To describe a successful mentoring scheme designed for mid-career clinician scientists and to examine factors associated with mentee report of positive career impact.Mixed methods study including in-depth interviews and cross-sectional data collection via an online survey.Academy of Medical Sciences mentoring scheme set up in 2002 and evaluated in 2010.One hundred and forty-seven of 227 mentees took part in the study (response rate of 65%). Ten mentees, three mentors and eight stakeholders/scheme staff were selected to participate in in-depth interviews.Qualitative data: Interviews were transcribed, and free text was analysed to identify themes and subthemes in the narrative. Quantitative data: We examined the associations of reported positive career impact of mentoring by performing simple and multiple logistic regression analysis.Mentoring success was determined by a variety of factors including reasons for selection , mentee characteristics (e.g. younger age), experience and skills of the mentor (e.g. ‘mentor helped me to find my own solutions’) and the quality of the relationship (e.g. ‘my mentor and I set out clear expectations early on’).Our evaluation demonstrates that both mentor and mentee value mentoring and that careful planning of a scheme including preparation, training and ongoing support of both mentor and mentee addressing expectations, building rapport and logistics are likely to be helpful in ensuring success and benefit from the intervention. The National Health Service (NHS) in the UK is a unique forum for the pursuit of medical research that advances understanding and leads to novel diagnostics and interventions for disease. A key requirement for the effective translation of research is a pool of talented bioscience professionals equipped with the necessary skills to exploit this potential.15 Nonetheless, the future of academic medicine is often ‘on the critical list’.6 The development and implementation of an Integrated Academic Training Pathway by the National Institute for Health Research (NIHR) recognised this skills shortage by streamlining the funding schemes already available through major research funders such as the Medical Research Council, Wellcome Trust and others and provided young clinicians access to a clear roadmap into academic medicine.8 With this career pathway in place, it is now appropriate to consider what interventions may support young academics to progress.This need for well-trained clinician scientists is well documented.9 Major funders such as the National Institute for Health consider the calibre and past performance of mentors to be as important as their protégés, and mentoring is formally enquired about in applications for training fellowships.10 In the UK, such formal endorsement of academic mentoring has been slower to embed; the major UK funders all request that junior academics are mentored, but there is no formal audit about whether this is occurring.a This has meant that while there are pockets of good practice in some Higher Education Institutions,12 many aspiring early career academics have struggled to find suitable mentors to support them.Mentoring has been identified as a key mechanism to assist early career academics with career progression. In the US, formal mentoring is widely used and is part of organisational culture at all Academic Health Centers.b for early career academics13 to support what was seen to be a dwindling cadre of clinician scientists.4 The scheme is distinct in medical academia as its flagship component matches early and mid-career researchers, specifically academic Clinical Lecturers and Clinician Scientist Fellows, with independent mentors (Academy members) for one-to-one career development. A stakeholder describes the scheme as follows:Access to some very successful people who can give a broader perspective, the bigger picture, because the trouble is that with day-to-day clinical supervision and day-to-day work in laboratories in research you can lose the big picture very easily. It is external and it’s from well respected people.In this paper, we first describe mentoring as an intervention and go on to outline how the AMS scheme was set up. We then review the progress of the scheme to date and share the details of an evaluation recently undertaken, focusing on which factors are associated with reported positive career impact (mentee report).In 2002, the Academy of Medical Sciences (AMS)  set up a14 Modern mentoring models in Europe  are distinct from traditional mentor–protégé relationships that have been historically widespread in academic medicine and based on models of patronage.14 In developmental mentoring, the emphasis is on mentees to find their own solutions to the challenges of career advancement rather than straight advice giving or ‘gifting’ of opportunities that is common in patronage. This form of support is more effective in the longer term because mentees are equipped with new problem-solving skills unlike patronage which offers ‘a hand up’ for the duration of the relationship but does not foster self-sufficiency.16 Mentoring is separate from supervision/appraisal which enables the mentee to speak more freely than they might with a research supervisor.Mentoring is defined as ‘off-line advice from one person to another to assist the recipient in making significant advances in their personal, professional or career development’.c The scheme was devised by an expertd with experience of both scholarship and practical delivery of mentoring schemes within medicine. Objectives of the scheme were to offer support and inspire potential clinical academics to develop independent research careers by providing access to objective guidance and mentoring from independent mentors apart from the mentee’s home institution, but also to enable Academy Fellows to keep in touch with the realities of being a early career clinical academic. All Academy Fellows are eligible to be selected as a mentor, but participation is voluntary. The Academy operates an ‘opt out’ system; unless a Fellow specifically requests not to mentor, they may be selected by potential mentees. Mentors are not required to attend any specific training before being chosen but are strongly encouraged to attend a half-day workshop covering key mentoring skills . Mentors are also signposted to online resources about mentoring. The scheme was deliberately designed to be ‘light touch’ and not to require ‘mandatory training’ because it was felt that this additional burden would deter senior mentors from taking part.The Academy’s one-to-one mentoring scheme was set up in 2002. At inception, the scheme was only open to Clinician Scientist Fellows.e reviewed the quality and relevance of the scheme and recommended that the scheme continue and be expanded. As a result of this endorsement and a resultant uplift in funding, the Academy was able to broaden the scheme’s remit and invited research-active Clinical Lecturers to select a mentor from the Academy’s Fellowship. Therefore, all UK Clinical Lecturers and Clinician Scientist Fellows are both now eligible for the scheme. Increased funding has also enabled a broader career development scheme to be implemented, which involves local workshops on career support/mentoring and opportunities for networking, all led by a dedicated staff.Regular informal reviews of the mentoring scheme were conducted from its inception. The first independent assessment of the mentoring scheme was carried out in 2006. The Department of Health and the National Institute for Health Research Trainees Coordinating Centre (NIHR TCC)f Eighty percent of mentors hold or have held a clinical role.Since 2002, over 350 mentor–mentee pairs have chosen to take part in the scheme. Sixty-nine percent of mentees involved in the scheme to date have been men, and 47% of the mentees have been based in London or the South East. At the time of writing, 166 of the now 1091 Academy Fellows have been selected as mentors and have agreed to participate. Eighty-four percent of the mentors on the scheme are men, reflecting the Academy’s membership  and the gender balance of the professoriate of the UK medical academic community.g The evaluation consisted of both quantitative and qualitative analysis. The main evaluation of the scheme was conducted via an online questionnaire sent to all mentees and mentors who had taken part in the scheme to date. To inform development of questionnaires and guide appropriate choice of measures, semi-structured interviews were conducted with scheme mentors (n = 1), mentees (n = 3) and Academy staff running of the scheme (n = 3). Interviews were also conducted with stakeholdersh (n = 5). The sample for each of the surveys was provided by the Academy from their records of those involved in the one-to-one mentoring scheme. All mentors and mentees were given the chance to opt out of the evaluation by the Academy before their details were passed to the evaluating team; one mentor opted out at this stage. The survey was open between 27 October 2010 and 13 December 2010, and three invitation reminders were sent out to mentors and mentees during this time period.In 2010, the Academy commissioned an independent evaluation of the scheme.n = 7) and mentors (n = 2) were selected for an in-depth telephone interview to provide further qualitative data. Interviews were transcribed, and free text was analysed to identify themes and subthemes in narratives.From this sample of participants, a small sample of mentees , we disregarded those who had not indicated an impact either way (n = 9) and we compared the positive group with those who had reported no impact on their career (n = 48).We used simple logistic regression to examine the associations between the dependent variable of interest – mentees reporting that the scheme has had positive impact on their career progression – and a variety of other variables identified Then we grouped the independent variables of interest into six categories  and prodThe five categories for which a significant association with career impact was demonstrated were then used in a multiple logistic regression analysis to see whether the associations that were observed when each of the individual composite variables was considered in isolation were preserved when the influence of other composite variables was taken into account.One hundred and forty-seven of 227 mentees took part representing a 65% response rate. Most mentees held positions as either a Clinician Scientist Fellow (63%) or a Clinical Lecturer (30%). Fifty-six percent were based in London or surrounding areas, and 67% were men. Eighty-two of 166 mentors responded to the survey (50% response rate). Five mentors were deemed ineligible because they had not yet taken part in the scheme, giving an adjusted response rate of 51%. Of the 77 who remained, 91% were Professors and 29% were aged over 65 years. Eighty-one percent of mentors were men.Nineteen percent of mentees reported that the scheme has had a very positive impact on their career progression to date, and among this group, satisfaction with the scheme was high. Forty percent said that it has had a fairly positive impact. The remainder reported that there had been no impact of mentoring (35%) or ‘did not know’ (6%). No mentees reported a negative impact.inspiring greater confidence in their own abilities (59%);providing objective career advice and guidance (57%) to enable greater independence (60%); andfostering greater commitment to stay in academic medicine (53%).Respondents reported that their mentors helped them by:Across six categories, we examined the associations of a wide variety of variables with the dependent variable (reported positive career impact of mentoring). The full range of variables explored is detailed in There is also evidence that the scheme has had an impact on some of the mentors involved. Thirty percent strongly agreed that being a mentor has been worthwhile and 63% agreed somewhat. In particular, mentoring appeared to give some mentors a better understanding of the pressures faced by young academics, kept them up-to-date with the ‘bigger picture’ in their field and prompted them to think about their own career decisions and progress. Sixty-nine percent of mentors were willing to mentor again.n = 53) and a variety of other variables that are detailed in We examined the associations between mentor report of benefit from being a mentor  gives a greater degree of freedom to really say what you think no codes, no partial concealment … just honest opinion!It is enjoyable and provides a unique insight into the problems faced by my early career colleagues at a national level. I have got to know some very nice and interesting people! The process of mentoring someone else inevitably makes one reconsider your own position and reflect on what you want to do and achieve.Mentees, stakeholders and mentors alike commented on the particular benefits of the Academy scheme:It potentially offers access to a wider range of mentors than are available through institutional schemes. Also, I think it is very important to have a mentor from outside one's own institution to allow for impartiality and objectivity - the Academy's scheme offers this. (Mentee)Collection of the finest academics in medicine … so as a clinical academic trainee, you couldn’t wish for a better set of individuals. I think particularly at the end of the Clinical Lecturer’s stage, you have a lot of uncertainty around what you want to do and how you should progress and you really value the opportunity to talk to experienced individuals who will give you good advice. (Mentee)Mentors reported:The process of selecting a mentor was a clear area highlighted for potential improvement in the Academy scheme. Specifically, mentees requested more information about mentors and their interests and guidance in how to select a mentor. Both mentees and mentors agreed that it is the responsibility of mentees to make and maintain contact with their mentor. Contact between mentors and mentees, one to three times a year, was mainly face-to-face supported by email. Face-to-face meetings generally lasted 1–2 h and were mainly arranged as bespoke meetings.Both mentees and mentors emphasised the importance of the pair being based at different institutions in allowing the mentor to offer objective advice, although geographical distance was raised as a barrier to meeting by some mentees in the free text responses.Just over half of the mentors had received some training in mentoring either through the Academy or from elsewhere, and almost all mentors who had attended an Academy mentor development workshop found it very or fairly helpful. However, overall, mentors either did not see the need for training or did not regard training as important. Mentees, on the other hand, requested that mentors should have some training and would support training being offered to mentees as well.Mentors saw their role as encouraging mentees to talk openly, offering an alternative perspective and acting as a sounding board, and they saw a clear distinction between the role as a mentor and that of a supervisor. Mentees reported that the mentor’s role was to suggest specific career strategies, help mentees to find their own solutions and to be good listeners.Mentees were unsure when their mentoring relationship would end, and 28% of mentees would feel uncomfortable ending an unproductive relationship. Where mentoring relationships have ended, this tended to have been because the mentee had stopped contacting the mentor or had come to the end of a particular post.The distinct nature of the Academy scheme, which allows early and mid-career academics to be paired with senior scientists outside their immediate institution and reporting line, has had clear benefits for the participants to date. Impact of mentoring appears to be determined by mentee’s age, the frequency and duration of meetings, mentee’s expectations of the mentoring relationship, the quality of the mentee–mentor interaction and the perceived role of the mentor, and all of these variables remained significantly associated with reported positive career impact when entered into a multiple logistic regression analysis.The current data are limited in various ways. As the data are anonymous, it was not possible to pair mentor and mentee data. The response rate means that it is possible that response bias may have been introduced; it may be that those who did not respond had a different experience of the mentoring scheme from the participants in this study. We have collected data from mentors and mentees relatively early in their relationship; given that mentors and mentees only meet once or twice a year, it may be that we need to examine outcomes five years or more into the mentoring relationship in order to see the true benefit of the interventions. Finally, the data are cross-sectional, meaning that we cannot infer a direction of causality for the associations we report.18 but these data do not support this.Several key variables appear to be associated with a successful mentoring relationship. First, age seemed to matter with younger mentees reporting greater career impact than those aged over 45 years. This may be for a variety of reasons – it may be that the younger mentees joined the scheme more recently and the scheme has evolved over time, although this association persists when we controlled for ‘I am currently being mentored’ (data available from authors on request). It may instead be that those earlier in their careers are less likely to be critical of their senior mentors. Finally, it may be that younger scientists have more career flexibility and therefore benefit more from mentoring than those who are older and more ‘differentiated’ career-wise. Gender and career stage did not seem to matter; several commentators have suggested that women may preferentially benefit from mentorship,19 while others report no difference.20Mentees who went into the mentoring relationship with a clear idea of what they hoped to gain, and who had received personal recommendations of a specific mentor, reported greater career impact – this is intuitive. In terms of mentor skills, serving as a sounding board and encouraging the mentee to think for themselves and find their own solutions appeared to be the most valued. The quality of the mentoring relationship emerged as an especially important association with reported career impact. Mentees who reported having adequate time with their mentors and who described a warm reciprocal relationship were more likely to report benefit. Meeting at least once a year and sessions that lasted at least an hour were also important; this information is useful for guiding new mentor–mentee pairs on our scheme. One interesting question that our study did not address relates to how formal mentoring schemes need to be in order to provide benefit. The majority of mentors on the scheme did not favour mandatory training, and instead this scheme has evolved as ‘light touch’. Future studies would profitably explore the impact of various features of a more formalised scheme such as training, contracts, ongoing supervision, etc. Currently, opinion is divided with some authors suggesting better outcomes from informal relationships,The AMS runs a successful mentoring scheme that pairs senior academics from across the UK with postdoctoral scientists from a wide variety of institutions. Feedback from both mentors and mentees is positive.Academic medicine has always benefitted from a strong culture of informal ‘advice-giving’ from senior academics towards those seeking to develop their career. Mentoring harnesses this informal goodwill within a structured intervention to maximise the personal, professional and career development of early and mid-career academics. It is a low-cost, evidence-based intervention, which is easy to learn and as such has appeal in the current austere financial climate.Our paper has highlighted that mentoring schemes are likely to be enhanced by careful planning and education of mentee and mentor. Training needs to allow for discussion about expectations of the mentoring relationship, as well as more practical logistics such as frequency of meetings.


Glenoid loosening is the most common cause of failure in primary total shoulder arthroplasty (TSA) and often occurs years after the initial surgery. It is rare for a glenoid component to fail acutely. Several case reports of complete glenoid dissociation appear in the literature. It is important to report these failures to identify technical errors or component design flaws to improve outcomes in TSA. In this case report, we present an unrecognized acute failure of a cemented hybrid glenoid component at the time of surgery. Total shoulder arthroplasty (TSA) is an increasingly common procedure in the treatment of omarthrosis, with more than 50,000 total shoulder arthroplasties being performed annually in the United States . This nuSeveral case reports of complete glenoid dissociation appear in the literature –9. AmongThe patient is a 65-year-old man referred to the senior author's clinic with persistent right shoulder pain and severely restricted range of motion approximately 10 months after a primary anatomic TSA. The indication for the primary procedure was longstanding severe osteoarthritis with an intact cuff. The Exactech Equinoxe  shoulder system was used for the index procedure, with a pegged, caged glenoid (model number 314-02-04). Utilizing a standard deltopectoral approach, a 15 mm press fit humeral stem with a 47 × 18 humeral head, offset with a 4.5 mm replicator plate and a large beta curvature, pegged glenoid with cage was implanted. No complications had been noted at the time of the primary procedure. The patient had an uneventful, atraumatic postoperative course, but he failed to progress. His range of motion was severely restricted to about 15 degrees in all planes 8 months after surgery. A CT arthrogram was ordered to evaluate if there was a rotator cuff tear but instead found that the glenoid component had dissociated and was sitting posterior to the humeral head . The polThe patient was subsequently referred to the senior author and underwent revision TSA. The glenoid component was retrieved from the posterior capsule. The component failure occurred at the interface between one of the pegs and its metallic cap as well as at the cage component, which had disengaged from the polyethylene . InspectSix months later, the patient was progressing well. He regained 90 degrees of pain-free forward flexion (110 passive) with good rotation. He planned to continue to work on strength and range of motion with physical therapy. However, after the 6 month visit the patient was lost to follow-up. A good faith effort was made to contact the patient, but he could not be reached.Glenoid loosening is the most common cause of failure of contemporary TSA, comprising 32% of all complications and 7% of all indications for revision [A variety of glenoid designs exist which have had varying degrees of success. A cemented, pegged all-polyethylene glenoid is presently the gold standard –17, thouNonetheless, the differences in stiffness between bone and polyethylene (Young's modulus <1 GPa) can create a stress riser across the cement mantle leading to aseptic loosening of all-polyethylene glenoid components. This is the most common point of late failure with this type of implant. To address this issue, glenoid components have been modified to hybrid designs with a central cage component in which bone graft is placed to promote bone ingrowth. The cemented pegs impart rotational stability and initial fixation while the central cage promotes bone ingrowth to improve late fixation. These implants have shown some promise with mid-term results –23. The It is rare for a component to suffer an acute mechanical failure. Most of the acute glenoid failures described in the literature have been due to late traumatic events. Polyethylene inserts have dissociated from their metal base plates –9. One rThe cause of acute glenoid failure in the subject of this case report was likely multifactorial and in all likelihood related to inadequate glenoid exposure. The glenoid component failed through the cement-implant interface as well as within the implant itself. The metal cage and one of the three metal peg caps dissociated from the polyethylene and remained seated in the patient's glenoid, while the remainder of the component dissociated posteriorly. The cement mantle failed acutely, which may have been due to premature cement hardening before the glenoid was impacted or improper component positioning secondary to poor exposure. Less likely, the cement may not have adequately set when compression was released and the shoulder reduced. However, with this implant, it has been reported that holding compression while the cement sets is an unnecessary step.The dissociation of the metallic peg caps and cage from the polyethylene is not without precedence. In the two revised failures of the Exactech Equinoxe system noted previously, the central cage locking mechanism failed as well. Especially for this implant, glenoid exposure is paramount. The component requires “straight line” glenoid impaction. It must be directly perpendicular to the face of the glenoid to prevent damage to the locking mechanism of the central peg as it engages the drilled hole. Failure to do so may disengage the central peg from the polyethylene . This teGlenoid component failure remains the most common cause of TSA failure and has been the focus of recent implant design modifications. The design rationale behind this hybrid glenoid component is intriguing, but it remains to be seen if it is a durable solution for glenoid failure. Further investigation with long-term outcome and comparative studies are necessary. Regardless of which TSA system is being used, it is imperative that the surgeon achieve adequate exposure for proper component placement. Poor glenoid exposure was likely the biggest factor in the component failure presented here. Delay in detection of failure in TSA can lead to poor outcomes. Postoperative monitoring of the patient's clinical progress and close radiographic evaluation of implant position, glenoid tilt, and peri-implant osteolysis will aid in catching failures early. Ultimately, good implant design, surgical technique with adequate exposure, and vigilant postoperative surveillance give patients the best chance for a successful long-term outcome with TSA.


In endothelial cells, α-thrombin, a pro-inflammatory mediator that stimulates phospholipase Cβ, increases the cytosolic Ca2+ concentration and elicits clustering of IP3R3s. These responses, and the resulting Ca2+-dependent phosphorylation of myosin light chain, are prevented by depletion of either EB3 or mutation of the TxIP motif of IP3R3 responsible for mediating its binding to EB3. We also show that selective EB3 gene deletion in endothelial cells of mice abrogates α-thrombin-induced increase in endothelial permeability. We conclude that the EB3-mediated interaction of IP3Rs with microtubules controls the assembly of IP3Rs into effective Ca2+ signaling clusters, which thereby regulate microtubule-dependent endothelial permeability.The mechanisms by which the microtubule cytoskeleton regulates the permeability of endothelial barrier are not well understood. Here, we demonstrate that microtubule-associated end-binding protein 3 (EB3), a core component of the microtubule plus-end protein complex, binds to inositol 1,4,5-trisphosphate receptors (IP  •3 receptors (IP3Rs) bind to microtubule end-binding protein EB3IP•3R clustering and Ca2+ signals in endothelial cellsEB3, in turn, promotes IP•3R-EB3 interaction thereby contributes to endothelial barrier disruptionIP•In vivo EB3 deletion in endothelial cells protects from vascular hyperpermeability 3 receptors control clustering of IP3 receptors in endothelial cells and Ca2+ signaling, and thus permeability of endothelial barrier in inflammatory diseases.End binding proteins (EBs) mediate interactions between growing microtubules and intracellular structures. Geyer et al. demonstrate that interactions between EB3 and IP Activatractions , causes ractions . An increability .The roles of actin polymerization and disassembly of AJs in increasing endothelial permeability have received considerable attention, but microtubules may also contribute to the response by poorly understood mechanisms . Microtu2+ store. Inositol 1,4,5-trisphosphate receptors (IP3Rs) within ER membranes allow rapid release of Ca2+ from the ER  suppressing microtubule dynamics suggest an important role of microtubule cytoskeleton in organizing IP signals . Perturbia IP3Rs , prevent2+ waves , slows dembranes , disruptto IP3Rs , and abory cells . Many ofeability .3R3 that allows IP3Rs to associate with growing microtubule tips. Depletion of EB3 or disruption of the interaction with an IP3R3 point mutation prevents both clustering of IP3R3s and Ca2+ signals elicited by activation of PAR-1. Further, selective deletion of the EB3 gene in endothelium in mice inhibits the increase in endothelial permeability elicited by activation of PAR-1. Thus, microtubule-associated EB3 plays an obligatory role in organizing IP3-induced Ca2+ signaling, and, in turn, regulating endothelial permeability.Here, we demonstrate a direct interaction between EB3 and the S/TxIP motif within IP3 and release of Ca2+ from intracellular stores. Depletion of EB3, using small interfering RNA (siRNA) , which prevents binding of endogenous EBs to microtubule tips by forming non-functional dimers with endogenous proteins , α-thrombin stimulates PAR-1, a GPCR that causes activation of PLCβ, formation of IP (siRNA) A, signiflar Ca2+ A and 1B.proteins , also in signals B and S1C2+ indicators expressed in the lumen of the ER, G-CEPIA1er and the ratiometric indicator GEM-CEPIA1er (2+ content of the intracellular stores. Depletion of EB3 affected neither the organization of the ER nor the free Ca2+ concentration within the ER ([Ca2+]ER) (2+]ER was 583 ± 77 μM and 489 ± 53 μM (n = 6–16 cells) in control and EB3 siRNA-treated cells, respectively   C–1E. ER and increase in [Ca2+]c in cells expressing GFP-IP3R3(T804A) relative to those expressing GFP-IP3R3 (3R3(T804A) also formed fewer clusters than GFP-IP3R3, they barely responded to α-thrombin, and the few clusters that formed were short lived  kinase, facilitate cell contraction and destabilization of AJs  for these analyses because they are amenable to measurements of trans-endothelial electrical resistance (TEER), which directly report, in real time, changes in the integrity of AJs  was restricted to endothelial cells , a measure of endothelial vascular permeability to liquid, in naive lungs and after activation of PAR-1  in endothelial cells. The enhanced binding of EB3 to IP3R3 correlated with the requirement for EB3, rather than EB1, for α-thrombin-induced Ca2+ signaling. The requirement for EB3 may also result from its binding to additional, as-yet-unidentified proteins mediating clustering of IP3R3s. Indeed, residues surrounding the S/TxIP motif have been recently shown to recognize EB3  that dimerizes with native EB3 and prevents binding to microtubules confirmed that the requirement for EB3 is associated with its ability to bind to the growing microtubule tips. The concept of EB3 binding to IP3R3 is further supported by our FRET analysis showing a close and specific apposition of IP3R3 and EB3 at microtubule tips.The S/TxIP motif, located immediately downstream of the IPnize EB3 . Inhibit3R3 and IP3R2 are the major IP3R subtypes in endothelial cells. IP3R2s are concentrated in perinuclear regions and clustered in unstimulated endothelial cells. The perinuclear distribution of IP3R2  were generated using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies).Sources of the expression constructs, antibodies, and siRNAs are provided in 2, ∼90% purity) was synthesized by the Research Resources Core at UIC. Sources of other materials are provided in the relevant sections in DAPI was from Sigma. Human α-thrombin was from Fisher Scientific. The PAR-1-activating peptide -tagged EB proteins and their covalent immobilization on Ni-NTA columns for pull-down experiments using lysates from HEK cells expressing GFP-tagged proteins are described in HPAECs and HLMVECs (Lonza) were grown in EGM-2 medium supplemented with 10% fetal bovine serum (FBS) and EGM-2 MV Bulletkit or EGM-2 Bulletkit (Lonza), respectively. Cells were used between passages 2 and 6. CHO-K1 and HEK293 cells (ATCC) were grown in DMEM with 10% FBS (Gibco). Cells were transfected at ∼80% confluence using X-tremeGENE HP according to the manufacturer’s protocol (Roche) and used after 24–48 hr. For siRNA-mediated inhibition of protein expression, cells were treated with 70 nM siRNA  using Ge2, 5.6 mM glucose, and 25 mM HEPES (pH 7.4), and, after ∼10 min, cells were used for experiments at 25°C. Fura-2 fluorescence was excited at 340 and 380 nm and collected at 510 ± 80 nm using an Axiovert 100 inverted microscope (Carl Zeiss) equipped with Plan-Apo 60× with the numerical aperture (NA) 1.4 oil immersion objective, Lambda DG-4 switcher illumination system (Sutter Instruments), AxioCom Hsm camera (Zeiss), fura-2 filter set (Chroma), and AxioVision Physiology Acquisition module. Images were collected at 2-s intervals. Fluorescence ratios (F340/F380) were calculated within a circular region of interest (radius 3 μm) for each cell after subtraction of intracellular background fluorescence, determined by quenching fura-2 fluorescence by addition of 3 μM ionomycin with 5 mM MnCl2. [Ca2+]c was calculated from F340/F380 ratios by reference to Ca2+ standard solutions (Life Technologies). Measurements of cytosolic IP3 concentrations are described in HLMVECs grown on glass-bottomed dishes (Becton Dickinson) were loaded with fura-2 AM  for 20 min at 37°C in culture medium without supplements. The medium was then replaced with medium comprising: 150 mM NaCl, 4 mM KCl, 1 mM MgCl2+] within the ER ([Ca2+]ER) was measured in HLMVECs transfected with the GEM-CEPIA1er ratiometric indicator  equipped with a 63× 1.4 NA Plan-Apochromat oil immersion objective and a diode-pumped solid-state laser. Cells were excited at 405 nm, and emission was collected by two photomultiplier tubes (456–476 and 510–530 nm). Images (5–10/cell) were collected at 1-s intervals, averaged over four frames to optimize signal-to-noise ratios, and fluorescence ratios (R = F466/F510) were calculated. For calibration, the plasma membrane was permeabilized , Rmin and Rmax were then determined in the absence of β-escin in medium with (Rmax) or without (Rmin) Ca2+. The medium comprised: 140 mM KCl, 10 mM NaCl, 1 mM MgCl2, 20 mM HEPES, 3 μM ionomycin, and 3 μM thapsigargin (pH 7.4), and either 0.3 mM EGTA (Rmin) or 10 mM CaCl2 (Rmax). [Ca2+]ER was then calculated from the observed fluorescence ratio (R):D for Ca2+ = 558 μM  with BIG detector was used to capture dual-color images of m-Cherry-er with G-CEPIAer. For comparisons of relative changes in [Ca2+]ER D, a cros3R2 or IP3R3 , transfected with siRNA, and used after 72 hr . Changeskf,c, isolated lungs were perfused with RPMI medium at constant flow (2 ml/min), temperature (37°C), and venous pressure (4 cm H2O) as previously described (2O) and end expiratory pressures (2 cmH2O). Lung weight change was recorded with a force-displacement transducer . A 20-min equilibration perfusion established isogravimetric conditions, before measuring the gravimetric filtration coefficient  by comparing the rate of lung weight-gain during a baseline isogravimetric period with the rate after a change in hydrostatic pressure of at least 6 cm H2O for 20 min. The filtration rate was determined from the slope of the weight-gain curve between 15 and 20 min after PAR1-AP infusion. kf,c was calculated from:Animal care and handling were performed according to an approved protocol of the University of Illinois at Chicago Animal Care Committee. The transgenic mice are described in escribed . The prekf,c via a left atrial cannula perfused with buffer (50 mM Tris-Cl (pH 7.8), 0.2% Triton X-100, and protease and phosphatase inhibitor cocktails; 0.4 ml/min). Fractions were collected at 1-min intervals. Fractions 2 and 3, which were positive for VE-cadherin  and negative for smooth muscle actin, were used for assessment of EB3 expression by western blot.Endothelial lysates were collected after each measurement of ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.Comparisons between groups were made using ANOVA with the Tukey post-test method or Student’s t test. Significance values are shown by Y.A.K. conceived the project; M.G., F.H., Y.S., S.M.V., and Y.A.K. conducted experiments; M.G., C.W.T., and Y.A.K. analyzed and interpreted data; M.G., C.W.T., A.B.M., and Y.A.K. wrote the paper.


Glucagon promotes hepatic glucose production through Ca2+ signaling in the fasting condition. In this study, we investigated the role of CD38 in the glucagon signaling of hepatocytes. Here, we show that glucagon induces cyclic ADP-ribose (cADPR) production and sustained Ca2+ increases via CD38 in hepatocytes. 8-Br-cADPR, an antagonistic cADPR analog, completely blocked glucagon-induced Ca2+ increases and phosphorylation of cAMP response element-binding protein (CREB). Moreover, glucagon-induced sustained Ca2+ signals and translocation of CREB-regulated transcription coactivator 2 to the nucleus were absent and glucagon-induced glucose production and expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (Pck1) are remarkably reduced in hepatocytes from CD38−/− mice. Furthermore, in the fasting condition, CD38−/− mice have decreased blood glucose and hepatic expression of G6Pase and Pck1 compared to wild type mice. Our data suggest that CD38/cADPR-mediated Ca2+ signals play a key role in glucagon-induced gluconeogenesis in hepatocytes, and that the signal pathway has significant clinical implications in metabolic diseases, including type 2 diabetes.CD38 is a multifunctional enzyme for the synthesis of Ca Our data show that CD38 plays a critical role in glucagon-induced glucose production by producing cADPR in hepatocytes.In this study, we investigated whether CD38 is activated to produce a Ca2+ concentration ([Ca2+]i), resulting in gluconeogenesis in hepatocytes (HCs). Firstly, we tested whether our prepared HCs are optimal condition by measuring [Ca2+]i upon treatment with ATP, a well-known standard agonist−/− mice resulted in a rapid increase in [Ca2+]i, and the increased [Ca2+]i was sustained  were involved in glucagon-induced Ca2+ signals. Glucagon-induced Ca2+ increase was blocked by 8-Br-cADPR, an antagonistic analog of cADPR, but not by 8-Br-ADPR, an antagonistic ADPR analog, or Ned19, an NAADP mimic antagonist , an IP3 receptor antagonist. XeC inhibited glucagon-induced Ca2+ signaling  were suppressed in CD38-deficient HCs , CRTC2 from R&D Systems and Actin from Millipore. 8-Br-ADPR from Biolog Life Science Institute  and Ned19 from Enzo Life Sciences , and all other reagents were obtained from Sigma-Aldrich . Xestospongin C was from Santa cruz biotechnology.−/−; B6.129P2-Cd38tm/Lud) were purchased from Jackson Laboratory . Mice were bred and kept in animal housing facilities at Chonbuk National University Medical School under SPF conditions. All experimental animals were used under a protocol approved by the institutional animal care and user committee of the Chonbuk National University Medical School (CBU 2014-00031). Standard guidelines for laboratory animal care were performed in accordance with the Guide for the care and use of laboratory animals published by the National Institutes of HealthCD38 knockout mice  with Williams’ Medium E (Life Technologies) containing 1% L-glutamine and penicillin/streptomycin and supplemented with 10% fetal bovine serum (Life Technologies). Four hours after plating, the medium was replaced with fresh medium. To experimental treatments, HCs were incubated overnight in serum-free Medium 199 (Life Technologies) before the addition of glucagon (100 nM).Primary hepatocytes (HCs) were isolated from 8- to 12 weeks old male C57BL/6 J mice as described previouslyGlucose production assays were carried out as described2O with protease and phosphatases inhibitors. Samples were then mixed KOH (1:2), boiled for 25 min, and washed with 70% ethanol. The pellet was dried and dissolved in 100 μl H2O, and the glycogen content was assessed using the glycogen assay kit  according to the manufacturer’s instructions.Glycogen content were measured carried out as describedLiver tissues were fixed in 10% formalin for 24 h and embedded in paraffin. Sections (5 μm) were stained for glycogen using PAS stain (Sigma-Aldrich) according to the manufacturer’s instructions. The sections were counterstained with hematoxylin and examined by light microscopy.Mice were fasted overnight and injected intraperitoneally with pyruvate (2 g/kg body weight). Tail vein blood was taken for glucose measurement at 0, 15, 30, 60, 90 and 120 min after pyruvate injection. Blood glucose values were determined using a LifeScan automatic glucometer.Total cellular RNAs from whole liver or from primary HCs were extracted using the RNeasy kit . cDNA was synthesized by reverse transcription from 50 ng total RNA using a cDNA Reverse Transcriptase Kit . The PCR reaction was carried out in 384-well plate using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Real-time PCR primers for mouse G6Pase, Pck1, PGC1a and GAPDH were as follows: G6Pase ; Pck1 ; PGC1alpha ; GAPDH .2+]i in HCs were determined as described previously+]i were determined at 488 nm excitation/530 nm emission by air-cooled argon laser system. The emitted fluorescence at 530 nm was collected using a photomultiplier. The image was scanned using a confocal microscope (Nikon). For the calculation of [Ca2+]i, the method of Tsien et al.2+]i = Kd(F − Fmin)/(Fmax − F), where Kd is 345 nM for fluo-4, and F is the observed fluorescence level. Each tracing was calibrated for the maximal (Fmax) by addition of ionomycin (8 μM) and for the minimal intensity (Fmin) by addition of EGTA 50 mM at the end of each measurement.Changes of Ca+i in HCs3, and vortexed. The resulting KClO4 precipitate was removed by centrifugation at 20,000 × g for 10 min. Supernatant was adjusted to pH 8.0. To remove all contaminating nucleotides, the samples were incubated with the following hydrolytic enzymes overnight at 37 °C: 0.44 unit/ml nucleotide pyrophosphatase, 12.5 units/ml alkaline phosphatase, 0.0625 units/ml NAD glycohydrolase, and 2.5 mM MgCl2 in 20 mM sodium phosphate buffer, pH 8.0. Enzymes were removed by filtration using a Centricon-3 filter (Amicon). To convert cADPR to β-NAD+, the samples (0.1 ml/tube) were incubated with 50 μl of cycling reagent containing 0.3 μg/ml Aplysia ADP-ribosyl cyclase, 30 mM nicotinamide, and 100 mM sodium phosphate, pH 8.0, at room temperature for 30 min. The samples were further incubated with the cycling reagent (0.1 ml) containing 2% ethanol, 100 μg/ml alcohol dehydrogenase, 20 μM resazurin, 10 μg/ml diaphorase, 10 μM riboflavin 5′-phosphate, 10 mM nicotinamide, 0.1 mg/ml BSA, and 100 mM sodium phosphate, pH 8.0, at room temperature for 2 h. An increase in the resorufin fluorescence was measured at an excitation of 544 nm and an emission of 590 nm using a SpectraMax gemini fluorescence plate reader (Molecular Devices Corp.). Various known concentrations of cADPR were also included in the cycling reaction to generate a standard curve.cADPR was measured by some modification of the cycling method described previouslyData represent means ± standard error of the mean (SEM) of at least three separate experiments. Statistical analysis was performed using Student’s t-test. A value of P < 0.05 was considered significant.How to cite this article: Rah, S.-Y. and Kim, U.-H. CD38-mediated Ca2+ signaling contributes to glucagon-induced hepatic gluconeogenesis. Sci. Rep.5, 10741; doi: 10.1038/srep10741 (2015).


While hydrogen peroxide (H2O2) affects Ca2+-activated CaMKII in vitro, Angiotensin II (Ang II)-induced CaMKIIδ signaling in cardiomyocytes is Ca2+ independent and requires NADPH oxidase-derived superoxide, but not its dismutation product H2O2. To better define the biological regulation of CaMKII activation and signaling by Ang II, we evaluated the potential for peroxynitrite (ONOO−) to mediate CaMKII activation and downstream Kv4.3 channel mRNA destabilization by Ang II. In vitro experiments show that ONOO− oxidizes and modestly activates pure CaMKII in the absence of Ca2+/CaM. Remarkably, this apokinase stimulation persists after mutating known oxidation targets , suggesting a novel mechanism for increasing baseline Ca2+-independent CaMKII activity. The role of ONOO− in cardiac and neuronal responses to Ang II was then tested by scavenging ONOO− and preventing its formation by inhibiting nitric oxide synthase. Both treatments blocked Ang II effects on Kv4.3, tyrosine nitration and CaMKIIδ oxidation and activation. Together, these data show that ONOO− participates in Ang II-CaMKII signaling. The requirement for ONOO− in transducing Ang II signaling identifies ONOO−, which has been viewed as a reactive damaging byproduct of superoxide and nitric oxide, as a mediator of GPCR-CaMKII signaling.Ca Similarly, nitric oxide (NO) cannot on its own account for Ang II effects as SOD would not be inhibitory. However, O2.− is not a robust oxidizing species and does not react with methionine, which is oxidized in CaMKII in response to Ang II2.− mediates Ang II signaling.Angiotensin II (Ang II) induces NADPH oxidase-dependent generation of partially reduced oxygen species , which is generated by the diffusion-limited reaction of O2.− and NO8− was shown to react with pure Ca2+/CaM-activated CaMKII in vitro to generate Ca2+-primed autonomous activity10− or contaminants from its synthesis or spontaneous decomposition account for CaMKII responses; (c) determine if CaMKII regulation strictly depends on Ca2+/CaM activation; and (d) address whether ONOO− is required for Ang II stimulation of CaMKII in living cells. Therefore, we tested the hypothesis that ONOO− participates in Ang II-induced CaMKII signaling by conducting experiments with purified CaMKII and cardiac and neuronal cells.One possibility is that Oin vitro experiments with ONOO− and purified CaMKIIδ reported increased autonomous activity following Ca2+ priming10− (27.64% ± 1.53%). To extend these studies, the effect of ONOO− on CaMKIIδ methionine oxidation was determined and compared with a control consisting of neutral pH decomposition products and potential residual species used in the synthesis of ONOO− (nitrite (NO2−), nitrate (NO3−), H2O2). First, recombinant human CaMKIIδ was activated by Ca2+ and CaM in the absence of Mg2+ and ATP and then treated with ONOO−. CaMKIIδ M281/282 oxidation state was assayed by immunoblot using a site-specific redox antibody. A low signal for M281/282 oxidation was observed for no ONOO− treatment and treatment with low concentrations of ONOO− (0.2 and 2 μM), while 20 and 200 μM ONOO− enhanced methionine oxidation of CaMKIIδ 8−, as opposed to decay products or contaminants from synthesis, ONOO− was added to neutral buffer for 2–5 minutes before exposing these products to CaMKIIδ. In contrast to ONOO−, decayed ONOO− (dONOO−) resulted in no CaMKIIδ methionine 281/282 oxidation . Remarkably, immunoblotting showed that CaMKIIδ M281/282 oxidation induced by ONOO− was similar in the presence and absence of Ca2+ and CaM (−-induced oxidation does not require Ca2+-CaM binding to permit CaMKII oxidation in the autoregulatory domain (ARD).Based on cellular results showing Ca and CaM . While p2+/CaM may lead to activity independent of Ca2+/CaM priming. The effect of ONOO− on CaMKIIδ apoenzyme was therefore determined by studying enzymatic activity under low Ca2+  conditions. Despite preventing canonical activation by omitting CaM and chelating Ca2+, ONOO− (but not dONOO−) induced a small, yet significant increase in autonomous activity in freshly isolated enzyme . Thus, these experimental data show that ONOO− induces a small, yet significant increase in apoCaMKIIδ activation without a requirement for Ca2+ and CaM in either the oxidation step or to increase basal activity by prior disinhibition of the ARD.This result led us to consider that CaMKII oxidation in the absence of Caactivity . Specifi−-induced apoCaMKII activity in vitro is less than seen with priming in saturating Ca2+/CaM . Nevertheless, this increase in apoCaMKII activity could be biologically relevant. First, because maximal activation is likely never attained in cells, normalization to maximal activity is not a representation of physiological activity. Second, a small degree of apoCaMKII activation could lead to autophosphorylation, which would amplify Ca2+-induced activation. Third, basal activation is sustained and so not limited by the incidence of action potentials. Therefore, enhanced modest activation could be functionally significant over a sustained period of time. Finally, increased basal activity plus the enhanced autonomous activity previously reported following Ca2+/CaM priming10−-induced signaling by CaMKII. Therefore, we explored the molecular basis of ONOO− action on apoCaMKII.The ONOO2+-CaM-primed CaMKII activity by H2O2, ONOO− and NO11011− effect on wildtype human apoCaMKIIδ activity using double (M281V/M282V) and triple (M281V/M282V/C290V) mutants of these known oxidation sites in the ARD. To minimize complications with CaMKIIδ oxidation during storage and freezing, we expressed and purified the wildtype, double and triple mutants simultaneously and used fresh non-frozen enzyme to perform the comparisons.Because prior experiments demonstrated roles for M281/282 and C290 in facilitation of Ca− activation of CaMKII was similar for wildtype and the double and triple mutants  and H2O2 is much more stable than ONOO−, which decomposes in seconds. Therefore, the novel Ca2+-independent increase in apoenzyme activity described here must involve a residue that has not been previously implicated in redox regulation of Ca2+/CaM-primed CaMKII. Apparently, this site is readily oxidized by ONOO−, but less reactive H2O2 is not effective unless the enzyme is partially activated by mutagenesis of the ARD.This surprising result led us to examine the effect of Hin vitro studies using human CaMKIIδ are consistent with the Ca2+-independent activation described previously in cardiomyocytes− may contribute to Ang II-induced activation of CaMKII in cells, at least in part through Ca2+-independent modifications. Thus, to examine the mechanisms underlying Ang-II redox signaling, we turned to the Ang II-induced CaMKII activation shown previously to downregulate the Kv4.3 K+ channel, which is produced by mRNA destabilization induced by AUF1 protein binding to the Kv4.3 3′ untranslated region (3′ UTR) in response to CaMKII phosphorylation25Contrary to prior reports, the above −. First, the impact of the ONOO− scavenger FeTPPS  porphyrinato Iron (III)) was determined. 50–100 μM FeTPPS blocked the Ang II-induced reporter responses, increased the baseline reporter signal and altered cell morphology, suggesting possible toxicity. To address this limitation, the ONOO− scavenger UA was used2.− to yield ONOO−2.− for this signaling− with UA show that NO is not sufficient for this Ang II effect. Instead, these results suggest a requirement for ONOO− in cardiac and neuronal cell Ang II-Kv4.3 mRNA responses.Because this suggests shared signaling, the four cell systems were also used to test whether the Ang II-Kv4.3 effect requires ONOO− becomes protonated under physiological conditions to peroxynitrous acid (ONOOH), which in turn undergoes homolytic scission to the oxidizing species hydroxyl radical (.OH) and the nitrating species nitrogen dioxide (.NO2). An index of ONOO− generation and signaling is tyrosine nitration, which is induced by .NO2 and not the individual reactions of O2.−, H2O2 or NO. Therefore, to independently test whether Ang II signals via ONOO−, tyrosine nitration was assayed by immunoblot from H9C2 cell extracts by quantifying the formation of nitrotyrosine (NO2-Tyr) adducts. In these experiments, the strongest signal was from a 53 kD protein, which may be derived from desmin, a key target of tyrosine nitration in the heart−, not only did Ang II induce tyrosine nitration in H9c2 cells but most importantly, this effect was abolished by either inhibition of NO synthesis with L-NAME or by the ONOO− scavenger UA  indicates the diversion of NO signaling away from guanylate cyclase activation and (b) yields potent oxidizing and nitrating species. Thus, the reaction of ONOO− with proteins often inactivates or impairs function− can act as an intermediate in G-protein coupled receptor (GPCR) signaling by activating a functionally-significant downstream kinase defines a regulated and central biological action for this reactive species. Indeed, our results suggest that other receptor-NADPH oxidase effects that have been attributed to O2.− or its dismutation product H2O2 may in fact be mediated by ONOO−.These data reveal a novel signaling function for ONOO2.− and its dismutation product H2O2. Because Ang II and CaMKII oxidation are associated with cardiac pathology23− is required for Ang II-induced CaMKII activation, suppressing ONOO− generation and steady state concentrations in the heart may be of clinical benefit. It will also be interesting to determine whether this novel mechanism contributes to the clinical efficacy of Ang II AT1 receptor blockers (ARBs) and ACE (angiotensin converting enzyme) inhibitors. Furthermore, because Ang II-NADPH oxidase-Kv channel signaling occurs in neurons7− is required for Ang II-Kv channel signaling in neuronal cells  plus 8% horse serum and 4% FBS. The following day cells were transfected using Lipofectamine Plus reagent (Invitrogen) with 0.5 μg DNA per well following the manufacturer’s protocol. After 4 hours, the medium was replaced with the fresh medium supplemented with 1 mM dibutryl-cAMP. Two days later, cells were treated with vehicle or Ang II for 6 hours in serum free medium. Neuro-2A cells were plated on 24 well plate at a density of 2 × 105 per well in MEM (Invitrogen) with 10% FBS, 1 mM sodium pyruvate and 25 mM HEPES, pH 7.5. The following day, the cells were transfected using FuGENE HD Transfection Reagent (Promega) at a 4:1 ratio to DNA (0.75 μg per well) following the manufacturer’s protocol overnight. Then the medium was replaced with MEM 1% FBS for two days. Cells were then treated with vehicle or Ang II for 6 hours in serum free medium. H9C2 cells were plated on 24 well plate with density of 3.75 × 104 per well in DMEM (Invitrogen) with 10% FBS. Cells were cultured 4 days and then were transfected using same method as used with Neuro-2A cells. The following day the medium was replaced with serum free medium and treated with vehicle or with Ang II for 24 hours.Neonatal Sprague-Dawley rat ventricular cardiomyocyte cultures were generated by a protocol approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Culture, transfection and Ang II treatments in serum-free medium (which yields nonbeating cardiomyocytes) were performed as previously described2Inhibitors were purchased from Sigma-Aldrich. 100 μM L-NAME (NG-Nitro-L-arginine methyl ester), D-NAME and 300 μM uric acid (UA) were applied for 30 minutes prior to treatment with 100 nM Ang II. IP and SDS-PAGE followed published protocolsH9C2 cells treated with vehicle or Ang II (100 nM) in the presence or absence of L-NAME or UA as described above. Cells were harvested in lysis buffer  and proteins resolved by SDS-PAGE. Nitrotyrosine was detected by immunoblotting after overnight incubation with a 1:1000 dilution of a monoclonal antibody , followed by incubation with an Horse radish peroxidase-conjugated secondary antibody and chemiluminescent detection using the Immun-Star substrate system (BioRad). Densitometric analysis was performed using ImageJ software (NIH) and the resulting data was normalized to β-actin levels. Statistical evaluation was performed by one-way ANOVA with Tukey’s multiple comparisons test.−1 cm−1) in 500 mM NaOH.Peroxynitrite was synthesized from nitrite and hydrogen peroxide using a quenched-flow reactor as described previously−-induced CaMKII oxidation. CaMKII oxidation in the presence of Ca2+/CaM was carried out in a reaction mixture containing 100 mM Tris, pH 7.0, 0.3–1 μM CaMKIIδ, with 6–10 μM of CaM and 0.5 mM of Ca2+, or in the absence of Ca2+/CaM and presence of 5 mM EGTA. ONOO− was added last to the side of the reaction tube and rapidly mixed with the kinase by vortexing. For control reactions using the decayed ONOO− product, the reaction was preincubated in the Tris pH 7.0 buffer 2–5 minutes to induce decomposition before addition of kinase. The ability of 100 mM Tris pH 7.0 to maintain the reaction pH was experimentally verified.Recombinant human CaMKIIδ , and either 1 mM CaCl2 and 10 μM CaM for maximal activation as described previously262+/CaM) for measuring activity independent of Ca2+/CaM. The reaction proceeded at 30 °C for 3 minutes (within the linear range) before spotting onto P81 filter paper and thoroughly washed in 75 mM phosphoric acid. The Ca2+/CaM independent activity was compared between the mutants and across the oxidative agents, as specific autonomous activity (Ca2+/CaM-independent activity divided by Ca2+/CaM-dependent activity). For activation assays comparing mutant proteins, CaMKII wild type and mutants were expressed in Hi5 cells using baculovirus. Hi5 cells expressing wildtype, double mutant  and the triple mutant  were collected by centrifugation. The cells were lysed using a microfluidizer in buffer containing 50 mM HEPES, 100mM NaCl, 0.1 mM EDTA, and protease inhibitor with 500μM AEBSF HCl, 300 nM Aprotinin, 6 μM E-64, 20 μM Leupeptin. The lysate was then spun at 30000 rpm for 30 min and the supernatant was used to load onto a NiNTA resin. After binding, the resin was washed and the protein was eluted using buffer containing 50mM HEPES, 100 mM NaCl, 0.1 mM EDTA, 25 mM Imidazole. The elute was then passed through HiTrap desalting column and exchanged to buffer containing 50 mM Tris, 100 mM NaCl, 0.1 mM EDTA. The protein was quantified and 1 μM concentration of the protein was used for experiments as described above.For activity measurements, 1 μM hCaMKIIδ wild type and mutants were oxidized as described above in 200 uM ONOO− or dONOO− at room temperature, samples were boiled in SDS-PAGE loading buffer and separated on a 8.5% acrylamide or 10% NuPage gel, which was transferred onto a Trans-Blot Transfer Medium Pure Nitrocellulose Membrane (0.2 μm) (Bio-Rad). Following blocking with bovine serum albumin or milk, the membrane was incubated overnight at 4 °C with anti-rabbit polyclonal against M281/282 oxidized-CaMKII antibody (1:2000) (Millipore) and anti-goat polyclonal antibody against total CaMKIIδ (Santa Cruz Biotechnology). Membranes were washed for 5 minutes 4 times with PBST or TBSt and then incubated 1 hour in room temperature with IRDye 800CW Gt Anti-goat IgG (H+L) antibody and IRDye 680LT Anti-rabbit IgG (H ± L) antibody  (Li-COR Bioscience). Infrared fluorescence was detected by Odyssey Imaging System.For M281/282 oxidation blots, 5–10 minutes after treatment with ONOOData are quantified as the mean ± standard error of the mean (s.e.m.). Statistical significance was determined by Student’s t-test for experiments with two experimental conditions and one-way ANOVA followed by Dunnett’s, Bonferroni or Tukey’s post-test for experiments with more than two experimental conditions.How to cite this article: Zhou, C. et al. Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling. Sci. Rep.6, 23416; doi: 10.1038/srep23416 (2016).


Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca2+. Furthermore, Cdc25 phosphatase is also phosphorylated by Cmk1, inducing cell cycle arrest in response to an increase in Ca2+. Moreover, cmk1 deletion shows a high tolerance to chronic exposure to Ca2+, due to the lack of cell cycle inhibition and elevated Prz1 activity. This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription. These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.Calcium signals trigger the translocation of the Prz1 transcription factor from the cytoplasm to the nucleus. The process is regulated by the calcium-activated phosphatase calcineurin, which activates Prz1 thereby maintaining active transcription during calcium signalling. When calcium signalling ceases, Prz1 is inactivated by phosphorylation and exported to the cytoplasm. In budding yeast and mammalian cells, different kinases have been reported to counter calcineurin activity and regulate nuclear export. Here, we show that the Ca Calmodulin (CaM) is a small ubiquitous adaptor protein that belongs to the EF-hand family. In CaM, conformational change is driven by Ca2+-binding to its four EF-hands, and thereby triggering the capacity of CaM to interact with different proteins, including the Ca2+/CaM-dependent kinase (CaMKK) family of proteins and phosphatase calcineurin   ,4. When Ca2+/CaM . Thus, C  for CaMKK. Cmk1 activity is dependent on Ca2+ and CaM, and increases when threonine 192 is mutated to aspartic acid (Cmk1-T192D), which is consistent with the conclusion that Cmk1 activity is regulated in a manner homologous to mammalian CaMK1  in its activation loop (Thr-189) when cells are starved of nitrogen or glucose ,13. Howe2+/CaM-dependent phosphatase calcineurin, Ppb1 in S. pombe, is known to be one of the central elements of calcium response in a wide variety of cell types and organisms. Like CaM-dependent kinases, calcineurin is involved in various Ca2+-mediated processes in human cells, including lymphocyte activation, cardiac hypertrophy, apoptosis, angiogenesis and memory development   ,21; the , Hrr25) ; the CDK, Hrr25) ; dual sp, Hrr25) ; MAPK p3, Hrr25) ,29. Crz1In fission yeast, a genetic screen to isolate negative regulators of Prz1 transcription factor identified seven genes , two neg2+. We found that Cmk1 is a target of the Prz1 transcription factor, which in turn is involved in negatively regulating Prz1 activation in Ca2+ signalling. In addition, we have identified a second CaMKK in fission yeast, the Ckk2 kinase, which is involved in the activation of Cmk1 in response to Ca2+.Here, we focus on the physiological function of Cmk1 in response to an increase in cellular Ca2+ sensitivity assays, 10-fold serial dilutions of exponentially growing cells were spotted onto agar plates and treated with the indicated dose of CaCl2.Strains were manipulated using standard techniques ,32 and acmk1 coding sequence was polymerase chain reaction (PCR)-amplified from S. pombe cDNA using specific primers and cloned into the XhoI/ BamHI site of the nmt (no message in thiamine)-driven expression vector pREP3x to create the pREP3x-cmk1. Then, the PstI/NotI fragment of the pREP3x was ligated to the pREP1 vector to obtain pREP1-cmk1. To analyse the effect of the different mutants, single point mutations were introduced using pREP1-cmk1 as a template and the oligonucleotides listed in Supplementary Table S2.The 2VO4 and 50 mM NaF. Cells were incubated when required, with the calcineurin-inhibitor FK506 (Sigma F4679) (0.5 μg/ml) for 1 h.Whole-cell extracts were obtained from exponentially growing cells by mechanical disruption in standard NP-40 lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 2.5 mM EDTA, 0.002% NP-40, 50 mM NaF, protease inhibitor tablet ). The GFP-Prz1 protein was immunoprecipitated from cell extracts with 1 μg of monoclonal anti-GFP antibody  and using 30 μl of protein A/G Sepharose beads . Protein was resolved by SDS-PAGE using 10% gels with an acrylamide/bisacrylamide ratio of 99:1. Proteins were transferred to nitrocellulose membranes and the membranes were blocked with 5% milk in Tris-buffered saline with 0.05% Tween. The antibody probes used for immunoblotting were: monoclonal anti-HA ; polyclonal anti-GFP ; polyclonal anti-PSTAIR , polyclonal anti-phospho-Akt, which recognises phosphorylated consensus R-XX-S/T  and polyclonal anti-cdc25 (1/1000). Horseradish peroxidase–conjugated anti-mouse or anti-rabbit antibodies  were used as secondary antibodies. Membranes were developed by enhanced chemiluminescence . The λ-phosphatase (NEB) was used for phosphatase treatment. Cell extracts were incubated at 30ºC for 1 h in the presence or absence of λ-phosphatase. As a control, λ-phosphatase activity was inhibited with 5 mM NaTM qPCR Supermix Universal . qPCRs were denatured at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The oligonucleotides used for qPCR amplification are listed in Supplementary Table S2. The act1 gene encoding actin was used to normalise. For purposes of plotting graph, data were also normalised to wild-type without treatment values. The data shown are representative of three independent experiments.Total RNA was isolated using RNeasy plus mini kit according to the manufacturer's instructions . RNA quality and concentration was determined using a Nanodrop 1000 Spectrophotometer . cDNA was synthesised with the High-Capacity cDNA Reverse transcription kit  using random primers. Relative quantification of cDNA was carried out in duplicate on an Mx3000P qPCR  using Express SYBR® GreenERnmt promoter. The cells were incubated with 0.5 mM D-luciferin at 30°C and CDRE transcriptional activity was measured as described previously  AGCCTC/GAGGCT motif , which has the lysine 60 from the ATP-binding site mutated to alanine, in GFP-prz1 cells in the presence of Ca2+. We observed that Prz1 nuclear localisation was altered, displaying a cytoplasmic pattern when Cmk1 was overexpressed, while Prz1 was maintained in the nuclei with Cmk1-KA expression  which recognises proteins containing phospho-serine/threonine preceded by arginine at the -3 position. This substrate specificity is characteristic of many Arg-directed kinases, including Cmk1 . We immunoprecipitated endogenous GFP-Prz1 from wild-type cells treated or untreated with Can Figure . When the Figure . This reGFP-prz1 and a plasmid overexpressing Cmk1 or an empty plasmid as a control. As Figure We next examined the interaction between Cmk1 and Prz1 by co-immunoprecipitation. Cmk1 protein was immunoprecipitated from cells containing endogenous 2+ homeostasis. Therefore, we screened the growth of wild-type and Δcmk1 cells at different concentrations of Ca2+. As shown in Figure Δcmk1 cells displayed a resistant phenotype to high Ca2+ concentrations. This observation is consistent with its role as a negative regulator of Prz1 , rendered it insensitive to Ca2+ . Therefore, we examined the double mutant Δppb1 Δcmk1 at high Ca2+ concentrations and, as expected, deletion of ppb1 abolished the Δcmk1 Ca2+ resistance  arrests cell cycle progression Figure  (9). We 2D arrest kinases ,42–43. Ts Figure  and B.cdc25–9A cells. This result is consistent with our previous report  for CaMKK to aspartic acid (Cmk1-T192D) converts Cmk1 into a constitutively active form  and overexpression of Ssp1 did not display the phosphorylation pattern of Cmk1 . Regarding the Ca2+ stress response, Δssp1 cells were highly sensitive to Ca2+, in contrast to the resistance of Δcmk1 cells . Taken together, these results indicate that Ssp1 is not the kinase involved in Cmk1 regulation in the Ca2+ stress response.Previous studies have postulated that Ssp1 is fission yeast CaMKK ,44. AccoIn addition to Ssp1, we found a second sequence homologous to mammalian CaMKK: SPC1919.01, named Ppk34 for the uncharacterised putative protein kinase 34 in a systematic deletion analysis of fission yeast kinases . Due to 2+ stress of single ckk2 or double ckk2 cmk1-deleted cells and we observed that Δckk2 and Δcmk1 Δckk2 cells were resistant to Ca2+ stress as Δcmk1 cells are  does not affect Prz1 nuclear localisation. Second, Prz1 activity is higher in cmk1 deleted cells and lower when Cmk1 is overexpressed. Third, Cmk1 binds and phosphorylates Prz1 in vivo. The overexpression of Cmk1 causes a change in the electrophoretic mobility of the Prz1 protein which is not observed when the inactive Cmk1-KA is overexpressed. Furthermore, phosphorylation in the consensus site of Cmk1 kinase is detected in the inactive Prz1, whereas this phosphorylation is absent in Δcmk1 cells. Lastly and importantly, the cellular Ca2+-resistance of Δcmk1 cells is suppressed by prz1 deletion or by deletion of an upstream factor of Ca2+ signalling, such as calcineurin, pbb1, indicating that the lack of negative regulation of Prz1 and thus, the increase in transcription of its targets involved in the calcium response, confers Ca2+-resistance.Our central goal was to elucidate the role of Cmk1 kinase in response to Ca2+ levels through the expression of its targets. It was tempting to speculate that Cmk1 would be involved in controlling intracellular Ca2+ levels. Thus, we also studied whether Ca2+ uptake and cellular Ca2+ concentrations were affected by the absence of Cmk1. We have monitored a change in intracellular free Ca2+ concentrations with the Ca2+ sensor, yellow Cameleon-nano15 , indicating that changes in intracellular Ca2+ levels are not sufficient to provide Ca2+-resistance despite the slight decrease in Ca2+ levels observed in Δcmk1 cells.Prz1 activation tends to normalise intracellular Can-nano15 ,58. UponIn fission yeast, overexpression of Prz1 induced slow growth and small cell morphology similar to that of constitutively active calcineurin ,59. We fIn fission yeast, it has previously been reported that the constitutively active form of Cmk1 blocks cell cycle progression, although the mechanism remains elusive . We founThis result contributes to generalising a function already defined for members of the CaM-dependent kinase family, such as Chk1, Srk1 and MAPK-K2, which, in response to different genotoxic or environmental stresses, blocks cell division through phosphorylation of Cdc25 ,41,60.2+ signalling also provokes phosphorylation of Cmk1. As indicated, Cmk1 activity depends on Ca2+/CaM and full activation is reached upon phosphorylation at T192: the CaMKK consensus site , Cmk1 and probably Ckk2 are inactive Figure , and und2 Figure . Active n Figure  and inhin Figure . Thus, w2+ inhibiting NFAT nuclear translocation by directly phosphorylating calcineurin. CaMKII mediated phosphorylation of calcineurin has been widely reported (2+ activates two cytoplasmic signals with opposite effects on calcineurin activity. The increased calcineurin-mediated NFAT nuclear translocation was the dominant effect of elevated Ca2+, with CaMKII acting as negative regulator. Together calcineurin and CaMKII determine NFAT nuclear translocation, hypertrophy and pro-survival signalling (2+ signals mediate the bidirectional responses of growth cone, attraction or repulsion are induced by CaMKII and calcineurin, respectively. Interestingly, a relatively large local Ca2+ elevation preferentially activates CaMKII which induces attraction; while a modest local Ca2+ signal predominantly acts through calcineurin producing repulsion (In mammalian cells the kinase CaMKII, a member of the CaM-dependent kinases, is also activated in response to Careported . Specifignalling . Additioepulsion .2+ stress activates two cytosolic signalling cascades with opposite effects on Prz1 nuclear activity; the Pbb1 and the Ckk2/Cmk1 cascades. It would be interesting to study whether Cmk2 kinase is also involved in Pbb1 inhibition by direct phosphorylation and to determine whether there is a balance between Cmk2 and Pbb1 depending on the cytosolic Ca2+ level.Our results also indicate that CaMoreover, due to the functional analogy, it should be interesting to study whether the CaMKK/CaMK1 axis regulates NFAT expression in addition to CaMKII in pathologies such as Alzheimer's disease, muscular hypertrophy or cancer progression and metastasis, where hyperactivation of NFAT has been detected ,55,64–66Supplementary Data are available at NAR Online.


A general concept for thinking about causality facilitates swift comprehension of results, and the vocabulary that belongs to the concept is instrumental in cross-disciplinary communication. The causal pie model has fulfilled this role in epidemiology and could be of similar value in evolutionary biology and ecology. In the causal pie model, outcomes result from sufficient causes. Each sufficient cause is made up of a “causal pie” of “component causes”. Several different causal pies may exist for the same outcome. If and only if all component causes of a sufficient cause are present, that is, a causal pie is complete, does the outcome occur. The effect of a component cause hence depends on the presence of the other component causes that constitute some causal pie. Because all component causes are equally and fully causative for the outcome, the sum of causes for some outcome exceeds 100%. The causal pie model provides a way of thinking that maps into a number of recurrent themes in evolutionary biology and ecology: It charts when component causes have an effect and are subject to natural selection, and how component causes affect selection on other component causes; which partitions of outcomes with respect to causes are feasible and useful; and how to view the composition of a(n apparently homogeneous) population. The diversity of specific results that is directly understood from the causal pie model is a test for both the validity and the applicability of the model. The causal pie model provides a common language in which results across disciplines can be communicated and serves as a template along which future causal analyses can be made. Life is all about cause and effect. A general concept for thinking about causality facilitates swift comprehension of results, while the vocabulary that belongs to the concept is instrumental in cross-disciplinary communication. Although in and of itself it does not lead to different results than those obtained with more specific models, a general model provides a quick grasp of the commonalities of seemingly diverse situations. Rothman's “causal pie model” Rothman , 2012 haTo paraphrase Rothman , the ligsufficient cause is a constellation of component causes, the causal pie, that leads to an outcome. A component cause can be a component of more than one sufficient cause. If and only if all the component causes that make up a causal pie of some sufficient cause are present does the outcome occur. As a result, the effect of a component cause depends on the presence versus absence of the other component causes that make up some causal pie. These are called complementary component causes, which jointly make up the complementary set of a component cause. In the absence of any one of the complementary component causes, a component cause in itself has no effect. Referring to the example above, if there is no connection to the power grid, varying the position of the switch does not alter the light being on or off.The causal pie model, depicted in Figure This model is deterministic in the sufficient causes: If all component causes of some causal pie are present, then the sufficient cause is present and the outcome occurs. This does not prohibit the model from having statistical properties: If one (or more) of the component causes is described statistically, the (joint) distribution function of the (mixed) statistical model describes the outcome as a random variable.The level of detail required for an analysis determines the biologic outcome of interest, the number of sufficient causes that can exist for the same biologic outcome, and what component causes make up the sufficient causes. For instance, a stroke that is caused by high blood pressure may be classified as an outcome different from a stroke that is not caused by high blood pressure. The latter clearly does not include sufficient causes that contain high blood pressure as a component cause, while the outcome “any stroke” would.As discussed above, causality adds up to more than 100%. Variation in the occurrence of an outcome due to variation in a specified component cause is bound by 100%. To illustrate this, consider another example Rothman : The disVariation explained is a matter of prevalence of component causes given some context. Although the percentage of variation explained might be of interest, of even greater interest are statements that are generally true, not just on planet Earth or on the other planet. Such a statement is that 100% of the incident cases of PKU is caused by a mutant gene and 100% is caused by diet. On planet Earth, variation in the prevalence of PKU is explained by genes. Yet, it is another component cause, diet, that is targeted as a treatment. This illustrates why general insights into causality are the more interesting kind of insights.The causal pie model has a number of implications for (evolutionary) biology and ecology: It charts under what circumstances component causes have an effect and therefore can be subject to natural selection, and how component causes modulate the effect of and natural selection on other component causes; it shows which partitions of outcomes are useful and which are not; and it provides a way of viewing the composition of a(n apparently homogeneous) population. Each of these points is developed below, and examples are given and framed in terms of the causal pie model.other component causes have an effect and become subject to natural selection. We outline some generalities regarding this thinking, and give examples, which are framed in terms of the causal pie model.The causal pie model explains when and why biologic factors (component causes) have an effect. When component causes produce an effect, they could affect Darwinian fitness and hence become subject to natural selection. Component causes also influence whether The presence of all component causes of a causal pie constitutes a sufficient cause, which implies that the outcome occurs. Each of the component causes is then subject to the full force of natural selection on the outcome, for without any one of them, the outcome would not occur Fig. . The forIn the absence of any one of its complementary component causes, the emergence of a component cause has no effect and therefore is not subject to natural selection (barring effects through other sufficient causes). However, component causes are part of the complementary sets of all the other component causes in a causal pie. A component cause that does not give rise to an outcome immediately does change the set of component causes against which yet other component causes arise . It thus affects the chance that another component cause will arise in the context of its complementary set and hence make the outcome occur. In the long term, this gets picked up by evolution. Below, we give a wide-ranging sample of applications of these principles.Wade and Kalisz  recognizWade and Kalisz's realization is a special case of the general principle that selection on a component cause depends on the presence or absence of its complementary set. The effect of the component cause “gene” depends on its complementary component cause “environment”. The complementary set can certainly be environmental, but can be essentially anything that completes a causal pie. If the interaction of many different genes is necessary to bring about some effect, as in the next example, genes act as each other's complementary component causes.Genetic mutations can be semi-neutral: While they do not have an immediate phenotypic effect, they pave the way for non-neutral change over evolutionary time Wagner . This haIn terms of the causal pie model, new mutations arise in the context of the existing mutations, which determine whether the complementary set is present. The first few mutations are insufficient to have a phenotypic effect, but they do change the context against which new mutations arise. At one point, the new mutation arises in the presence of the other mutations necessary for phenotypic change, that is, its complementary set of a sufficient cause for phenotypic change. All of the mutations are equally causal to the outcome. Yet, all mutations before the one that leads to phenotypic change seem neutral, because their complementary set for phenotypic change is lacking.of refers to an empirical observation of what is selected, while selection for means that there is selection due to a causal effect of some trait on vital rates . For instance, in Figure Correlated traits of particular interest are trade-offs  may be distributed probabilistically over the habitat or over time. As the causal pie model is also probabilistic, it fits the examples above well. The probability distribution of either outcome depends on the probability distributions of the complementary component causes, which determine the preferred behavior.Trade-offs depend on the presence of complementary component causes. There is selection toward optimal outcomes given the complementary component causes that are present: Evolution will have the tendency to optimize within the limitations of the trade-off. For instance, in the molecular example in the first paragraph of this section, the trade-offs depend on the presence of scavengers for oxygen radicals and on the vulnerability of the macromolecules to oxygen radicals. Given the vulnerability of macromolecules to oxygen radicals, and given a certain concentration of scavengers, evolution will optimize metabolism. At the same time, there is selection on the complementary component causes: Evolution will have the tendency to push the boundaries of the trade-off, such that the trade-off becomes less restrictive. To stay with the example of oxidative damage, evolution will tend toward using less vulnerable macromolecules (if possible), and toward increasing scavenging capacity, thus changing the trade-off that it faces.The evolutionary theory of aging is sometimes defined in terms of “age-specific genes”, genes that are active at a particular age or during a particular age range and not before or after. The idea is that natural selection acts less forcefully against detrimental genes the later they are expressed Medawar . HoweverThe causal pie model shows that it is not logically correct to say that substance S is not deleterious “in any normal sense”. The causal pie is made up of two component causes – a certain concentration of substance S, and a substance S-sensitive gene – which together form a sufficient cause for deterioration and death. Completing the causal pie, substance S is as causal to the detrimental effect as is the substance S-sensitive gene. Without substance S, there would be no senescence, just like without the substance S-sensitive gene, there would be no senescence. As a result, the force of natural selection on deterioration that results from the expression of the deleterious gene extends to both the gene and substance S Wensink . TheorieGenerally speaking, partitions are useful only when they are based on qualities that are mutually exclusive. If this is not the case, something could belong to more than one partition. Because the whole point of partitioning is to divide things up, making partitions based on qualities that are not mutually exclusive should not be attempted, because it will fail.In the causal pie model, multiple component causes need to interact for an outcome to occur. It is not useful to partition outcomes as caused by one versus the other component cause, as an outcome could be caused by both component causes at the same time. It can be useful, however, to make a partition between outcomes in which a particular component cause plays a role, versus outcomes in which it does not.Although they acknowledge a role for vulnerability of an organism to extrinsic threats, Carnes et al.  set out Mortality, however, is a prime example in biology of interacting component causes, some extrinsic, some intrinsic to the organism. Therefore, it is not possible to characterize mortality either extrinsic or intrinsic. A typical example is predation, where the presence of a predator (component cause 1) interacts with a state of vulnerability of the prey (component cause 2) and an environment that co-determines if prey and predator effectively meet, for instance a quality of vegetation (component cause 3). These three component causes could be partitioned in more detailed component causes if required, such as the cardiopulmonary capacity or fur color of predator and prey.Component causes of mortality can be partitioned in extrinsic and intrinsic, but mortality itself cannot, for the simple reason that extrinsic and intrinsic mortality are not mutually exclusive. Acknowledging a role for vulnerability in “extrinsic mortality” does not take away this objection. None of the component causes is more causative to the outcome, as without any one of them, the outcome would not occur. One could propose that there can be no predation without a predator and that therefore the (extrinsic) presence of a predator must be the most causative cause for death by predation. However, neither is there predation without a prey. Being prey is something “intrinsic”. There is no such thing as “most causative cause”. A partitioning that could have some utility is a partition of mortality in which an extrinsic component cause plays a role, versus mortality in which it does not. The term “extrinsic mortality” retains some meaning in this respect, although one could wonder whether purely intrinsic mortality exists at all.Populations are heterogeneous, and an observation at the population level may be quite different from individual-level processes  with a delay and why apparently healthy organisms may differ greatly in their vulnerability to stress. Together with trade-offs, causal pie models of aging may also contribute to explain the recently revealed diversity of aging patterns across the tree of life  the component causes may be ascertained only at a statistical level. More involved models, such as structural equation modeling Pearl , are theTo reveal underlying causal relations in a data set, the causal pie model offers no alternative to the appropriate tools of data analysis, such as structural equation modeling [Shipley  and GracAlthough we use different words in various disciplines, we describe the same phenomena. The causal pie model can help to make bridges, so that various disciplines can draw on and be inspired by each other's finding. The range of examples that we give is a good demonstration of the utility of the causal pie model in this respect. We propose that the causal pie model provides an effective framework for thinking about causation, that it helps to avoid mistakes, and that it provides a simple common language in which results can be communicated across disciplines.


These RBE values increased to 5.8 and 5.6 in the absence of interference of indirect action initiated by addition of 2-M dimethylsulfoxide. DSB conversion was ∼1–4% of the total non-DSB damage due to gamma rays, which was lower than the 10% estimate by experimental measurement. Five to twenty percent of total non-DSB damage due to helium ions was converted into DSBs. Hence, it may be possible to increase the yields of DSBs in cancerous cells through DNA repair pathways, ultimately enhancing cell killing.Clustered DNA damage other than double-strand breaks (DSBs) can be detrimental to cells and can lead to mutagenesis or cell death. In addition to DSBs induced by ionizing radiation, misrepair of non-DSB clustered damage contributes extra DSBs converted from DNA misrepair via pathways for base excision repair and nucleotide excision repair. This study aimed to quantify the relative biological effectiveness (RBE) when DSB induction and conversion from non-DSB clustered damage misrepair were used as biological endpoints. The results showed that both linear energy transfer (LET) and indirect action had a strong impact on the yields for DSB induction and conversion. RBE values for DSB induction and maximum DSB conversion of helium ions (LET = 120 keV/μm) to  When cells are irradiated with ionizing radiation, both direct and indirect actions of the radiation may result in DNA damage . Among t2, which initiates a chain of events that finally results in DNA damage ip (E) was defined as the conversion probability of repair pathways for DNA damage clusters composed of i lesions, and iY was the yield of total non-DSB clusters per Gy per gigabase pairs (per Gy per Gbp) composed of i lesions with helium ions of energy E. The enzymatic DSBs for 60Co gamma rays was calculated by using the formula for dose-weighted DSBs [60Co photons, and Φ(E) was the energy fluence of secondary electrons. For electrons with energies higher than 1 keV, the unrestricted LET (stopping powers) from the National Institute of Standards and Technology was used [The DSB conversion for helium ions was calculated by using the formula that enzymatic DSB = ted DSBs :(1)Yi=∫was used .2) to that under anoxic conditions (2% O2).The biological effects of oxygen concentrations on cells can be quantified in terms of the OER, the ratio of hypoxic dose to aerated dose needed to achieve the same biological effects . OER may60Co gamma rays and helium ions. Total damage due to 60Co gamma rays in the presence of DMSO decreased by 35%, whereas 2SSBs and DSB+s decreased by ∼80%. Total damage due to helium ions did not decrease, but the constituents of DNA damage changed. In the presence of DMSO, BD and SSBs increased by 20% and 3%, respectively. However, the yields of other types of complex damage, namely, 2SSBs, DSB+s and DSB++s decreased by 44%, 40% and 63%, respectively. These data suggest that indirect action affected the constituents of the complexity of DNA lesions induced by low- or high-LET radiation, contributing significantly only to the DSB yields for low-LET radiation.To gain insight into the effects of indirect action on DNA damage, the results for various types of DNA damage in the presence and in the absence of DMSO are summarized in Table 60Co gamma rays was 6.84 per Gy per Gbp under anoxic conditions . The amount of total DNA damage induced by 60Co gamma rays increased by up to 6% going from 2% O2 to 21% O2; unsurprisingly, the yields of non-DSB clusters also increased. However, the ratio of non-DSB clusters varied with the number of lesions within a cluster . The ratio of non-DSB clusters due to 60Co gamma rays composed of three or more lesions (n ≥ 3) was more sensitive to the oxygen concentration than the ratio of clusters composed of two or more lesions (n ≥ 2) and the ratio of the total non-DSB clustered damage . The OER for non-DSB clusters composed of three or more lesions (n ≥ 3) was ∼1.3, whereas that for simpler clustered damage (n ≥ 2) was ∼1.15, highlighting the importance of oxygen in cluster complexity. MCDS predicted that clusters composed of two or more lesions (n ≥ 2) comprised 26% of the total non-DSB clusters and that clusters composed of three or more lesions (n ≥ 3) comprised 6% at 21% O2, indicating that most clusters were composed of one or two lesions. In contrast to those due to 60Co gamma rays, the total DNA damage induced by helium ions remained constant (∼250 per Gy per Gbp), and the yields of DSB induction were also approximately invariable (from 23.5 to 24.1 per Gy per Gbp) when the O2 concentration increased from 2% to 21%. Moreover, the OER of non-DSB clusters composed of six or more lesions (n ≥ 6) or less-complex damage (n ≥ 4) induced by helium ions  and six or more lesions (n ≥ 6) comprised 27% and 9% of total non-DSB clusters at 21% O2, and non-DSB cluster ratios increased as the oxygen concentration increased. However, the complexity of non-DSB clusters induced by high-LET radiation was less sensitive to the change in oxygen concentration.Oxygen also played an important role in DNA damage yields. MCDS results showed that DSB induction by nditions ) and 8.160Co gamma rays and helium ions (LET = 120 keV/μm). As expected, the repair outcomes for cells irradiated with 60Co gamma rays were more favorable than those for cells irradiated with helium ions, as most of the damage induced by 60Co gamma rays was BD ; the probabilities of total damage only are shown in Tables 3–5.Table ns LET = 20 keV/μmns LET = 20 keV/μm60Co gamma rays and helium ions are shown in Tables 60Co gamma rays were ∼90% or above and those of mutation and DSB conversion were in the ranges of 1–6% and 1–4%, respectively , cells irradiated with helium ions did not have enzymatic DSBs, whereas MCER predicted that up to 5–20% of total damage converted into enzymatic DSBs. The difference in measured and predicted DSB yields may be ascribed to the loss of small fragments in the PFGE assay for high-LET radiation, and hence the yields of DSBs were not accounted for in PFGE assays [MCER predicted that DMSO reduced the mutation frequency for cells irradiated with 0% Table . This raE assays .60Co gamma rays and RBE values of 4.0–5.3 for cell survival [Table survival . Experimsurvival . Furthersurvival . Figure Our results showed that the complexity of non-DSB damage had profound effects on DNA repair. Table In summary, this study shows that the degree of complexity of DNA damage induced by high-LET radiation is significantly higher than that induced by low-LET radiation. Rates of mutation and DSB conversion from misrepaired non-DSB clusters induced by high-LET radiation are also higher than those arising from low-LET radiation. These rates can be greatly reduced with radical scavengers such as DMSO. Additionally, oxygen increases the complexity of non-DSB clustered lesions arising from both low- and high-LET radiation. Taken together, these results suggest that indirect action contributes significantly to the yields and complexity of DSBs converted during repair of non-DSB clusters due to high-LET radiation and can be used as tools in further radiotherapy.This research was supported by grants CMRPD1C0642&0662, CIRPD0061 and MOST103-2314-B-182-062 to F.-H.C. Funding to pay the Open Access publication charges for this article was provided by MOST103-2314-B-182-062.