Tn) and subsequently tropomyosin molecules. Experimental studies have shown that a slight increase in intracellular calcium concentration leads to a rapid increase in sarcomeric tension. Though it is widely accepted that the rapid increase is not possible without the concept of cooperativity, the mechanism is debated. We use the hypothesis that there exists a base level of cooperativity intrinsic to the thin filament that is boosted by mechanical tension, i.e. a high level of mechanical tension in the thin filament impedes the unbinding of calcium from Tn. To test these hypotheses, we developed a computational model in which a set of three parameters and inputs of calcium concentration and sarcomere length result in output tension. Tension as simulated appeared in good agreement with experimentally measured tension. Our results support the hypothesis that high tension in the thin filament impedes Tn deactivation by increasing the energy required to detach calcium from the Tn. Given this hypothesis, the model predicted that the areas with highest tension, i.e. closest to the Z-disk end of the single overlap region, show the largest concentration of active Tn’s.We developed a model of cardiac sarcomere contraction to study the calcium-tension relationship in cardiac muscle. Calcium mediates cardiac contraction through its interactions with troponin ( Cardiac contraction is the culmination of multiple subcellular processes beginning with calcium induced activation of the contractile machinery. Interestingly, small increases in intracellular calcium concentrations lead to disproportionately large increases in tension development within the cardiac muscle, a phenomenon known as ‘cooperative activation’. Although the concept of cooperative activation is widely accepted, the mechanism is highly debated. Many complex computational models have been developed in an attempt to understand the underlying mechanisms. However, no single mechanism has been able to properly account for the range of experimental data. We propose that the interaction between the mechanics and chemistry in the contractile unit is an essential component of the cooperative activation. We have developed a simple computational model composed of only five parameters and a single ordinary differential equation that describes the cooperative nature of myocardial contraction through mechanochemical interaction. This model can reproduce experimental data relating to cooperative activation and provides a promising tool for future research. Ca2+) regulate muscle contraction. Although the mechanism was unknown at the time, Ringer found that rat hearts did not contract forcefully unless Ca2+ was added to the solution perfusing the muscle. Since Ringer’s discovery the relationship between Ca2+ concentration ([Ca2+]) and tension in muscle has been widely studied. Experiments have shown a steep rise in steady state tension with relatively little increase in intracellular [Ca2+]. The desire to understand the steepness of the [Ca2+]—tension relationship has led to the hypothesis that cardiac muscle contraction is highly cooperative  and muscle tension is described by the Hill-relation, having zero active tension at low [Ca2+] and approaching a level of saturation at high [Ca2+]. If a single Ca2+ ion would bind to a single Tn complex without cooperativity, for a physiological muscle contraction, the required increase in [Ca2+] would be unphysiologically large. By comparing the relatively small, physiological change in [Ca2+] with the resulting large change in muscle tension, Ca2+ binding to Tn requires an increase of cooperativity by the so-called Hill coefficient n. In experiments by Dobesh et al. -tension relationship. XB-XB interactions had the highest impact on peak force, RU-RU interactions had the greatest impact on the steepness of the [Ca2+]-tension relationship, and XB-RU interactions had the greatest impact on Ca2+ sensitivity. The model as developed by Rice and colleagues  and sarcomere length. We investigated whether the mechanochemical mechanism can boost cooperativity from the intrinsic level with a Hill coefficient of 3 described by Sun et al. . Under static conditions we considered the chemical reactions involved to be in equilibrium.On the basis of the abovementioned mechanochemical principle, we designed a computational model of cardiac sarcomere mechanics that we call the MechChem model. The MechChem model is intended to predict the tension in a thin filament as a function of  and sarcomere length as reported by Dobesh et al [Ca2+]-tension relation is found for each sarcomere length, requiring the estimation of 3 parameters. Furthermore, it is investigated which parameters values of the MechChem model could remain while changing sarcomere length.In investigating the static behavior of cardiac muscle contraction, muscle tension as simulated with the MechChem model is compared with simulations based on conventional chemical binding of sh et al . When fiXB’s is initiated by the binding of Ca2+ to TnC which we will refer to as the binding of Ca2+ to an in-register pair of Tn, referred to in the equations as Tn2. The related chemical equilibrium reaction is represented byFormation of n indicates the coefficient related to the intrinsic cooperativity of Ca2+ binding to Tn2. When Ca2+ binds to Tn, Tm moves away from the position in which it blocks the XB-binding sites on the actin monomers. We assume that the unblocking of XB-binding sites, or activation of a Tn, automatically implies XB formation in the environment of that Tn. After formation, the XB exerts a longitudinal force FXB on the thin filament which is guided as tension along the thin filament to the Z-disk (S(x) in the thin filament is increasing by a step FXB at each location where a XB has formed. Variable x represents the distance from the beginning of the single overlap region moving toward the Z-disk (xmax) of x represents the location on the thin filament where the single overlap region ends. The single overlap length depends on SL and the assumed filament lengths as shown in Parameter e Z-disk . Thus, te Z-disk . The maxS(xj) just distal to XBj at position xj it holds:Looking from the middle of the sarcomere towards the Z-disk, for tension jmax indicates the number of XB’s attached to the thin filament in the single overlap region. S(x) increases with each attached XB until distance x exceeds the end of the single overlap region where no XB's can be formed. The total tension of the thin filament equals the tension acting on the Z-disk from that thin filament.The symbol The following assumptions are key to the MechChem model implementation presented in this section:Ca2+ binding to Tn moves Tm azimuthally on the thin filament, unblocking the XB-binding sites on nearby actin monomers.XB binding and force development.Unblocking of binding sites implies XB’s can form solely in the single overlap region.XB force acting on the thin filament.The total tension in the thin filament at the Z-disk is the sum of the individual Tn to release Ca2+ increases linearly with the mechanical tension in the thin filament.Tension hinders the deactivation of the thin filament. The energy required for Ca2+ to an in-register Tn pair (Tn2) is intrinsically cooperative, characterized by the base cooperativity coefficient n (according to  and substituting the result into P(x) as a function of [Ca2+] and KTnCa  in the thin filament. Thus, the equilibrium constant is multiplied by the exponential of the product of tension S(x) and a constant Cs, representing the effect of the added affinity for Ca2+ by an increase of tension. The latter relation is based on the general physical principle that in equilibrium the ratio of state concentrations is proportional to the exponential of a constant multiplied by the energy difference between both states. Analogously, the ratio of ion concentrations on both sides of a membrane depends on the voltage difference across the membrane. So, we express the dependence of the equilibrium concentration constant, KTnCa on tension S(x) by:Because in the MechChem model binding of KTnCa0 represents the equilibrium constant in the absence of tension in the thin filament. The physical dimension of constant Cs is the inverse of tension. Replacing KTnCa(x) in P(x) in The symbol XB’s is proportional to P(x). Assuming that all attached XB’s exert the same force during steady state isometric contraction, we find that the XB–induced force density fXB(x) acting at location x is proportional to P(x) (We assume that the density of attached  to P(x) .Cf represents force density with full Tn activation, having the physical dimension of force per unit length along the thin filament. After elimination of P(x) by substitution of S(x)/dx of tension with respect to x equals force density fXB(x), we find the following differential equation for S(x) with boundary condition S(0) = 0:Constant n = 3 according to the findings by Sun et al. . The three parameters EC50, nH, and Smax, represent the [Ca2+] at the 50% level of maximum tension, the Hill coefficient, and maximum tension, respectively. Thus we usedWe have fitted the Hill-type model with the experimental data of Dobesh et al.  for compCa2+ ions from the extracellular space through channels and pumps [Ca2+] can be manipulated, and the muscle will contract and generate tension in response.Under normal conditions, cardiac muscle cells are enclosed by membranes that regulate the influx of nd pumps . SubmersSexp) at various SL’s and [Ca2+]’s. We tested our model at a range of [Ca2+]’s between 0.001 and 10 μM for five SL’s ranging from 1.85 to 2.25 μm. Single overlap length xmax increases with SL according to the formulation of Rice et al -tension relationships in KTnCa0, Cf and Cs per curve. Similarly, in EC50, nH and Smax. The lower panels, 4C and 4D, show the fitting errors per curve as a function of the [Ca2+] for the MechChem and Hill-type model, respectively. The black lines indicate the median value of the error. For the MechChem model, there is no clear common pattern, and the error does not significantly differ from zero. For the Hill-type model, the error values show a clear common pattern, showing a consistent underestimation at the beginning of the upslope and the very last data points and a consistent overestimation at the location where the curves bend towards the maximum value. Thus, in contrast with the MechChem model, the shape of the tension curve, as simulated with the Hill-type model, is clearly different from the measured data. The MechChem curves are asymmetric with a relatively sharp upward bend for low [Ca2+] and a moderate bending toward the saturation level.The experimental data of Dobesh and colleagues in skinned cardiac muscle   is compaRSME for the MechChem model is smaller than that for the Hill-type model. The parameter Cf varied by a maximum of 6.5% at different SL’s, whereas KTnCa0 and Cs decreased by 43.2% and 70.6%, respectively. Interestingly, for the MechChem model, parameter values KTnCa0 and Cs exhibit strong interdependency. Our analyses showed that for a change in KTnCa0 by a factor of a (a>1), Cs changed according to Ca2+]-tension relationship.In KTnCa0 or Cs changes with SL. We assume that KTnCa0 is the parameter that changes with SL because the necessary increase in KTnCa0 is much less than that in Cs. Additionally, a large body of research has shown that the sensitivity of Tn to bind Ca2+ changes with SL -tension relationship  decreases with longer SL’s indicating the increasing sensitivity of tension development in the thin filament to Ca2+. The experiments of Dobesh et al. ’s . These values were chosen to show the results in the middle range of the experimental conditions. The MechChem model predicts that the density of bound XB’s increases with the position x along the thin filament. The plateaus shown in Tn’s, i.e. all binding sites on that section of the thin filament are exposed for XB binding.Ca2+ binding to an in-register pair of Tn’s, i.e., cooperativity in the absence of mechanical tension, is characterized by a base cooperativity coefficient n = 3 according to experimental data reported by Sun et al. -tension relation appeared in good agreement  and SL increasing from 1.85 μm to 2.25 μm, the simulated peak tension increased by 49% and the measured value by 47%. Simulations also produced an increase in Ca2+ sensitivity, shown by a decrease of KCaTn0 from 16.6 down to 11.8 μM, implying a leftward shift of the [Ca2+]-tension curves with increasing SL. The shape of the MechChem curves . One pr binding . However binding . It has tivation . Converssitivity . It is pRU-RU, XB-XB or XB-RU interactions. These models generally result in Hill-type [Ca2+]-tension relations that fit accurately to experimental data. However, the curves are slightly, but systematically different from the MechChem curves . RU-RU cooperativity is accounted for in the MechChem model through the intrinsic cooperativity coefficient n.As already indicated in the introduction, various models have been developed on the basis of nearest neighbor cooperativity, based on m curves . It is n highest . With thTm molecules under Tn causes a simultaneous unblocking or blocking of all Tm molecules along the filament. Conversely, the “cooperative realignment of binding sites”, a model developed by Daniel and colleagues  will trigger relaxation. Due to the buildup of tension within the thin filament as shown in Tn’s closest to the mid-line first. We propose that the areas of highest tension (closest to the z-disk) are the latest to deactivate.Due to the interaction between mechanics and chemistry within the model, neither mechanics nor chemistry fully account for the activation or subsequent deactivation of the thin filament. While we propose that high tension in the thin filament hinders deactivation, a decrease in intracellular - tension relationship. The introduction of dynamics such as the XB cycle requires additional assumptions to be made and additional unknown parameters added. However, the next logical step for the model is the implementation of the XB cycle that will be studied in the isometric twitch. The MechChem model is a mean field approximation, so it is highly simplified. Specific spatial details such as individual binding sites are not accounted for, but the simplification reduces computational cost considerably.The current model results are limited to the [Ca2+]-tension curves resembled those obtained in steady state isometric muscle experiments. Thus, our results support the hypothesis that high tension in the thin filament impedes deactivation by increasing the energy required to detach calcium from the troponin complex. Furthermore, we found that the tension in the thin filament was relatively low toward the beginning of the single overlap region close to the mid-line of the sarcomere but increased steeply in the overlap region closer to the Z-disk. Model simulations suggest that the concentration of calcium bindings to the troponin complexes and active XB’s are low at the free end of the thin filament and saturated closer to the Z-disk. Future experimental studies are needed to test the latter property, indicating the validity of our hypothesis on the cooperative effect of tension in the thin filament on force generation by the cardiac sarcomere.A novel mechanochemical model of tension generation by the sarcomere has been developed based on long range cooperativity imposed by mechanical tension in the thin filament and intrinsic cooperativity resulting from the interaction between the calcium-binding sites on the in-register troponin complexes. Simulated . Cs =  (S-1) Cf =  (nm-1S) KTnCa0 =  (μM).The cooperativity constant (TIF)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S1 Model Code(ZIP)Click here for additional data file.


Several studies including ours have demonstrated an involvement of PD-1 and other T cell inhibitory pathways in AML progression7. Strategies of blocking immune suppression for leukemia treatment are attractive due to their relatively simple administration and better tolerability profile. In fact, clinical studies applying anti-PD-1 in AML therapy have been initiated and are currently in early phase trials8. AML is a highly heterogeneous disease with multiple steps involved in the treatments. Determining the status of the immune response at each disease stage in individual patients is crucial for decision making of subsequent clinical management. In addition, immune characterization has great potential to identify predictive biomarkers of responsiveness to immunotherapy, thus providing pivotal information to optimize clinical trial design using strategies modulating anti-leukemia immunity for AML treatment.Acute myeloid leukemia (AML) is a devastating blood cancer with 5-year survival of only 25%. Novel effective leukemia therapy is an urgent unmet need. Immunotherapy is a promising strategy for cancer treatment. Targeting inhibitory mechanisms, such as the programmed cell death protein 1 (PD-1) pathway to unleash the patient’s own anti-tumor immune response has achieved major success9. A better understanding of the anti-leukemia immune response within the bone marrow of AML patients is likely to be a key to develop immune-based therapeutic approaches for leukemia. In this study, we investigated the T cell immune response within the bone marrow in AML.Due to limited accessibility, the majority of clinical studies in AML have been restricted to evaluation of peripheral blood samples. In contrast, the immune response in bone marrow of AML patients is poorly understood. AML is derived from myeloid hematopoietic progenitors and rapidly grows in bone marrow before mobilizing to peripheral blood in a majority of patients. Therefore the development and progression of AML largely relies on the bone marrow microenvironment10. Two patients (9.1%) were categorized with favorable risk, ten (45.5 %) with intermediate risk, and the other ten patients (45.5 %) were in the adverse risk category. Peripheral blood and bone marrow aspirates were collected from each patient prior to any leukemia treatment.Samples collected from a cohort of 22 patients with newly diagnosed AML were used in this study. Clinical and demographic information are summarized in Supplementary Table + T cells among lymphocytes is significantly lower in bone marrow than in peripheral blood , and the percentage of TEM in bone marrow is significantly higher than that of peripheral blood , a recently identified co-inhibitory receptor, in the CD8 T cell response in AML7. To evaluate whether the effect of inhibitory pathways is different between bone marrow and peripheral blood, we assessed the expression of PD-1, TIGIT, and TIM-3 on T cells derived from both sites. We observed a significantly increased frequency of PD-1-expressing CD8 T cells in bone marrow compared with that of peripheral blood  in AML progressionood Fig. . There wood Fig. . PD-1 isood Fig. . Eomes a126–134 is the most studied epitope in AML15. We found a significantly lower production of both IFN-γ and TNF-α by CD8 T cells from bone marrow compared with that from peripheral blood , we observed a significantly higher expression of PD-1 on cells derived from bone marrow compared with that of peripheral blood  upon in vitro stimulation with anti-CD3 and anti-CD28 Fig. . In addiood Fig. . Furtherood Fig. . These iTo evaluate the correlation between the PD-1 expression and clinical outcome, we defined high-PD-1 vs. low-PD-1 subgroups in the cohort of AML patients. The mean value of PD-1 expression on CD8 T cells in bone marrow of the 22 AML patients evaluated in our study was used as the cutoff here. Upon analyzing the rate of complete remission (CR) after induction treatment and the overall survival, we found no significant difference between the high-PD-1 and low-PD-1 subgroups Supplemental Figure 1(PPTX 76 kb)


Mentoring programmes are commonplace and delivered in a range of different ways in the United Kingdom and North America. To better understand the type of programmes available and to inform future evaluations, we developed a typology of formal mentoring programmes for young people in secondary schools in the United Kingdom. Telephone interviews with 23 programme managers from purposively sampled mentoring organisations were conducted and analysed using thematic and framework analysis. The typology was consulted on with five experts in mentoring. The final typology differentiates mentoring programmes by three overarching categories: type of mentor , programme setting  and programme aim. The findings suggest that although mentoring programmes are heterogeneous, it is possible to group programmes into ‘personal and developmental’ and ‘academic and employability’ mentoring programmes and to differentiate between 12 overall mentoring models. The typology helps understand what is being delivered and how, which is a necessary precursor to any evaluation of health, educational, relational and social outcomes. •Formal mentoring programmes for young people are heterogeneous.•Programmes can be distinguished by taking account of the type of mentor, programme setting and aim.•Programmes can be grouped into ‘personal and developmental’ and ‘academic and employability’ programmes.•Twelve distinct models of youth mentoring programmes have been identified in the United Kingdom. Youth mGiven the plethora of mentoring programmes in existence, it is important to assess whether formal mentoring programmes in the UK are effective and cost-effective in improving young people's health, wellbeing, educational, employment and training outcomes.In the following sections, we review what formal mentoring programmes are, what is known about the effectiveness of mentoring programmes and previous ways that have been used to make sense of the multiplicity of what is considered as ‘mentoring’ programmes.1.1As Freedman noted in 1991, “Mentoring is flexible, accommodating whatever attributes people want to give it” , p. 37. Given this broad and flexible definition, there is a range of different types of programmes that are referred to as ‘mentoring’ and that these programmes can vary widely . The Big1.2The earliest robust evaluation of a formal mentoring programme, employing a randomised control trial design, concerned the BBBSA community-based programme which paired young people from typically single-parent households with a mentor . The evaWhen looking at mentoring programmes in the UK, a few individual programmes have been subject to robust evaluation, with no randomised control trial yet undertaken of a mentoring programme for young people of secondary school age. Past evaluations include a national evaluation of youth justice board mentoring schemes for young people who already had or were at risk of offending. This evaluation concluded that programmes resulted in re-enrolling approximately 45% participating young people back into education or training . AnotherA range of systematic reviews have been undertaken to scrutinise the available evidence on mentoring with regard to a range of different outcomes including academic, health, relational, and social outcomes of young people. Whereas only some reviews focussed solely on randomised control trial evaluations , others Despite some promising findings, not all reviews reported evidence of statistical significance in the outcome domains and effect sizes reported have generally been described as moderate or small  and someWhen trying to make sense of the available evidence, it is important to consider that systematic reviews differ in the type of studies that they included as ‘mentoring programmes’. This might in part be due to the lack of a unifying definition of mentoring or mentoring programmes [18]. For instance, DuBois's systematic review explicitly stated that the definition of mentoring was kept broad and that some programmes “fell at the conceptual boundaries of traditional conceptualisations of youth mentoring” , p. 67. For example, one factor that has been established to impact on the effectiveness of a programme is the overall length of the relationship between mentor and mentee, also referred to as match length. Research based upon the BBBSA evaluation revealed that the impact of mentoring on outcomes of the young people with a mentor become increasingly stronger with a longer match length . In factThe lack of robust evidence on youth mentoring's effectiveness coupled with the fact that most of the extant evidence is for programmes in the USA , means t1.3In order to evaluate the effectiveness of any intervention, a clear and detailed understanding of the intervention and intervention context is required . Among oThe aim of a good classification is to (i) identify the key dimensions that define the topic of interest and (ii) to order entities of the topic of interest into one or more mutually exclusive and independent categories . CompareMultiple studies have attempted to form typologies in the field of mentoring, for instance, to differentiate between types of relationships between mentor and mentee  differenA few other typologies have been developed to differentiate mentoring programmes from one another . HoweverBased on interviews with young people in Scotland, Summarising the attempts to conceptualise mentoring through typologies, Hall  concludeIt is important to note that these dimensions were derived from reviewing past classifications, which in turn were mainly developed based on programmes in the USA. Given the individual nature of mentoring programmes and contexts in which mentoring programmes operate in, it is uncertain how mentoring programmes in the UK can be best classified.1.4Contrary to many existing classifications, we were interested in deriving a typology that used mutually-exclusive categories, to be able to find a way of grouping programmes to not only aid the future evaluation of different programme models, but also to disentangle the effects that different mentoring programmes might have. Providing a framework for separating different mentoring programmes from one another might also guide future research on the effectiveness of certain groups of programmes. Past research has highlighted that mentoring programmes differ widely and that by grouping different programmes together, their possible effect size might be undermined or inaccurate.As past attempts at classifications of mentoring have largely been based on mentoring relationship characteristics, types of mentors, USA-based programmes, informal and formal mentoring practices, and on broader target groups, the purpose of this study was to develop a typology of currently active mentoring programmes that are offered and available for young people in secondary schools in the UK .We were interested in young people within secondary schools as adolescence is regarded as a crucial time in which trajectories towards health and wellbeing in adult life can be modified . We wereFrom what is known about programmes in the United Kingdom, we expected there to be a variety of different programmes in existence, both within and outside of schools. We did not have a prior hypothesis about how we would group programmes.22.1Mentoring organisations were eligible for inclusion in the study if they provided one or more formal mentoring programmes to young people enrolled in secondary schools in the UK. Young people were assumed to be 11–16 years old which is equivalent to Years 7–11 within English secondary schools. To capture the range of potential organisations and programmes, the definition of mentoring used in the study was kept broad and inclusive. Mentoring was defined as ‘any programme between an identified young person and other individual(s), aimed at the support of the young person’ and that is referred to by the provider organisation as ‘mentoring’. Mentoring organisations that provided mentoring programmes solely for another group of the population (e.g. primary schools) or that were not operating in the UK were not eligible for inclusion.2.2A pilot search established that most organisations listed as part of national mentoring networks websites had an internet presence. Consequently, a website search was undertaken to identify UK organisations that provided formal mentoring programmes for young people. The search contained three elements: (i) search of two national mentoring network websites operating in England and Scotland; (ii) search of relevant charitable organisation and trust registers; and (iii) a Google search to identify programmes in Wales and Northern Ireland where no mentoring networks were known to exist. Details of websites searched and search results can be found in Appendix A. A total of 815 organisations were identified and information on these organisations and their mentoring programmes were captured in an Excel spreadsheet. Through looking at the available information of each programme, we identified 163 organisations that met the eligibility criteria for our study. To ensure the development of a comprehensive typology, maximum variation sampling based on country  and the type of mentoring programme  was used to recruit a purposeful sample of organisations.2.3Programme managers of selected organisations were invited to take part in semi-structured telephone interviews to obtain a thorough account of their mentoring programme(s) and to explore views on how mentoring programmes differ. Programme managers were selected as they were regarded as having insight into the details of their mentoring programme(s), programme history and wider experiences of delivering and managing mentoring programmes.Experts in the field of mentoring in the UK were invited to take part in telephone interviews to provide their views on the draft typology. Academic and practitioner experts were identified through the literature and consultation with other researchers. An important criterion in the selection process was that these experts were not involved in the interviews used to draft the typology.2.4All participants provided written consent and ethics approval was granted by the University of Bristol Faculty of Health Sciences Research Ethics Committee in 2015 (reference number 24341). Given the relatively small numbers of what can be considered mentoring ‘experts’ in the UK, experts in this study were asked for their consent to be acknowledged by name.2.5The research was carried out in two main stages as displayed in Programme managers were asked in the interview (Step 2) whether they would like to take part in the consultation on the draft typology (Step 6). During the first interview (Step 4), experts were asked whether they would be interested in taking part in a second, follow-up interview (Step 8). As part of the consultation process, all participants were assured that that there were no right or wrong answers and were encouraged to critique the draft typology.All telephone interviews were facilitated using a flexible and semi-structured topic guide and audio-recorded using a digital password encrypted audio recorder (Olympus DS-3400). Interview recordings were transcribed verbatim and anonymised. Data were stored on a secure, password protected, electronic database.2.6Anonymised transcripts were read and re-read and analysed thematically . A selecAdditional to the inductive approach to the data analysis, the framework approach was employed to aid the typology development . A centr3Twenty-three programme managers and five experts took part in the research. An overview of participants and the stages of research is presented in 3.1Programme managers had various career backgrounds; most had previously worked with young people for example as learning mentors, or in a sports or youth work context. Participants varied in the amount time that they had been working for their respective organisations (from a few months to 17 years). Most had worked for the organisation for more than five years.The participants who were experts in the consultation had various job roles and were either practitioners or academics. Most experts indicated that they had been involved in the mentoring field for over 10 years. To our knowledge, most of the experts were based in the UK when the research was undertaken.3.2Of the 23 managers that took part, 21 were involved in organisations that provided and delivered one or more formal mentoring programmes to secondary school students. One interviewee worked in an organisation that provided informal mentoring and one respondent worked in an organisation that provided online mentoring infrastructure. Two thirds of the organisations provided mentoring as part of a wider service for young people, involving non-mentoring services. Whereas most organisations delivered the mentoring programme themselves, two organisations commissioned another organisation to deliver the programme.A total of 35 formal mentoring programmes were described by participants of which 28 were provided for UK secondary schools students and described in sufficient detail to be included in the analysis. An overview of the characteristics of mentoring organisations and programmes is presented in Whereas some internally-run school-based programmes were open to all young people, most externally-run programmes referred to a set of eligibility criteria that either the schools or individual young people had to meet to access the programme. These criteria included school-level characteristics  and individual-level characteristics . Most programmes worked with mentors of a range of ages, ethnicities, gender and professional backgrounds who typically acted in a voluntary capacity.3.3“It's an alphabet soup, there are hundreds of them [mentoring programmes].”(Manager M5)“I mean they [mentoring programmes] differ dramatically in terms of aims and objectives, mentoring can be applied for so many different things.”(Manager M13)“And then, one-to-one mentoring which happens completely independently almost, where you might meet up in a coffee shop, discuss things. There is mentoring groups who are going into schools along like tutors, […] I think, it's, it's evolving constantly and it's taking a lot of different forms.”(Manager M12)Participants were asked about differing features between mentoring programmes provided to secondary school students in the UK. Interviewees acknowledged the multiplicity of formal mentoring programmes in existence and gave insight into a few different programme types underlying the fact that mentoring programmes take many different forms.Participants spoke at length about differences in mentoring programmes and mentioned many ways in which they thought programmes differed from one another. Two participants expressed feeling unsure about other mentoring programmes and this was not further discussed in the interview. A total of 20 categories which distinguished between programmes were explicitly mentioned by participants: an additional 13 were identified in the data analysis. A list of all 33 categories is presented in the Appendix B and a brief description of each category is given in Appendix C.Categories included those capturing information about the mentoring organisation , characteristics of mentor and mentee , mentoring relationship details , programme delivery  and details of mentoring sessions .Each of the categories and the 28 mentoring programmes were examined for general patterns or programme models that were able to classify mentoring programmes into more than one exclusive category. Generally, mentoring programmes were provided in a variety of different delivery pathways and settings and no two mentoring programmes were described in the same way. Programmes typically overlapped on a few of the categories but differed on other categories. For instance, so called peer-mentoring programmes delivered within schools were similar in that they had they utilised the same setting and the same type of mentor (older students from within the same school), but they were used for different purposes. Whereas some programmes focussed on facilitating the transition to and from secondary school, other programmes focussed on older students helping the younger ones in providing support to increase academic skills and results. Other differences persisted regarding the overall duration of the programme, how regularly mentor and mentee would meet, etc.(i)Programme's overall aimDespite their heterogeneity, analysis of the data found that programmes can be best distinguished from one another by looking at the following three over-arching categories: the setting, the type of mentor(s) and the programme's overall aim. These factors clearly distinguished between programmes and were seen to impact on a variety of other programme components such as its delivery, underlying assumptions and expectations. Each of these categories with supporting quotations from participants will be described in turn.“I personally think that one of the best ways to differentiate is by outcome. So what is it you are trying to achieve with your mentoring programme?”(Manager M23)The foremost category used to distinguish between programmes was the aim of programmes. This was also referred to as the focus or intended outcome of the mentoring programme and was the category that was mentioned the most.“Because we come from a kind of employability and professional friend type scenario, we always bringing it around to the kind of jobs and the kind of careers and the courses that they should be looking at whereas some schemes will just be talking about the personal issues, the, the problems, the home life, you know, that kind of thing.”(Manager M22)Aims of mentoring programmes were regarded to impact on the overall process of mentoring from the start, in the recruitment and training of mentors, to the end as they influenced the types of outcomes that a mentoring programmes aims at and uses to assess its potential impact. Furthermore, the aim of mentoring was seen to impact on the basis of the conversation, communication and generally on what goes on in the mentoring sessions between mentor and mentee as described by the following participant:“When we are looking at funding that people go for different types of mentoring, so there is that, you know, the social, kind of socially disadvantaged route or, you know, real problems route, and we don't really, we don't do that. We don't focus on that. So I think those are the two main areas of differences.”(Manager M22)As one interviewee stated, the type of mentoring provided also corresponded to the funding that is sought and obtained.“How do they differ? I suppose that, that the way that we kind of, the way that we, I, split it up in here I think is probably the clearest distinction. Where you have got academic, where the results are focussed on the academic and underachievement and trying to push people on in terms of their grade and their performance in tests and their learning, improving their learning, to that more, I suppose, dealing with kind of social issues, and dealing with issues of a more, of a more personal nature, personal development issues […] that has to do with relationships and bullying and, kind of, just things that are affecting the young people in terms of their lives outside of the classroom. I think that's probably the clearest distinction that I have been able to make.”(Manager M19)(ii)Type of settingWhereas some programmes aimed to improve and help the current situations a young person was in (i.e. help with learning and academic achievement in school), other programmes were aimed towards supporting a young person's future choices (i.e. help to plan career and university options). Furthermore, programmes differ with regard to the focus of their aim, whether this was quite specific (i.e. help with GCSE learning) or rather broad (i.e. to help develop life skills). One participant, part of an organisation that provided multiple mentoring programmes, gave insight into the way a distinction was made between programmes.“The last thing is, you know, where does it actually take place? Do you go to the kids or do the kids come to you?”(Manager M3)The category that was further able to distinguish between programmes was the predominant setting where the mentoring programme was taking place, i.e. whether mentoring was provided within the school context, in the local community or predominantly online.“It depends on the facilities in school. And they do vary quite a bit. So some of the schools corner off part of the learning centre and then they got the access to like computer and things, and the use of the bigger classrooms for three at the time. I think they basically have got those community rooms, but it is never sort of like rooms in isolation.”(Manager M7)This indicated the context in which the mentoring occurs which was regarded to determine the boundaries, capabilities and expectancies of programmes. For example, whereas mentoring within the school context was regarded as bounded by the school timetabling, safeguarding regulations, institutional rules and regulations and practical considerations like available spaces and facilities, mentoring offered in the community was seen to be more flexible and less bounded.“They meet in, in what's called our [name for learning centre] […] But there are different, sort of little areas within there, where you can have some privacy because obviously in terms of confidentiality they have to be, and safety, they have to be in a place where they can be seen but not overheard. And a lady who works with us […] she is always in there when the meetings are taking place.”(Manager M19)“The majority of our schemes do meet at school, they would meet their student in reception area, they would then go to, a library or the school canteen or if they did use an office, it would be a kind of public office so, you know, have a door open, that kind of thing. So, that's the usual way it works […] it's in a private but public place.”(Manager M22)Regarding mentoring programmes taking place within schools, interviewees highlighted that, for safeguarding as well as practical reasons, mentors worked in spaces such as the canteen, learning centres or open offices and emphasised the ‘private but public’ nature of the setting. This was contrary to many community programmes which were less closely monitored and where mentor and mentee sometimes met in isolated areas.“Yes, it's out in the community. And it could take the form of any sort of activity that you can imagine. So, that could be as simple as going out for coffee, or it could be, going out for dinner, going to the cinema, horse-riding, kayaking, quad-biking, the list is endless. And, and it's all focussed around, initially anyway focussed around trying new experiences, and building a relationship of the back of that.”(Manager M9)The setting also influenced the type of activities that were undertaken as part of mentoring. Whereas community-based programmes typically focussed on engagement in specific activities, such as sports, arts and crafts, most school-based programmes took place in a classroom setting and involved the mentor and mentee sitting down and talking together.“So mentors always will text in and out to us, you know, just like they are a social worker or something, if they do a house visit, then they always need to text the management […] so, say they wanted to go to an isolated beach, I would know the area, I'd do a risk assessment for it, and then, you know, they would clearly let me know the times and they also let the parent know the times.”(Manager M14)Participants described processes by which mentoring in the community was monitored and procedures to make sure that mentoring was in line with safeguarding policies by undertaking risk assessments for mentors working one-to-one with a young person.“They would meet outside of the school environment. Particularly- again, some of these young people have challenging relationships within schools to then to reinforce that with the mentor, we don't see it as a positive. So they would normally happen out of school days, so after 3 o'clock, early evening, night time, and part of that is also to engage the young person with positive activities in the local communities that they don't normally have access to so youth clubs, gyms, other activities, groups that they would not normally have the opportunity to go.”(Manager M2)“It's not that the school environment is a better setting, it is that you are very unlikely to persuade the at-risk young people to give up their afternoon or evening to go voluntarily in- to travel to go and see someone and be mentored. You wanna- you wanna work with the hard kids, yeah? You gonna need to go to them. They are not going to come to you.”(Manager M3)(iii)Type of mentorThe setting of mentoring also impacted on the timing of the mentoring session. Mentoring in the community typically happened after school or on weekends, whereas mentoring within the school would happen as part of school, sometimes provided within school lessons or at lunch times. Views differed on whether mentoring should happen in school time or outside of school and that this would influence the type of young people that were going to engage with the programme.“We are not advisers through experience. So generally, many of the things we talk to young people about, we haven't experienced ourselves.”(Manager M17)Another category that was clearly able to distinguish between programmes was the type of mentor that provided the mentoring and whether this was an older student, a (paid) school staff member, an adult volunteer or paid adult. Whereas adult volunteers were usually seen to differ in characteristics from the young person that took part in the programme, paid adults were seen to sometimes be of similar characteristics, having experienced similar challenges as those taking part in the programme.“And particularly when I speak to the young people and I offer them this programme, I explain that its voluntary on their part, but I also make a point of telling them that the mentors, that I would be, you know, be matching them with, are volunteers, you know? They are not getting paid to spend time with you, they want to spend time with you, because they feel like they can make a difference. And that's always really received really well, because they, you know, especially the young people who are in, in the looked after care system, they constantly deal with changing faces, they constantly deal with people who are paid to do a job, and I think […] they relate really well to someone who is actually, you know, they've got their own life, and they are with them because they want to be.”(Manager M9)Benefits and limitations were perceived with paying mentors for their work. It was acknowledged that whereas paying mentors for mentoring might attract different kind of mentors to the programme, the young people appreciated being mentored by individuals who voluntarily chose to spend their time with them.3.4Depending on subcategories within these categories, a total of twelve mentoring models were identified. As a second step to the development of the final typology, these mentoring models were then grouped into one of two overall categories of programmes.Mentoring programmes that focussed broadly on helping and supporting young people with their character, personal and social development were allocated to ‘Personal and Developmental’ Mentoring (PDM). Programmes that focussed on helping and supporting the young person with regard to their educational learning, future career choices and employability were allocated to ‘Academic and Employability Mentoring’ (AEM). PDM and AEM can be seen as broader mentoring groupings, each encompassing six mentoring models.In total, 15 of the 28 formal mentoring programmes fitted exclusively into PDM, whereas 13 fitted in AEM. The final typology is presented in 3.5“When I look at the granular models that you provide, it's definitely comprehensive. I don't see that there is anything missing”(Expert E3)As outlined in “The major question mark I have over the classification is, are all of the schemes actually mentoring schemes or are they just calling themselves mentoring schemes?”(Expert E1)“For me, some of them [mentoring models] are a mix of befriending and coaching […], others are more mentoring approaches I would recognise”(Expert E1)However, it became clear that participants differed in their conceptualisation of mentoring and what was considered to be a mentoring programme. Overlaps of some models with befriending, counselling and coaching were drawn.“And I would ask the question, is a paid professional a mentor, or is it a paid professional doing some support?”(Expert E2)Moreover, some participants did not see programmes with paid adults as mentoring programmes.“There is no way I would think of a couple of sessions as mentoring. That's not mentoring.”(Expert E4)Consequently, colour-coding had been included in the typology to include this feedback. Another expert participant indicated that mentoring programmes that only entail a few sessions were not regarded as mentoring.“In our experience, mentoring schemes don't fit into boxes this neatly.”Most of the participants felt that the typology was representative of programmes that they were aware of. Two participants highlighted that they felt that mentoring programmes cannot be classified.“I do believe that we do need these models, we do need to be more classified, in order to be able to evaluate our work better.”(Expert E3)“The classification I think will be very helpful […] in helping people to understand that […] in setting up mentoring and delivering mentoring, that having a clear understanding and looking at the different classifications, will help the implementation of a better run project. Which you can then, measure.”(Expert E2)“I think the classification is, as indicated earlier, very useful in helping […] myself and my team to better reflect on and evaluate what we are delivering in our context.”Participants viewed the typology as a useful aid in navigating and understanding different programmes. They also alluded that the typology could help practitioners to describe their programmes which in turn should aid the programmes' evaluation.“You are not being overly prescriptive. […] you are not […] dictating what mentoring should be or should not be, and that context, which is good.”(Expert E4)Furthermore, participants appreciated that the typology itself was not prescriptive of what mentoring should be.The typology was also perceived to act as a starting point for more work in this field and to aid facilitation between organisations that follow similar mentoring models. Appendix D details the main feedback and changes made to the typology at the different stages of the consultation process.4Formal mentoring programmes for young people in UK secondary schools can be classified into twelve distinct mentoring models within two broad groupings of ‘personal and developmental’ and ‘academic and employability’ mentoring programmes. The typology derived from the study differentiates programmes according by three overarching categories: a programme's setting, type of mentor and programmes' overall aim. This study found that formal youth mentoring programmes are heterogeneous and that differences exist in the way ‘mentoring’ is conceptualised.This is the first typology that has been created for the classification of formal mentoring programmes for young people in secondary schools in the UK. Contrary to other typologies , this tyThe methodology used to derive the typology differs from previous attempts  and ensuThe main distinction used within our typology, the overall aim of a programme, is similar to Karcher's work which distinguished what was referred to as ‘instrumental’ and ‘developmental’ mentoring . This inThe consultation process revealed that some participants did not perceive all mentoring models to be ‘mentoring’ which was the main reason for some models to be colour-coded. Participants drew links between mentoring, befriending and coaching. The difficulty of defining what ‘mentoring’ is and consists of is in line with the previous literature . MoreoveIt was further recognised by participants that mentoring programmes might aim to achieve multiple aims, crossing the boundaries between a focus on academic and employability to make long-lasting change in other domains of life as well. We acknowledge that programmes might aim to impact on both, directly and indirectly, and that the aim of the relationship between mentor and mentee might change over time, depending on the specific needs of the young person taking part. For example, whereas the mentoring relationship might focus on a more practical-oriented task in the beginning, through the establishment of a trusting relationship, this might shift and might then focus on sharing personal information.Developing the typology allowed us to learn about common programme practices of mentoring programmes within the UK. Most programmes were delivered on a one-to-one, face-to-face basis however there were also programmes using group mentoring and online mentoring which have been referred to as newer models of youth mentoring in the literature. Moreover, some programmes combined mentoring with other activities as has also been reported from programmes available in the USA . It furtLooking in detail at programmes models available in the United Kingdom highlights that some contain features which would seem to be at variance with what evidence has suggested is best. For instance, research has reported that mentoring carried the greatest impact for those formal relationships that last one year or longer, yet the4.1Due to the eligibility criteria and sampling framework, the generalisability and representativeness of our study is limited to mentoring programmes within the UK. Despite a pilot search undertaken confirming that most mentoring organisations had a web-presence, the search was not exhaustive and might not have included smaller programmes or recently set up programmes. Moreover, another limitation of the research is that characteristics of non-participations at each stage of the non-participation were not captured. All mentoring programmes described in interviews were able to be assigned to one and only one of the twelve mentoring models but this does not mean that the models are equally common and that the number of models is representative of all UK programmes. Further, in cases where multiple programmes were provided by participants, some interviews did not allow thorough coverage of each programme, thereby limiting the typology to the description of the programmes that were given in interviews. Compared to other countries of the UK, programmes based in England had a proportionally higher representation indicating that the mentoring models established in England might not be available in the other countries of the UK. Although the typology was seen by experts and programme managers to be representative, there are no objective data available which could be used to confirm or refute this. The typology does not allow for any inferences made regarding each model's (cost-) effectiveness or efficacy.However, involving programme managers to establish the typology was not only best suited to the needs of the research but also involved a novel approach. The use of maximum variation sampling and three sampling waves allowed for a range of programmes to be selected thereby increasing the generalisability and representativeness of the typology. A consultation process was implemented to allow participants to help improve and validate what was found which has not been done in past studies. Reliability of data was ensured by following the same research principles in each interview and all audio-recordings being transcribed by the same researcher.4.2This research emphasises the heterogeneity of programmes and what is understood to be mentoring, emphasising (i) the need for mentoring organisations to thoroughly describe what their mentoring programme(s) entail and (ii) the need for a definition of mentoring that takes into account current developments in mentoring, such as mentors being paid . The difWhereas many different programmes are typically grouped together in existing systematic reviews, synthesising the evidence of mentoring programmes based on grouping them by their different mentoring models can provide a more useful approach to this. The typology itself can help practitioners and researchers to help allocate their programmes into one of the mentoring models. Doing so can help practitioners to identify similar mentoring programmes. The typology is useful to researchers as it provides guidance about the types of programmes in existence which can help when seeking to evaluate ‘mentoring’ as an intervention. This ultimately paves the way for a more systematic evaluation of the effectiveness of mentoring programmes as interventions for young people. Future research is required to test the typology's generalisability within the UK and internationally. This might be achieved by surveying existing mentoring programmes which will also shed light on the prevalence of each mentoring model. Given the small number of online mentoring programmes or programmes using group mentoring, further research is needed into these newer models of mentoring, their prevalence and process of change. Another distinction that has not been brought up in our work but that has been made in the mentoring literature is between homogenous and heterogeneous groupings of mentor and mentee . It woulGiven the importance of the length of relationship and quality of relationship on the outcomes of mentoring that has been alluded to in the mentoring literature , it woul5Although mentoring programmes are heterogeneous in the ways that they are delivered, it is possible to identify key characteristics and distinguish between different mentoring models based on their overall aim, setting and the type of mentor used. This work emphasises the need for formal mentoring programmes to be well defined and described. The typological approach to mentoring programmes helps academics and service providers understand what is being delivered, for whom, and how, which is a necessary precursor to any evaluation, service design or commissioning process.The Centre for the Development and Evaluation of Complex Interventions for Public Health Improvement (DECIPHer), a UKCRC Public Health Research Centre of Excellence. Joint funding (MR/KO232331/1) from the British Heart Foundation, Cancer Research UK, Economic and Social Research Council, Medical Research Council, the Welsh Government and the Wellcome Trust, under the auspices of the UK Clinical Research Collaboration, is gratefully acknowledged.The work was undertaken with the support of None.


N-glycosylation pathways are dependent of N-acetylglucosaminyltransferase I (GnTI), a key glycosyltransferase opening the door to the formation of complex-type N-glycans by transferring a N-acetylglucosamine residue onto the Man5GlcNAc2 intermediate. In contrast, glycans N-linked to Chlamydomonas reinhardtii proteins arise from a GnTI-independent Golgi processing of oligomannosides giving rise to Man5GlcNAc2 substituted eventually with one or two xylose(s). Here, complementation of C. reinhardtii with heterologous GnTI was investigated by expression of GnTI cDNAs originated from Arabidopsis and the diatom Phaeodactylum tricornutum. No modification of the N-glycans was observed in the GnTI transformed cells. Consequently, the structure of the Man5GlcNAc2 synthesized by C. reinhardtii was reinvestigated. Mass spectrometry analyses combined with enzyme sequencing showed that C. reinhardtii proteins carry linear Man5GlcNAc2 instead of the branched structure usually found in eukaryotes. Moreover, characterization of the lipid-linked oligosaccharide precursor demonstrated that C. reinhardtii exhibit a Glc3Man5GlcNAc2 dolichol pyrophosphate precursor. We propose that this precursor is then trimmed into a linear Man5GlcNAc2 that is not substrate for GnTI. Furthermore, cells expressing GnTI exhibited an altered phenotype with large vacuoles, increase of ROS production and accumulation of starch granules, suggesting the activation of stress responses likely due to the perturbation of the Golgi apparatus.Eukaryotic  N-linked glycosylation is an extensive eukaryotic post-translational modification of secreted proteins consisting of the covalent attachment of an oligosaccharide onto asparagine residues belonging to the consensus sequence Asn-X-Ser/Thr/Cys, where X represents any amino acid except proline4. The process starts in the endoplasmic reticulum (ER) with the biosynthesis of a lipid-linked oligosaccharide (LLO) precursor which involves the action of a conserved set of enzymes named Asparagine-Linked Glycosylation (ALG). First steps of the LLO synthesis occur on the cytosolic face of the ER membrane where N-acetylglucosamine (GlcNAc) and mannose (Man) residues are added step by step onto a membrane-anchor dolichol pyrophosphate (Dol-PP) to form a Man5GlcNAc2-PP-Dol. Then, the Man5GlcNAc2-PP-Dol is flipped into the lumen of the ER6 where its extension occurs by the addition of several Man and glucose (Glc) residues into a complete oligosaccharide precursor Glc3Man9GlcNAc2-PP-Dol7. This LLO is thereafter transferred by the oligosaccharyltransferase (OST) complex onto the asparagine of the N-glycosylation consensus sequence of the nascent polypeptides8. The N-glycan is then trimmed by the action of α-glucosidases I and II, and eventually an ER-mannosidase into Man8-9GlcNAc2 oligomannoside. During these ER events, interactions between the glycoprotein and ER-resident chaperones ensure the glycoprotein folding10. Correctly folded glycoproteins then enter the Golgi apparatus where their N-glycans are processed through the action of an α-mannosidase I into Man5GlcNAc2. Subsequently, N-acetylglucosaminyltransferase I (GnTI), α-mannosidase II and finally N-acetylglucosaminyltransferase II (GnTII) give rise to the canonical GlcNAc2Man3GlcNAc2 core common to mammals, insects and land plants12. This core undergoes further maturation into organism-specific complex-type N-glycans that are involved in several physiological functions like cell-cell interaction, intracellular communication and signaling14.N-linked glycans of the green microalgae C. reinhardtii were recently characterized15. Predominant N-glycans carried by C. reinhardtii endogenous proteins are oligomannosides ranging from Man2GlcNAc2 to Man5GlcNAc2. In addition, mature N-glycans were identified as oligomannosides Man4GlcNAc2 and Man5GlcNAc2 presenting methylation on mannose residues and substituted by one or two xylose residues15. The absence of terminal GlcNAc residues on these N-linked glycans suggested that mature N-glycans in C. reinhardtii are derived from GnTI-independent Golgi events15. In silico analysis of the C. reinhardtii genome allowed the identification of numerous candidate genes encoding enzymes of the protein N-glycosylation pathway, such as the ALG, OST subunits, glycosidases and glycosyltransferases involved either in the ER or Golgi N-glycan biosynthesis steps15. However, in agreement with biochemical analyses, no GnTI candidate is predicted in C. reinhardtii genome suggesting that the N-glycosylation pathway in this organism is different from that found in land plants, insects, mammals16 and diatoms for which a functional GnTI has been recently characterized17.The structures of N-glycosylation pathways between C. reinhardtii and land plants, as well as between C. reinhardtii and diatoms, we have undertaken the expression of either a diatom or a plant GnTI in C. reinhardtii to investigate the effects of such a complementation on the physiology and N-glycan pathway in C. reinhardtii. Our findings demonstrate that the expression of GnTI induces stress responses in C. reinhardtii and reveal a truncated ER N-glycosylation pathway in C. reinhardtii.Considering the difference in the GnTI) or from the diatom Phaeodactylum tricornutum (PtGnTI). Both have been shown to be able to process N-linked glycans. AtGnTI (At4g38240) encodes for a Golgi enzyme that is responsible for the transfer of a terminal GlcNAc residue onto Man5GlcNAc2N-glycan19. PtGnTI has been shown to restore in vivo the biosynthesis of complex-type N-glycans in CHO Lec1 mutant that lacks endogenous GnTI activity17. These sequences fused to a tag sequence encoding for a V5-epitope were used for nuclear expression in C. reinhardtii22. RT-PCR analyses show that both plant and diatom GnTI were expressed in C. reinhardtii transformed lines . As illustrated in Fig. N-glycosylation in cw92 and transformed cells were then investigated by mass spectrometry profiling. N-linked glycans were released from total protein extracts using PNGase F, labeled with 2-aminobenzamide (2AB) and analyzed by liquid chromatography coupled to electrospray ionization mass spectrometer (LC-ESI-MS). Although the expression of GnTI induced altered phenotypes in C. reinhardtii, the comparison of the N-glycan structures between cw92 cells and the transformed lines did not reveal any modification of the N-glycan profiles  or the oligomannoside Man5GlcNAc2N-linked to secreted proteins. Man5GlcNAc2 oligomannoside was previously identified in C. reinhardtii cw92 proteins and was assigned to a branched Man5GlcNAc2 by analogy with data published regarding the Golgi N-glycan processing in eukaryotes15. The non-effect of GnTI on the N-glycan profiles from C. reinhardtii raised questions regarding the structure of this Man5GlcNAc2, which therefore needs to be reinvestigated. Here, we made use of Ion Mobility Spectrometry–Mass Spectrometry (IMS-MS), a reliable analytical method which allows the separation of isomers including oligosaccharide isomers25. In this analysis, the ion mobility of Man5GlcNAc2 coupled to 2AB oligosaccharide (sodium adduct) prepared from C. reinhardtii proteins was compared to the ion mobility of branched sodiated 2AB labeled Man5GlcNAc2 obtained from the bovine ribonuclease B. As illustrated in Fig. 5GlcNAc2-2AB from C. reinhardtii and from bovine ribonuclease B exhibited different ion mobilities  which suggested that they possess distinct structures. This data indicated that Man5GlcNAc2 in C. reinhardtii is different from the branched Man5GlcNAc2 that results from the trimming of oligomannosides Man8-9GlcNAc2 in the ER and the Golgi apparatus in plants and mammals.Different scenarios may explain the inability of exogenous GnTI to affect the protein C. reinhardtii Man5GlcNAc2 isomer was further investigated using electrospray ionization-multistage tandem mass spectrometry (ESI-MSn)  taking advantage of specific ion transitions reported in ref. 2+ of permethylated Man5GlcNAc2-2AB (m/z 882) prepared from proteins of PtGnTI#1 transformed line and from bovine ribonuclease B were compared -Man residues instead of α-Man. For further confirmation of the C. reinhardtii Man5GlcNAc2 topology, the N-glycan mixture was submitted to exoglycosidase degradations using either Jack bean mannosidase, a non-specific α-mannosidase and Aspergillus saitoi mannosidase, an exoglycosidase specific for α-mannose residues27. Man5GlcNAc2-2AB from C. reinhardtii was efficiently converted into ManGlcNAc2-2AB by Jack bean mannosidase after removal of the four α-mannose units. In contrast, Man3GlcNAc2-2AB was the major end product after treatment with Aspergillus saitoi α-mannosidase that results from the removal of two terminal α-mannose residues from the linear trimannoside arm   yeast mutant  Fig. . A minorAc2 Fig. . The seq ER Fig. .Figure 6N-glycan structures linked to endogenous proteins in C. reinhardtii and showed they were mostly oligomannosides and for 30% novel mature structures containing xylose residues and methylated mannoses on Man4-5GlcNAc215. These structures resulted from a Golgi GnTI-independent processing of oligomannosides as the bioinformatics analysis of the genome has revealed that it lacks GnTI. Furthermore, no N-glycan harboring terminal GlcNAc residues has been identified in the whole glycan population15. This situation contrasts with most eukaryotic organisms in which the GnTI is a key Golgi enzyme in the N-glycosylation pathway opening the door to the biosynthesis of structurally diverse mature N-glycans12. In this canonical pathway, the addition of a first GlcNAc by GnTI is required for the sequential activity of a large repertoire of other specific transferases giving rise to complex N-glycans involved in numerous biological processes, such as intracellular communication and signaling14. Inactivation of GnTI in those organisms induces strong developmental phenotypes. For instance, GnTI-null embryos of mouse die at about 10 days after fertilization indicating that mature N-glycans are required for morphogenesis in mammals33. Also, inactivation of GnTI reduces the viability in worm and fly35. In plants, GnT I mutants exhibit a stress phenotype, thus suggesting a role for mature N-glycans in specific physiological processes37. In rice, gntI mutants, impaired in the N-glycans maturation showed severe developmental defects, resulting in early lethality, associated to reduced sensitivity to cytokinin36.In previous work, we characterized GnTI genes are predicted in different microalgae genomes. In the diatom P. tricornutum, PtGnTI was demonstrated to encode for an active glycosyltransferase17. Therefore, the capacity of C. reinhardtii to express GnTI from Arabidopsis and from P. tricornutum was analyzed to investigate whether C. reinhardtii N-glycan biosynthesis can be complemented with this key transferase and shift into a GnTI – dependent pathway. Moreover, in a biotech context, the production, in microalgae, of recombinant glycoproteins for therapeutic applications will require the engineering of their endogenous N-glycosylation pathway for the production of biopharmaceuticals exhibiting human-compatible N-glycans. Therefore, implementation of a GnTI - dependent pathway is a prerequisite for any production of glycosylated biopharmaceuticals in C. reinhardtii.N-glycan profiles from proteins secreted in transgenic lines did not show any modification of the N-glycan population by comparison with the cw92 cells. Among the different scenarios that may explain this result, the absence of UDP-GlcNAc nucleotide sugar in the Golgi apparatus was first considered. The transport of the cytosolic nucleotide sugars across the Golgi membrane is performed by Nucleotide Sugar Transporters (NSTs). Searching for NST orthologues in the C. reinhardtii genome allowed the identification of 23 candidate genes of which the deduced amino-acid sequences harbor the characteristic Triose Phosphate Translocator (TPT) domain (Pfam 03151) present in NSTs . However, as most of the characterized NSTs has been shown to transport at least two distinct substrates, determination of their specificity for nucleotide sugars based on sequence homologies with other Golgi NSTs remains difficult without additional biochemical evidence38. Therefore, further experimental work is needed to determine whether a specific Golgi UDP-GlcNAc transporter exists in C. reinhardtii. In relation to this, we cannot ruled out that the cytosolic abundance of UDP-GlcNAc may not be sufficient to supply the Golgi apparatus which would consequently limit GnTI in C. reinhardtii.Transgenic lines expressing the Arabidopsis or diatom GnTI were obtained and GnTI protein was immunodetected as expected in the microsomal fraction. However, further experiments are required to confirm the GnTI localization within the Golgi apparatus. Mass spectrometry analyses of N-glycan population in C. reinhardtii transformed lines may also result from the absence of the appropriate glycan substrate. Man5GlcNAc2 oligomannoside was previously identified in C. reinhardtii15 and considered as being the branched isomer substrate for GnTI by analogy with mammals and plants N-glycan pathways. Its structure was reinvestigated by mass spectrometry and enzyme sequencing and compared to a branched Man5GlcNAc2 standard from mammals26. Ion mobilities determined by IMS-MS and fragmentation patterns resulting from ESI-MSn clearly show that C. reinhardtii and mammalian oligomannosides differ in their shape and sequence. Moreover, the fragmentation pattern determined by MSn and enzyme digestion with an α-mannosidase are consistent with the presence of a linear Man5GlcNAc2 on C. reinhardtii proteins as depicted in Fig. C. reinhardtii genome40. These ER enzymes are involved in the completion of the biosynthesis of the LLO precursor Man9GlcNAc2-PP-Dol prior to its glucosylation -linked Man (I) or the first α-linked Man (II). MS4 experiments are more consistent with the structure I depicted in Fig. Inability of GnTI to affect the endogenous ion Fig. . As a coAc2 Fig. . In contses Fig. . HoweverAc2 Fig. . Such a MSn Fig. . This anGnTI exhibited an altered phenotype although no modification of the N-glycans has been observed. The presence of large vacuoles as well as the increase of ROS production observed in transformed cell lines suggested that transformation with GnTI induced the activation of stress responses such as autophagy and oxidative stress47. Previously, Pérez-Martin and collaborators showed that ER stress caused by tunicamycin or DTT triggers autophagy48. It can be hypothesized that AtGnTI or PtGnTI may localize in the ER of C. reinhardtii. Indeed, a previous study published by Schoberer and coworkers in 2009 demonstrated that overexpressed GnTI in tobacco recruit the ER protein Sar1p resulting in a GnTI-Sar1p association in the ER membranes49.Surprisingly, cells expressing GnTI or PtGnTI accumulate starch. In BY2 cells and chlamydomonas noctigama, Hummel and collaborators50 reported in 2010 that the disassembly of the Golgi apparatus using the secretion inhibitor Brefeldin A (BFA) caused the accumulation of plastid starch. This starch increase was also observed when the CopII-mediated ER to Golgi transport was inhibited in tobacco plants expressing a dominant negative version of the small GTPase Sar1p51. Moreover, it was proposed that disruption of the Golgi apparatus resulting in the loss of secretory activity may cause the redirection of free carbohydrates to the plastids where they would be converted into starch51.In addition, chlamydomonas cells expressing AtGnTI may affect Golgi organization or the endosomal system, which would consequently disturb the biosynthesis of glycans or glycoconjugates and the cell biology of C. reinhardtii. Additional work and phenotypic studies of transformants expressing a non-functional GnTI would allow the confirmation of this hypothesis.Therefore, we postulate here that the heterologous expression of −2 s−1 using TAP medium redirection of free carbohydrates to the plastids where they would be converted into starch52.CC-503 cw92 strain, later called cw92 cells, was obtained from the Chlamydomonas Culture Collection at Duke University  and grown in batch cultures at 25 °C, illuminated with 150 μmol mGnTI (At4g38240) and PtGnTI (gi: 307604450) fused to the V5 epitope17 were codon optimized and synthesized by Sloning Biotechnology. Optimized sequences were cloned as a XbaI/NdeI fragment in the pSL18 vector53. pSL18 (empty vector), pSL18-AtGnTI or pSL18-PtGnTI construct was introduced using the glass beads method54. To detect the transgene in genomic DNA, screening was performed by PCR using V5-reverse GGAGTCCAGGCCCAGCAGG and with AtGnTI-forward GCCCTAAGTGGCCAAGGC or PtGnT1-forward CCAGTCCAAGTGGCCGGGC as primers.The sequences encoding for At6 cells), using TRIzol (Invitrogen). gDNA contamination was removed by a Turbo DNase treatment (Thermo Fisher Scientific). Reverse transcription was performed on 2 µg of RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The transcription level of AtGnTI and PtGnTI transgenes was analyzed by PCR using the V5-reverse primer, with either AtGnTI-forward or PtGnT1-forward primers. Each PCR contained 2 µL of diluted cDNA (1/10), 0.8 mM of each primer, 0.25 mM dNTPs and 0.6 U GoTaq polymerase (Promega) and was performed in the reaction buffer provided by the manufacturer, according the following program: 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 62 °C for 30 s, 72 °C for 30 s and a final step of 72 °C for 5 min. The expression of the actin gene was monitored as a control using CrActin-forward CGCTGGAGAAGACCTACGAG and CrActin-reverse GGAGTTGAAGGTGGTGTCGT as primers.Total RNA were isolated from fresh cell pellets  and washed with a 20 mM potassium phosphate buffer at pH 7.4. All the following preparation steps were carried out at 4 °C. The cell pellet was broken with 2 mL of protease inhibitor cocktail 25X (Roche) dissolved in 10 mM potassium phosphate buffer (pH 7.4) using the FastPrep-24™ 5G15. Samples were then spun (300 g for 3 min) in order to remove intact cells and debris. The supernatant was collected and centrifuged  in order to eliminate pigments and chloroplasts. Finally, the supernatant was ultracentrifuged  to pellet the microsomal fraction. Immunodetection using anti-V5 antibodies has been performed as described in ref. 1.4 × 10N-glycosidase F (PNGase F) as described in ref. N-glycans were then labeled with 2-aminobenzamide (2AB) according to ref. Aspergillus saitoi α-mannosidase (ProZyme) enzymatic treatments were performed on 4.5 µL of 2AB-labeled N-glycans according to the manufacturer’s instructions.Total proteins were extracted and deglycosylated using Peptide Permethylation - The 2AB-labeled N-glycans were permethylated29 and cleaned-up according to ref. N-glycans were enriched and desalted on a 500 nL PGC trap column and separated on a PGC (3-μm particle size) column . A 30-min linear gradient (5–75% acetonitrile in 0.1% formic acid) at a flow rate of 400 nL min−1 was used. Separated N-glycans were analyzed with the QTOF analyser. Full autoMS scans from 290 to 1,700 m/z and autoMS/MS from 59 to 1,700 m/z were recorded. In every cycle, a maximum of 5 precursors sorted by charge state (1+ and 2+ preferred) were isolated and fragmented in the collision cell with fixed collision cell energy at 15 eV. Scan speed raise based on precursor abundance  and precursors sorted only by abundance. Active exclusion of precursors was enabled and the threshold for precursor selection was set to 1,000 counts. The ESI acquisition parameters in positive mode were: capillary voltage; 1.9 kV, drying gas temperature; 250 °C, gas flow (air); 11 L min−1; fragmentor voltage, 360 V; Skimmer1 voltage, 65 V and OctopoleRFPeak voltage, 750 V.The analyses were performed using the nano-LC1200 system coupled to a QTOF 6550 mass spectrometer equipped with a nanospray source and a LC-Chip Cube interface . Briefly, 2AB-labeled −1) and operated in ‘V’ resolution mode . The ESI parameters were in positive ion mode: capillary voltage, 3 kV; sample cone voltage, 70 V; source temperature, 90 °C; desolvation temperature, 250 °C; desolvation gas flow (N2), 700 L h−1. The data were acquired using a 50–2000 m/z range with 1 s scan time and 0.02 s interscan delay. Sample solutions were infused into the source at a flow rate of 400 µL h−1 with a syringe pump . The IMS conditions were optimized as followed: gas flow (N2), 90 mL min−1; IMS pressure, 3.05 mbar; wave height voltage, 40 V and T-wave velocity, 550 m s−1. The ion mobility spectra were fitted using the Origin 9.0 software (OriginLab).The ESI-IM-MS experiments were performed using a Waters SYNAPT G2 hybrid quadrupole/HDMS instrument equipped with an ESI LockSpray™source, the MassLynx 4.1 and the DriftScope 2.2 softwares . The SYNAPT HDMS system was calibrated using sodium formate cluster ions  using a Bruker HCT Ultra ETD II quadrupole ion trap (QIT) mass spectrometer equipped with the Esquire control 6.2 and Data Analysis 4.0 softwares . The ESI parameters were as followed: capillary and end plate voltages respectively set at −3.5 kV and −3.0 kV in positive ion mode, skimmer and capillary exit voltages set at 40 V and 200, respectively, nebulizer gas (N2), pressure, drying gas (N2) flow rate and drying gas temperature were 10 psi, 7.0 L min−1 and 300 °C, respectively. Helium pressure in the ion trap was 1.2 × 10−5 mbar. The data were acquired using a 200–2200 m/z range, using a scan speed of 8,000 m/z per second. The number of ions entering the trap cell was automatically adjusted by controlling the accumulation time using the ion charge control (ICC) mode  with a maximum accumulation time of 50 ms. The injection low-mass cut-off (LMCO) value was m/z 100 for Man5GlcNAc2 derivatives and 140 for LLO precursor respectively. The values of spectra averages and rolling average were 6 and 2. ESI-MSn experiments were carried out by collision-induced dissociation (CID) using helium as the collision gas, isolation width of 2 m/z unit for the precursor ions and for the intermediate ions using a resonant excitation frequency with an amplitude from 0.8 to 1.0 Vp–p. Sample solutions were infused into the source at a flow rate of 300 µL h−1 by means of a syringe pump .Permethylated 2AB-derivatives of C. reinhardtii cells were treated at room temperature 1 hour with 100 mM mannitol as cryoprotectant diluted in fresh culture medium. Pre-treated C. reinhardtii cells were transferred into an aluminium cryocapsule covered by soy lecithin dissolved at 100 mM in chloroform. Excess medium was absorbed by filter paper. After fixation on the loading device, samples were frozen according to a maximum cooling rate of 20,000 °C s−1 and a pressure of 2,100 bars. Samples were transferred to a freeze substitution automate  pre-cooled to −110 °C. Samples were substituted in anhydrous acetone with 0.5% uranyl acetate and 0.5% osmium tetroxide at −90 °C for 72 h. The temperature was gradually raised to −60 °C using a gradient of +2 °C h−1 and stabilized during 12 h, then gradually raised to −30 °C by using the same gradient during 12 h and gradually raised again to +4 °C. Then, samples were rinsed twice at room temperature with anhydrous acetone. Infiltration was then processed in acetone-Spurr’s resin . The Spurr’s resin was finally polymerised at +50 °C during 24 h. Ultrathin sections  were collected onto carbon-formvar-coated nickel grids. A classical staining using 0.5% uranyl acetate and 0.5% lead citrate was done before sections were observed in a Philips, FEI Tecnai 12 Biotwin transmission electron microscope operating at 80 kV, with ES500W Erlangshen CCD camera (Gatan).High pressure freezing was performed with the freezer HPM100 Leica-microsystems. Prior to freezing, 72 h old 7) were collected . The cell pellet was incubated in TAP medium containing 4% of paraformaldehyde (PAF) (v/v) during 30 min, under shaking, at room temperature. Then, cells were washed four times and finally resuspended in 200 µL of TAP. 20 µL of fixed cells were spotted between slide and coverslip  and were observed with a Leica DMI-6000B inverted microscope at magnification 400. Cell size was determined using fixed cells by measuring the cell longitudinal diameter with Image J software. The measurements were done in triplicates on a minimum of 200 cells (n > 200).Cells  production was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Cells were incubated at room temperature in the dark for 60 min in Krebs-HEPES buffer containing 5 µM diethyl dithiocarbamate, 25 µM deferoxamine, and the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl pyrrolidine hydrochloride . Spectra of the oxidized product of CMH (CM.) were recorded from frozen samples with a X-band spectrometer  with the following acquisition parameters: microwave power, 1 mW; modulation amplitude, 5 G; sweep time, 60 s; and 1 scans. After correction of the baseline, the total amplitude of the signal was measured and expressed in arbitrary units produced per 12 × 10C. reinhardtii according to a protocol adapted from28. Released oligosaccharides were then lyophilized and resuspended in 500 µL of water and purified on a carbograph column  using the manufacturer’s instructions.The LLO extraction was performed on microsomal fractions from Supplemental Figures


The academic scandal on a study on stimulus‑triggered acquisition of pluripotency (STAP) cells in Japan in 2014 involved suspicions of scientific misconduct by the lead author of the study after the paper had been reviewed on a peer‑review website. This study investigated the discussions on STAP cells on Twitter and content of newspaper articles in an attempt to assess the role of social compared with traditional media in scientific peer review.This study examined Twitter utilization in scientific peer review on STAP cells misconduct.Searches for tweets and newspaper articles containing the term “STAP cells” were carried out through Twitter’s search engine and Nikkei Telecom database, respectively. The search period was from January 1 to July 1, 2014. The nouns appearing in the “top tweets” and newspaper articles were extracted through a morphological analysis, and their frequency of appearance and changes over time were investigated.The total numbers of top tweets and newspaper articles containing the term were 134,958 and 1646, respectively. Negative words concerning STAP cells began to appear on Twitter by February 9-15, 2014, or 3 weeks after Obokata presented a paper on STAP cells. The number of negative words in newspaper articles gradually increased beginning in the week of March 12-18, 2014. A total of 1000 tweets were randomly selected, and they were found to contain STAP-related opinions , links to news sites and other sources , false scientific or medical claims , and topics unrelated to STAP .The discussion on scientific misconduct during the STAP cells scandal took place at an earlier stage on Twitter than in newspapers, a traditional medium. In recent years, the number of cases of scientific misconduct, including fabrication, falsification, and plagiarism has increased . MiscondThe three major historical scandals of scientific misconduct were German physicist Jan Hendrik Schön’s fraudulent superconductor breakthroughs at Bell Labs in the United States in 2002, South Korean researcher Hwang Useok’s fabrication of embryonic stem cells in 2005, and Japanese stem-cell biologist Haruko Obokata’s and her fellow researchers’ claims on stimulus‑triggered acquisition of pluripotency (STAP) cells in 2014 ,7. SuspiThe STAP cells scandal demonstrated how Twitter enables the rapid spread of information through sharing between multiple users, allowing numerous users to obtain the information simultaneously. Suspicions on research papers, thus, can be raised on Twitter by multiple people, making the social media site a useful tool for discussion and debate. In fact, previous studies have found that researchers engage in discussions on their studies via Twitter ,13. SeveThis study investigated discussions on STAP cells on Twitter and the content of newspaper articles in an attempt to differentiate social from traditional media. To identify changes in the tone of Twitter and newspapers over time, collected tweets and newspaper articles on STAP were decomposed through a morphological analysis. Furthermore, to ascertain social media’s role in resolving scientific misconduct, Twitter’s role in the STAP cells scandal was compared with that of newspapers, which represent the traditional media.Asahi Shimbun, Yomiuri Shimbun, Mainichi Shimbun, Sankei Shimbun, and Nihon Keizai Shimbun). The 5 major national newspapers published a combined total of 23,543 thousand copies every half-year on average [Searches for tweets and newspaper articles containing the term “STAP cells” were carried out for 6 months (26 weeks) from January 1 to July 1, 2014. Tweets were extracted through Twitter’s search engine. A top tweets search was performed every week; top tweets are tweets that have been retweeted or replied to by several users and selected through an algorithm developed by Twitter . The new average ,18. Arti average .Tweets and newspaper articles generated from the search were saved as HTML files by using the Web browser function. One HTML file contained a week worth of search, except from April 9 to 15, when the number of tweets was so high that weekly data could not be saved in one file. As such, HTML data were saved separately for each day. The syntax for designating cut‑off dates provided on each of the official sites was used in each search.Text data were extracted by stripping the HTML tags from the HTML files saved from the Web browser. The dates, account names, text of the tweets, and the titles and text of newspaper articles were extracted from the Twitter HTML and newspaper article files, respectively. Nadeshiko  was used to write a program that would eliminate tags and extract the text.To generate the relevant words for the morphological analysis, the dates, account names, URLs, and graphic characters were eliminated from the tweet text data. The following frequently used terms were also eliminated from both tweets and newspaper articles: STAP, stap, cells, Obokata, Haruko, RIKEN Research Center, and RIKEN. The free bulk text processing software Text Search and Substitute.NET (TextSS.NET) version 5.21  was used for text processing.Morphological analysis is one of the basic techniques used in Japanese text mining analysis. It is the process of segmenting a given sentence into a row of morphemes. A morpheme is a minimal grammatical unit, such as a word or a suffix . MeCab , covering the week before the publication of the STAP cell paper (Week 0) and the 13 weeks after (Weeks 1-13). The nouns identified in the tweets and newspaper articles through the analysis were extracted, and their frequencies were calculated.The top 100 nouns that appeared most frequently each week in tweets and newspaper articles during the 14 weeks were then extracted. Terms that were either positive or negative toward STAP cells were then selected from among these 100 frequently appearing nouns. Changes in the use of these positive and negative terms each week were also investigated. Authors Yuya Sugawara, a specialist in medical informatics, and Hiroto Narimatsu, a medical doctor, selected the positive or negative Japanese terms semantically.R version 2.13.0  was used for morphological analysis. The R package used for our morphological analysis was RMeCab version 0.97. RMeCab is a package developed to operate the Japanese morphological analyzer MeCab version 0.98 from R.To investigate the attributes of Twitter account holders who tweeted using negative terms on STAP cells, 100 from the total 558 accounts that sent STAP‑cells tweets from February 5 to 18, 2014, containing the negative term “unnatural” were randomly selected. Profile searches were performed on these 100 accounts using Twitter’s Profile Search feature . All theIn order to classify the content of tweets on STAP cells, 1000 tweets were randomly extracted from the total 134,958 tweets that were assembled over the 6-month search period; all the 134,958 tweets were assigned random numbers, and the 1000 tweets were extracted in the descending order of the assigned numbers. These 1000 tweets were classified into four types: STAP related; links to news sites, and so on; false scientific or medical claims; and topics unrelated to STAP specified by Yuya Sugawara and Hiroto Narimatsu.We conducted a morphological analysis of the tweets and newspaper articles that appeared during the first 13 weeks (3 months) following the publication of STAP cells paper on January 30. The frequency of appearance of nouns in these tweets and newspaper articles during the same period was also investigated. A total of 100 nouns that appeared most frequently were extracted and classified whether positive or negative toward STAP cells. The positive terms selected were “major discovery” and “ground-breaking,” whereas the negative terms were “unnatural,” “fabrication,” and “falsification.”Four of the account holders who published tweets on STAP cells containing the negative term “unnatural” were considered to possess certain specialist knowledge being a former biological researcher, a staff at a manufacturer of reagents for biological experiments, a science writer, and a science journalist. The others included 8 news sites, 9 bots , and 79 accounts for which the level of specialist knowledge could not be determined.Of the total 134,958 tweets generated during the 6-month period, 1000 tweets were randomly selected and found to contain STAP-related opinions , links to news sites and other sources , false scientific or medical claims , and topics unrelated to STAP . The examples of tweets that were evaluated to make false scientific or medical claims were as follows: “STAP cells will give us immortality,” “STAP cells are produced from the skin of newborn babies,” and “STAP cells can be made at home.”This study revealed that the discussion on misconduct in the STAP cells affair was taken up at an earlier stage on Twitter than by newspapers. Positive terms appeared both on Twitter and in newspaper articles during the first week after STAP cells study was published, suggesting that the tendency was for STAP cells to be viewed positively. From Week 2 February 5-11), however, the use of positive terms decreased and negative ones started to appear. On the Twitter, the frequency of negative terms increased during February 12-18 and starting again from March 19 to 25, 2 weeks after Obokata’s coauthor called for the retraction of the paper. In newspaper articles, the negative terms increased between March 19 and 25 and April 9 and 15; an explanatory press conference by Obokata was held during April 9 and 15. The highest use of negative terms in newspaper articles was observed during this week. The use of both positive and negative terms declined beginning during April 16-22. The story on suspicions were taken up by newspapers after the “unnatural” nature of STAP cells had been pointed out on Twitter and after RIKEN announced that it had found Obokata guilty of scientific misconduct, with the increased use of “fabrication” and “falsification” setting a different tone. This tone might have affected public opinion on the STAP cells paper. In both the Schön and Useok scandals, both of which constituted serious scientific misconduct, the retraction of papers took several years ,27. Obok-11, howeAccuracy of Twitter is not always guaranteed. The medical and scientific accuracy of the tweets in this study was questionable for 8.9% of the cases. Accuracy is a constant issue not only in social media but also in Web-based information in general. Caution is always required when using Twitter and other Web-based sites to identify the wrong information. The Japan Internet Medical Association has issued a guide on using Web-based medical information . This guThe greatest advantage of using Twitter for scientific discussion is the rapidity with which the debate proceeds. However, Twitter also contains inaccurate information and excessive arguments that may become abusive. The latter can have a restraining effect on researchers. Depending on how the functions of Twitter are used, it may be possible to distinguish inaccurate information and to avoid abuse to some extent. Hence, the benefits of the rapid discussion enabled by Twitter, as shown in this study, can be enjoyed while limiting the risks of its disadvantages.This study showed the advantages of carrying out a scientific discussion on Twitter, but the scope of the study was limited. First, only top tweets were analyzed and an analysis of all tweets might have revealed different views. Second, the content of the discussion on Twitter, particularly on whether a discussion on misconduct in the STAP cells study took place, was not scrutinized. Third, other forms of media such as blogs, weekly magazines, and television were not investigated. Inaccurate images and articles broadcast by other media may also have had an effect on Twitter. Moreover, we did not analyze the accuracy of newspapers. The accuracy of information in newspapers was not necessarily better than that of Twitter. It is possible that the newspaper articles contained inaccurate statements.The discussion of misconduct in the STAP cells study might have spread rapidly as it involved copying and pasting, a careless behavior familiar to the public. Specialized and complicated misconduct would be less likely discussed by the public on Twitter.The discussion on scientific misconduct involving the STAP cell study took place at an earlier stage on Twitter than in newspapers, representatives of the traditional media. Results of the study suggest that the Twitter debate might have contributed to the resolution of the STAP cell scandal.