free survival (PFS) rate was 58.1% in 31 patients who completed Nac (per-protocol) followed by R0/R1 resection, and 40.8% in the intension-to-treat (ITT) analysis that included all 49 eligible patients  was 35.5 months. The observed 2-, 3-, and 5-year OS rates for per-protocol patients (n = 31) were 80.7%, 54.8%, and 44.0%, respectively, and the MST was 49.2 months. The 2-, 3-, and 5-year OS rates for off-protocol patients (n = 18) were 55.6%, 38.9%, and 11.1%, respectively, and the MST was 27.6 months  are shown in Supplemental Table Se3. Of the per-protocol (Nac complete) resection patients (n = 31), 18 (58.1%) patients experienced disease recurrence; this rate was lower than that of the off-protocol (Nac failure) resections . The rate of distant metastasis was high among off-protocol resections (75% vs. 48.3%). Liver recurrence (disease progression) for per-protocol resections, off-protocol resections, and non-resection was found in 25.8%, 33.3%, and 50% of cases, respectively.3. In fact, the present study subpopulation that met the JASPAC01 trial criteria showed relatively better survival . A recent meta-analysis, including 18 studies that involved 857 patients, has reported that the MST associated with Nac for patients with R-PDAC was 18.2 months (range: 10–50.2 months)2. In our trial, the MST of all 49 patients in the ITT analysis was 35.5 months, suggesting that survival outcomes in the present study were better than those in the studies included in the meta-analysis.This trial showed that the 2-year PFS rate was 58.1% among 31 patients who completed Nac before receiving R0/R1 resection (per-protocol). In the per-protocol analysis, the primary endpoint yielded values that were better than expected. However, in the ITT analysis of 49 patients, including those who failed the protocol, the 2-year PFS rate was 40.8%, which was below the reference value. Trials of adjuvant therapy, such as the JASPAC01 study, have reported better results than those of the ITT analysis, due to the exclusion of patients with metastasis confirmed during surgery or those with poor PS after resection and at the time of adjuvant therapy initiationP = 0.029), but not for Nac patients with R-PDAC 5. The subgroup settings for R-PDAC in the PREOPANC trial might be under-powered for analysis (65 Nac vs. 68 upfront surgery); furthermore, the median OS was 14.6 months in the Nac group, which was a disappointing finding. Nevertheless, the Prep-02/JSAP-05 trial  reported in preliminary findings that a significant benefit was observed with Nac compared with upfront surgery 6. At present, no other trials have delivered high-quality evidence on the impact of Nac on R-PDAC compared with upfront surgery.At present, the evidence level for Nac in R-PDAC remains low and the optimal protocol remains unknown. At least eight trials have compared the role of neoadjuvant treatment with that of upfront surgery in the outcome of R-PDAC, and their preoperative protocols vary . Of these, the PREOPANC trial  was the first randomized phase III trial to publish findings on the use of Nac for PDAC (including both borderline resectable and resectable); a preplanned subgroup ITT analysis demonstrated superior OS for Nac patients with BR-PDAC , and the R0 resection rate among the resected cases was 93% (40/43), suggesting that preoperative adjuvant therapy with S-1 monotherapy can achieve local tumor control in R-PDAC.The estimated MST of the ITT analysis of our Nac S-1 monotherapy was 35.5 months, while the estimated MST in the ITT analysis of the Nac Gem plus S-1 (GS) patients in the Prep-02/JSAP-05 was 36.7 months, with no difference in survival. No data were available for comparing S-1 with GS in R-PDAC survival; however, the GEST study, which was a randomized three-arm phase III study for advanced pancreatic cancer, showed non-inferiority of S-1, but did not show superiority of GS to Gem alone for OS14. The present trial included patients with an Asian background. However, pharmacokinetic and pharmacodynamic profile examination was outside the scope of the present study; thus, the exact reasons behind protocol failures associated with gastrointestinal toxicity remain unclear. We speculate that patients and physicians might be concerned about undergoing surgery when even minor gastrointestinal toxicities are present, as pancreatic resection is a major surgery and requires a cautious approach. An advantage of S-1 therapy is that it is a single oral agent that does not require intravenous treatment or frequent outpatient visits, thereby preserving medical resources. Nevertheless, the risk of gastrointestinal symptoms is high. In addition, this treatment might not be suitable for use in non-Asian populations.In this study, Nac was well-tolerated from the viewpoint of hematological markers; however, the gastrointestinal toxicity rate was high . S-1 is associated with a risk of gastrointestinal toxicities, which are generally higher in Caucasian than in Asian populations due to differences in pharmacokinetics and pharmacodynamicsThe second leading factor for Nac failure in the present study was patient refusal to continue with treatment despite the absence of severe AEs. In fact, patients were more likely to select surgical resection than to continue with Nac. As surgical resection is the only curative treatment for R-PDAC, patients might be eager to avoid tumor progression, which would make them ineligible for surgery. In fact, patients who refused to continue Nac had a strong desire for resection; four of five patients proceeded to surgery after discontinuing Nac. One of the possibilities for the strong desire for resection may be the patients’ medical expenses for chemotherapy, and the other may be the inconvenience of outpatient chemotherapy. Future trials should present evidence to patients considering Nac discontinuation, and patients should be informed about the importance of completing Nac in the absence of AEs rather than immediately undergoing surgery.In conclusion, S-1 neoadjuvant therapy for R-PDAC is safe and promising. S-1 monotherapy can be used as neoadjuvant therapy for patients with R-PDAC. However, well-designed, randomized controlled trials are required to better understand the safety profile and efficacy of this approach.16 .This study was a multicenter, open-label, single-arm phase II trial of Nac S-1 in patients with R-PDAC, conducted by the Hokkaido Pancreatic Cancer Study Group (HOPS)2, 50 mg for a body-surface area of 1.25–1.5 m2, or 60 mg for a body-surface area of > 1.5 m2, administered twice per day for 28 consecutive days, followed by a 14-day rest period (one cycle). The length of Nac was 12 weeks, which was slightly shorter than the reported median PFS of S-112, to balance the chance of resection with adequate tumor suppression and patient selection. After completing Nac, all patients underwent dynamic MDCT for restaging. All patients eligible for pancreatic resection underwent surgery 2–6 weeks after completing Nac. Patients with distant metastasis or locally advanced disease were excluded from this study, with further treatment at the discretion of the attending physician. All patients with R0/R1 surgical resection received four cycles of adjuvant S-1 therapy, which followed the same protocol as Nac. After completing therapy, all patients were followed up once every 3 months during the first 2 years, and once every 6 months from year 3 onwards. Tumor markers and MDCT of the chest/abdomen/pelvis or gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid enhanced magnetic resonance imaging were monitored during the follow-up period, which ended 2 years after the enrollment of the last patient.The Nac protocol involved two cycles of 40 mg of oral S-1 for a body-surface area of < 1.25 m3 and < 12,000/mm3, neutrophil count ≥ 2000/mm3, hemoglobin ≥ 9.0 g/dL, platelet count ≥ 100,000/mm3, total bilirubin ≤ 2.0 mg/dL [≤ 3.0 mg/dL in patients with biliary drainage], aspartate transaminase and alanine aminotransferase ≤ 100 IU [≤ 150 IU in patients with biliary drainage], creatinine ≤ 1.2 mg/dL, and creatinine clearance estimate by Cockcroft-Gault equation ≥ 50 mL/min). Exclusion criteria: history of S-1 treatment; history of PDAC treatment; another simultaneous or metachronous (within 3 years) cancer; current use of flucytosine, phenytoin or warfarin; watery diarrhea; pulmonary fibrosis or intestinal pneumonia; and confirmed or suspected pregnancy in women.Central review of diagnostic imaging was performed according to the definition of the NCCN guidelines 2012 (version 2) by a radiologist (YS) and verified by a surgeon (TN) and a physician (HK). Inclusion criteria: cytologically or histologically confirmed PDAC; age ≥ 20 years; Eastern Cooperative Oncology Group PS of 0 to 1; sufficient dietary intake; and satisfactory levels of blood parameters .Preoperative treatment-related AEs were assessed using the Common Terminology Criteria for Adverse Events (version 3.0). Surgical resection was performed by laparotomy and regional lymph node dissection was required. Resection of the portal vein/superior mesenteric vein was allowed. A DP-CAR due to suspected tumor involvement in tumor proximity to the bifurcation of the celiac and splenic artery was allowedThe primary endpoint was 2-year PFS. Secondary endpoints included OS, resection, and response rates, measured according to RECISTv1.1, pathological outcomes, preoperative treatment-related AEs, and surgical morbidity rate. PFS was defined as the time from registration with the trial to the date of first recurrence or disease progression, either local, distant, or both, whichever occurred first. Recurrence was defined as a radiologically rather than elevation of CA19-9. OS was defined as time from registration to the date of death from any cause and censored on the date of the final confirmation of survival for surviving patients. It was estimated using the Kaplan–Meier method.10. Given this threshold (29%) and expected 2-year PFS (48%), the sample size was calculated as 46, based on the Southwest Oncology Group one arm binomial tool, with a significance level of 0.025 and power of 80%. In anticipation of loss to follow-up, we expected to enroll 50 patients in the present study.To calculate the desired sample size for the present study, the threshold and expected values of the 2-year PFS rates were set at 29% and 48%, respectively. These estimates were based on the JASPAC01 study findings, where 2-year RFS was 48% in the S-1 group and 29% in the Gem group in an adjuvant settingSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.


In the original publication, the name of fourth and seventh authors were incorrectly published as: Takashi Sangai and Hidetoshi Kawagichi. The correct version of names are: Takafumi Sangai and Hidetoshi Kawaguchi as given in this Correction.The original publication has been corrected.