Recruitment rates in multi-centre randomised trials often fall below target recruitment rates, causing problems for study outcomes. The Studies Within A Trial (SWAT) Programme, established by the All-Ireland Hub for Trials Methodology Research in collaboration with the Medical Research Council Network of Hubs in the United Kingdom and others, is developing methods for evaluating aspects of trial methodology through the conduct of research within research. A recently published design for a SWAT-1 provides a protocol for evaluating the effect of a site visit by the principal investigator on recruitment in multi-centre trials.Z-scores for proportions were calculated to determine within site recruitment differences. Odds ratios and 95% confidence intervals were calculated to determine between site recruitment differences.Using the SWAT-1 design, the effect of a site visit, with the sole purpose of discussing trial recruitment, on recruitment rates in a large multicentre trial in the Republic of Ireland was evaluated. A controlled before and after intervention comparison was used, where the date of the site visit provides the time point for the intervention, and for the comparison to control sites. Site A received the intervention. Site B and Site C acted as the controls. Recruitment rates were increased in Site A post-intervention . No differences in recruitment occurred in Site B or in Site C. Comparing between site differences, at 3 months post-intervention, a statistically significant difference was detected in favour of higher recruitment in Site A .This is the first reported example of a study in the SWAT programme.. It provides evidence that a site visit, combined with a scheduled meeting, increases recruitment in a clinical trial. Using this example, other researchers might be encouraged to consider conducting a similar study, allowing the findings of future SWAT-1s to be compared and combined, so that higher level evidence on the effect of a site visit by the principal investigator can be obtained.www.controlled-trials.com); date of registration: 25 March 2008.ISRCTN-96340041 ( Recruitment in large multi-centre randomised trials invariably presents a challenge for the research team. Actual recruitment rates often fall below those that were hoped for at the start of the trial -3. Poor Faced with poor or slow recruitment, trial researchers often undertake activities aimed strategically at improving recruitment. Examples of these include increased communication between the researchers and the study sites , incentiEstablished by the All-Ireland Hub for Trials Methodology Research in collaboration with the Medical Research Council Network of Hubs in the United Kingdom and others, the SWAT  Programme is developing methods for evaluating the effects of different ways of designing, conducting, analysing and evaluating studies through the conduct of research within research . The SWAThe aim of this study is to evaluate the effectiveness of a site visit combined with a scheduled meeting, with the sole purpose of discussing recruitment on recruitment rates to the ADCAR trial.The ADCAR trial is a multi-centre, three-site randomised trial that was conducted in the Republic of Ireland between May 2008 and April 2012. ADCAR evaluated the effectiveness of admission cardiotocography (ACTG) versus intermittent auscultation (IA) of the fetal heart rate (FHR) in low risk women on admission to the labour assessment room with signs of possible labour. Based on sample size estimates, a target study sample of 5,776 was required, and it was anticipated that this could be achieved within a 28-month recruitment time-frame. However, as the trial progressed, monthly overall recruitment rates averaged approximately one-third of target rates, with recruitment in some sites proportionately better than in others.The study uses SWAT-1 methodology , with a The intervention consisted of a site visit by the lead researcher, that is, the person responsible for the day-to-day management of the trial, combined with a scheduled meeting arranged with the sole purpose of discussing recruitment rates to the trial. The clinical manager at Site A was contacted with a request to facilitate the visit and meeting. This included advertisement of the meeting 2 weeks in advance, targeting senior midwifery and obstetric staff to attend the meeting and setting the meeting date and time to facilitate maximum staff attendance. The site visit took place on 22 June 2011. The meeting consisted of a 10-minute presentation inclusive of information on overall trial recruitment rates and site-specific recruitment trends followed by 20 minutes of discussion on challenges to recruitment and how these might be overcome. Table The control sites (Site B and Site C) did not receive a site visit combined with a scheduled meeting. Rather, routine trial activities, such as weekly visits by a member of the research team to collect completed trial documents and visits to facilitate information sessions for new or rotating staff, were maintained. These routine trial activities were ongoing at all three study sites throughout the duration of recruitment in the trial.Difference in recruitment in each site  from 1 month pre-intervention to 1 month post-interventionDifference in recruitment in each site  from 3 months pre-intervention to 3 months post-interventionThe primary outcome measures were as follows:Change in adherence to trial procedure  in each site  from 1 month pre-intervention to 1 month post-intervention.Change in adherence to trial procedure in each site  from 3 months pre-intervention to 3 months post-intervention.The secondary outcome measures were as follows:The 1 month and 3 month timeframes were chosen to assess short- and long-term effects of the intervention.https://nl.tenalea.net). Randomisations were logged in real time in a central database, which was password protected and accessible to the lead research assistant only. All extracted data were entered into a pre-designed data management table in preparation for analysis.The data of interest  were extracted from hard copy trial screening and register forms and from the randomisation service database. The randomisation service used in the trial was an automated telephone randomisation service . For between-site differences, odds ratios (OR) and 95% confidence intervals (CI) were calculated to compare Site A pre- and post-intervention recruitment and adherence with pre- and post-intervention recruitment and adherence in Site B + Site C combined.Target recruitment rates at each study site had been calculated using a 50% sample population eligibility and assuming that 50% of the eligible population would agree to participate. Due to variations in the sizes of study sites, target recruitment rates varied between sites. Calculations were performed from the time point that the SWAT-1 intervention was implemented  to the corresponding monthly dates before and after the intervention. For example, recruitment proportions 1 month pre-intervention were calculated based on recruitment between 22 May and 21 June inclusive; similarly, recruitment proportions 1 month post-intervention were calculated based on recruitment between 22 June and 21 July inclusive. We used the Ethical approval was granted for the ADCAR trial by the Faculty of Health Sciences Research Ethics Committee, Trinity College Dublin, the Coombe Women and Infant’s University Hospital Research Ethics Committee, Galway University Hospitals Clinical Research Ethics Committee and by the Healthcare Research Advisory Committee, Health Services Executive, North-East Region. A written signed consent form was obtained from all participants in the ADCAR trial prior to inclusion and randomisation. Ethical approval was not required specifically or in isolation for the site visit because this was a responsive intervention, embedded within the main trial as part of trial procedure to address slow recruitment.Figure Table The analyses demonstrated a 17% and 14% percentage point increase in recruitment in Site A, respectively, at 1 and at 3 months post-intervention. No statistically significant differences (increase or decrease) in recruitment occurred in Site B or in Site C post-intervention at 1 month or at 3 months.Comparing between-site differences in recruitment, a significantly lower rate of recruitment compared to the target rate was found in Site A  compared to Site B + C  at 1 month pre-intervention . At 1 month post-intervention, this difference was no longer observed  . These figures were retrieved from the trial screening and register forms completed just prior to the time-point of each woman’s potential recruitment. Comparing within-site adherence, the analyses demonstrated a statistically significant increase in adherence at 1 month and at 3 months post-intervention in Site A Table . There wComparing between site differences in adherence rates, a statistically significantly lower adherence rate in Site A compared to Site B + C was found at 1 month pre-intervention . This difference was not observed at 1 month post-intervention  . The median time to half randomisation target was non-significantly lower in the additional communication group compared to the usual group , which suggests that an additional communication strategy may be of some, albeit non-statistically significant, incremental benefit in helping sites achieve recruitment targets sooner. In our SWAT-1, the additional communication strategy involved a personalised site visit, suggesting perhaps that face-to-face additional communication might confer more benefit in assisting boost recruitment rates in a trial compared to non-face-to-face strategies.In this paper, we provide a SWAT-1 evaluation to investigate the effects of a site visit combined with a scheduled meeting, with the sole purpose of discussing trial recruitment, on recruitment rates in a large multi-centre trial. The results provide evidence of benefits from such a site visit for increasing recruitment rates and adherence to trial procedure at 1 and at 3 months after the visit. Other studies have also suggested some benefit for additional communication strategies ,11. For There are some limitations associated with this implementation of SWAT-1 that need to be acknowledged when considering our results. Firstly, although recruitment rates had been less than expected at all three sites, month-end recruitment at the intervention site had fallen considerably and consistently in the months before the site visit and meeting. Therefore, this site was purposively chosen for the intervention, rather than randomly assigned. This lack of randomisation introduces the potential for bias. Secondly, there is a risk that other confounding factors might have influenced the results. For example, regular weekly visits by a member of the research team were ongoing for all three sites throughout the trial, including the intervention site, Site A. This makes it difficult to disentangle the effect that these routine activities might have had on the findings of our study. For example, one might question as to whether the targeted site visit alone caused the increase in recruitment at 1 and 3 months post-intervention at Site A or whether the targeted site visit increased the impact of the routine visits, such that this combination caused the effect. Thirdly, adherence to trial procedure was evaluated based on information available from completed trial screening and register forms but the accuracy of these findings is dependent on the completeness of reporting of these forms. We cannot be certain that forms were completed on all potentially eligible women; that is, those who did not receive the study information and those who did receive it but did not agree to join the study. Therefore, the results of this analysis should be treated with caution. Furthermore, just prior to implementing the intervention, Site A had taken a downturn in recruitment with a subsequent upturn, while Site B and Site C had taken an upturn with a subsequent downturn (Figure Recognising these limitations, our study does provide some evidence for the effectiveness of a site visit combined with a scheduled meeting on recruitment rates and adherence to trial procedure in a multi-centre randomised trial. More importantly, perhaps, our study demonstrates the potential value of using SWAT-1 to evaluate, pragmatically, what works when attempting to tackle the problem of poor recruitment to a trial. Based on our results, other researchers might also be encouraged to implement SWAT-1, or their own version of it, if they are facing similar problems of slow or poor recruitment in their trials. As the findings from further SWAT-1 s become available, the results could be combined in cumulative meta-analyses , which wThis paper provides the first reported example of a study in the SWAT programme, using the SWAT-1 design. It provides some evidence that a targeted site visit combined with a purposeful meeting increases recruitment rates and adherence to trial procedure in a clinical trial. We hope that this example of SWAT-1 might encourage other researchers to consider conducting a similar study to improve recruitment in their trials, allowing the findings of future SWAT-1 s to be compared, contrasted and combined so that higher level evidence on the effect of a site visit by the lead researcher or principal investigator can be obtained.


Western blot analysis revealed that while DRB or DMAT did not influence cytokine-induced IκBα degradation, they inhibited NFκB-dependent IκBα resynthesis, demonstrating that cytokine-induced NFκB activity is dependent on CK2. Both DRB and DMAT inhibited the constitutive phosphorylation of NFκB p65 at serine 529, while leaving cytokine-induced phosphorylations of NFκB p65 at serines 276 and 536 unaltered. In comparison, putative phosphorylation sites for CK2 on HDACs 1, 2, and 3 at serines 421/423, 394, and 424, respectively, which may stimulate NFκB transcriptional activity, were unchanged by cytokines and CK2 inhibitors. Whereas IL-1β and TNF-α stimulate IκBα degradation and NFκB activation, IFN-γ potentiates cytokine-induced β cell death through activation of STAT1. DRB and DMAT inhibited IFN-γ-stimulated phosphorylation of STAT1 at serine 727, while leaving IFN-γ-induced phosphorylation of STAT1 at tyrosine 701 unaffected. Inhibition of cytokine-induced β cell death by CK2 inhibitors was, however, not dependent on IFN-γ, and IFN-γ did not affect CK2-dependent IκBα turnover. In conclusion, it is suggested that cytokine-induced activation of NFκB in β cells is dependent on CK2 activity, which phosphorylates NFκB p65 at serine 529.This study aimed to assess the role of constitutive protein kinase CK2 in cytokine-induced activation of NFκB in pancreatic β cell death. The CK2 inhibitors DRB (5,6-dichloro-1-β- Diabetes mellitus is characterized by a decline in β cell mass and function leading to insufficient insulin secretion and hyperglycemia . In typeCK2 (formerly casein kinase 2) is a highly conserved serine/threonine kinase , which aNFκB is activated by a range of stimuli, including inflammatory cytokines, like IL-1β and TNF-α, and is generally regarded as one of the most important master regulators of the inflammatory response . NFκB isCK2 has been found to phosphorylate IκBα at a number of sites, including serines 283, 289, 293, and threonine 291, and this has been found, at least in certain context, to trigger the degradation of the protein and thereby activation of NFκB . NFκB p6In addition, NFκB activity in β cells is dependent on histone deacetylases (HDACs), which through deacetylation of histones promote NFκB binding to DNA and gene expression , 16. HDAIn most tissues, NFκB activation inhibits cell death. However, under certain circumstances, or in certain cell types as in β cells, activation of NFκB may promote cell death . IL-1β minsulitis in type 1 diabetes mellitus is well-established, its role in type 2 diabetes mellitus is disputed  and other test substances as specified in the text were present during the last 48 h of culture.Islets were prepared by collagenase digestion of the pancreas of male albino mice (NMRI) (approx. 18–22 g body weight)  fed ad libitum on a standard laboratory diet. Principles of laboratory animal care as approved by The Danish Animal Experiments Inspectorate were followed. Islets were kept in tissue culture for 48–72 h in a 5 % CO5 cells/well) or 3 cm dishes (1.25 × 106 cells/dish) in a 5 % CO2 incubator in RPMI 1640 medium (11 mM glucose) supplemented with glutamax, 10 % (v/v) fetal calf serum , 100 units penicillin/ml, 100 μg streptomycin/ml, and 50 μM mercaptoethanol. After 48 h of culture, the serum concentration was lowered to 0.5 % fetal calf serum for the remaining culture period with addition of cytokines  and other test substances as specified in the text. This low concentration of serum did not affect cell viability  assay, which measures succinate dehydrogenase activity . BrieflyPLUS kit . Briefly, groups of 25 islets were lysed in 200 μl lysis buffer  followed by measurement of histone-associated DNA fragments as outlined by the manufacturer.The proportion of apoptotic cells in treated versus control islets was determined by the Cell Death Detection ELISAg for 3 min. Protein concentration was then determined in supernatants using the BCA protein assay . 50 μg of protein was loaded and separated by electrophoresis on 7 % NuPage Novex Tris-Acetate gels in Tris-Acetate running buffer or 12 % NuPage Novex Bis–Tris gels in Mops running buffer as appropriate  and transferred to nitrocellulose membranes by electroblotting. After incubation of the membrane in blocking buffer , the membrane was exposed overnight at 4 °C to primary antibody diluted 1:1,000 in blocking buffer. The membrane was subsequently washed in Tris-buffered saline containing 0.1 % Tween 20, incubated with peroxidase-conjugated goat antirabbit IgG , and proteins detected by chemiluminescence using ECL plus Western blotting detection reagent . Proteins were then quantified relative to controls on the same gel with ImageJ software as described  ATP  as phosphate donor and 200 μM RRRDDDSDDD as phosphate acceptor as outlined by the manufacturer .After culture, INS-1E cells or mouse islets were washed in PBS, lysed in 100 μl lysis buffer benzene sulfonyl fluoride, 1 mM orthovanadate, 2 μM okadaic acid, 10 mM β-glycerophosphate, 10 mM NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 5 mM EGTA in 20 mM Mops, pH 7.2), and centrifuged at 11,000×3, 2 mg/ml of bovine serum albumin/ml, and 3.3 mM glucose. Islets were initially perifused for 45 min to obtain a basal release rate and then stimulated with 16.7 mM glucose for 60 min. The effluent medium was collected for periods of 5 or 10 min. Insulin was determined by radioimmunoassay [Insulin release from islets was determined by perifusion in a noncirculating system with beads of 0.25 ml Bio-Gel P2  as a supporting medium. 25 islets per chamber were perifused at 37 °C at a flow rate of 0.26 ml/min. The perifusion medium was Krebs–Ringer medium supplemented with 20 mM HEPES, 5 mM NaHCOunoassay .Groups of ten islets were collected in 1 ml of acidified ethanol  and incubated for approx. 20 h at 4 °C for extraction of insulin .n experiments. Statistical evaluation of the data was made by t test or ANOVA, followed by the Newman–Keuls test for multiple comparisons; not significant, P > 0.05.DRB, DMAT, and andrographolide were added in a small volume of DMSO, final conc. 0.01–0.1 %. Results are given as mean ± SEM for To evaluate the role of CK2 in β cell survival, INS-1E cells were exposed to increasing concentrations of the CK2 inhibitor DRB  and DMAT (5 μM), which per se did not affect the survival of mouse islets, improved survival of mouse islets during a 48 h exposure to cytokines Fig. c, d. In 2O2- and palmitate-induced demise of INS-1E β cells  failed to affect both Hlls Fig. , suggestDRB (50 μM) and DMAT (5 μM) caused an approximately 65 % inhibition of CK2 activity, measured as phosphorylation of the CK2 substrate RRRDDDSDDD, in cell extracts of both INS-1E cells and mouse islets  and DMAT (5 μM) on IκBα resynthesis were evaluated. As demonstrated in Fig. 0 μM and In comparison, NFκB p65 expression as determined after 30 min and 2 h was not affected by CK2 inhibitors and cytokines  Fig. c and DMAWhile NFκB phosphorylation may be important, NFκB transcriptional activity is also dependent on HDACs, which stimulate histone binding and NFκB transcriptional activity . HDAC1, Inhibition of CK2 has been described to attenuate the IFN-γ-regulated expression of several key genes in other tissues, and this is mediated, at least in part, via modulation of STAT1 phosphorylation on serine 727, necessary for maximal activity .In accordance DRB (50 μM) and DMAT (5 μM) inhibited cytokine-induced phosphorylation of STAT1 at serine 727, suggesting a potential significance of STAT1 in CK2-dependent β-cell death Fig. .Fig. 9EWhile IFN-γ stimulated phosphorylation of STAT1 at both tyrosine 701 and serine 727 Fig. , IFN-γ, P < 0.001) and to 69 ± 8 (3) % (P < 0.01) of controls after culture with DRB (50 μM) and DMAT (5 μM), respectively.Since glucose-induced insulin secretion has been described to require NFκB activity , 30, theP < 0.001) of controls without affecting islet insulin content , which per se did not affect the viability of mouse islets  or DMAT (5 μM) per se did not affect islet insulin content, but caused an inhibition of glucose-induced insulin secretion. The reason for this discrepancy from MIN6 β cells , 41 is nWhile IL-1β and TNF-α induce NFκB activity, IFN-γ may potentiate NFκB nuclear translocation and activity through activation of STAT1 , 21. ThuAccording to the present experiments, IFN-γ stimulated STAT1 phosphorylation at both tyrosine 701 and serine 727 and potentiated IL-1β plus TNF-α—induced β cell death. Indeed phosphorylation of serine 727 appeared to be dependent on CK2 activity. The significance of this phosphorylation of serine 727 for STAT1 activity is, however, at present not known, and although IFN-γ potentiated cytokine-induced cell death, STAT1 was not the primary target for CK2 in cytokine-induced β cell death.In conclusion, the present study has demonstrated that CK2 may have an indispensable function in cytokine-induced β cell death, which primarily may be exerted by NFκB activation and phosphorylation of NFκB p65 at serine 529.


In the highly competitive market, it is increasingly hard for ice cream stores to develop creative marketing strategies to retain existing customers and attract new ones. This study applies the means–end chain approach to identify the customer value, consequences, and attributes of ice cream and to suggest useful information for ice cream sellers to develop differential marketing strategies across various consumption situations . This study conducted one-on-one in-depth interviews with participants. The interview content was subsequently analyzed and coded to produce an implication matrix and a hierarchical value map, which was further used to determine customers’ value perceptions. The results indicate the terminal values of the highest strength comprised economy, pleasure, and efficiency. Pleasure was emphasized among consumers who were on a date or gathering with friends, whereas satisfaction was emphasized among consumers who craved ice cream. Based on the results, the study also provides suggestions to the industry and future researchers. According to Customs Administration, Ministry of Finance of Taiwan, the volume of ice cream imports has increased 56 % over the last decade, from 3399 tons in 2005 to 5316 tons in 2014. This lucrative business opportunity has intensified the competition in the ice cream market, motivating all ice cream stores to create uniqueness in their products to differentiate their market or use marketing strategies to attract new customers. For instance, Cold Stone allows customers to create their ice cream mix using a wide variety of cookies, nuts, Brownie cakes, and other ingredients with as many as 12000 combinations. Haagen-Dazs offers seasonal products and festival-themed products, such as ice cream hot pot and ice cream moon cake. Bigtom has its marketing focus placed on the low-fat and healthy nature of its ice cream. Movenpick claims to be dedicated to 100 % customer satisfaction by providing 100 % natural ice cream.Traditional marketing focuses more on product functionality, because it assumes customers to be rational decision makers who care about functional features and benefits Holbrook . With thProviding an emotional experience for consumers has become a way to create a competitive advantage. To deepen customers’ impressions, many businesses integrate story elements into their advertisements. For instance, a Taiwan High Speed Rail advertisement featuring a father buying fish in the market depicts parents’ longing to see their children; the Barley Black Tea—Youth Book edition is another story-based advertisement reminding consumers of the feeling of sharing joy with friends. By telling a story, these advertisements are intended to involve customers in a context where they can interact with the advertised product. Using consumption situational factors, businesses can create a successful experiential service for customers. This means consumption situational factors are positively related to consumers’ behavioral intention. A good consumption situation can stimulate impulse buying and increase customer loyalty, which in turn can lead to higher business performance  to explore the hierarchical relationship of value perceptions, consequences of consumption, and product attributes based on customer experiences of buying ice cream and further compare customer value between different consumption situations. The results can provide an insight into the relations between customer value hierarchies and help ice cream stores understand consumers’ actual needs and ultimate goals to offer services of higher customer value.The means-end chain approach was introduced by Gutman . This apAttributesAttributes are the characteristics of a product or service. Generally, they can be classified into concrete attributes and abstract attributes. Concrete attributes are the tangible and objective aspects of a product that can be directly measured, experienced by touch or perceived by eye. Color, size, and price are attributes of this kind. Abstract attributes are the intangible and subjective aspects of a product, such as services and reputation, which are determined subjectively or by individual feeling. Kotler and Armstrong  mentioneConsequencesAt the center of the hierarchical structure, consequences connect products attributes to personal values. They are the direct or indirect results of consumption at the physical or mental level. If the results of consumption are positive, the results are called benefits; if the results of consumption are negative, the results are called risks. Levitt  argues wValuesIn the means-end chain theory, values are defined as the desired end state Gutman . Values Olson and Reynolds  proposedHansen  categoriBelk  proposesThese situational factors affect not only consumers’ choice but also their product loyalty. The purpose of this study is to explore the consumption motivations and actual needs of consumers of ice cream specialty stores. Since customers’ motivations to buy vary across social situations, we will view different consumption situations as different social situations, including (1) on a date, (2) gathering with friends, and (3) simply craving for ice cream Liu .Data were collected by one-on-one interviews and the laddering method was used. Consumers may not be aware of the chain relationship between attributes, consequences, and values when making a purchase. In other words, consumers may not necessarily plan an approach to or the goal of buying beforehand. The laddering method which requires the interviewer to continuously ask further questions in response to the participant’s answer can better elicit the attributes, benefits, and values perceived by the participant.In qualitative research, sample size is usually not as important as the appropriateness and coverage of the sample . This matrix has three components. The first is the number in each column. Each number consists of an integer and a decimal. The integer denotes the number of direct links, while the decimal denotes the number of indirect links. The second component shows the in-degree and out-degree of each factor. In-degree indicates the number of times a factor is mentioned as resulting from another factor, while out-degree indicates the number of times a factor is mentioned as the source of or an antecedent to another element , and most participants were in the age group of 21–30 (52 %) and single (68 %). Besides, most participants had a college or university degree (70 %). In terms of occupation, students were the largest group (52 %), followed by the service industry (22 %). As to disposable income, most participants had no more than 50,000 (28 %); the group having 5001–10,000 and the group having 10,001–20,000 comprised 20 % respectively. Participants who purchased ice cream 2–3 times a month were the largest group (48 %). Most had average spending between NT $101–200 (65 %). Among the numerous ice cream brands, Cold Stone was most frequently visited by the participants (65 %), followed by Haagen-Dazs (13 %). Taste was the major appeal to most participants (63 %). To sum up, this sample comprised mainly of female consumers, consumers visiting ice cream stores 2–3 times a month, and consumers spending $101–200 in each visit.The interview results were transcribed and semantically analyzed to produce a factor implications table. Three coders participated in this study. They categorized words and phrases in the transcription independently. Through discussion, they deleted inappropriate words and phrases, gave a name to each factor, and divided the named factors into attribute, consequence, and value tiers. For the consequence tier, the research has adopted the concept of functional consequences and psychosocial consequences proposed by Olson and Reynolds . For theAs mentioned earlier, we measured reliability by interjudge reliability. The first judge agreed with the implications of 39 factors, the second agreed with the implications of 40 factors, and the third agreed with the implications of 41 factors. According to Eqs. , the intAfter content analysis, we obtained 161 attribute-consequence-value chains from the interview responses of 60 participants (each generating an average of 2.7 (=161/60) chains) and used these chains to build an implication matrix, as shown in Table Besides, the abstractness ratios in this matrix help distinguish between attributes, consequences, and values. Factors with a ratio of 0 are “attributes”, factors with a ratio between 0 and 1 are “consequences”, and factors with a ratio of 1 are “values”.We set the cutoff value at 3, meaning only factors with at least 3 direct links are included in the HVM. In the HVM, each number is obtained from the integer part of the corresponding number in the implication matrix. It denotes the number of times the direct link between two factors is mentioned. The higher the number, the more important it is to the participants. To capture the major value chain among numerous value chains, we used a thick line to represent the links in the top 50 % of links ranked by frequency (i.e. 6 or more times) and a thin line to represent the other links (i.e. fewer than 6 times). The HVM is as shown in Fig. In the HVM for the entire sample, different attributes have different value chains. The thick lines show the major stable chains. These chains include “promotion-good bargain-economy”, “taste-delicious-pleasure”, and “store location-convenient-efficiency”. From these major stable chains, we can infer economy, pleasure, and efficiency are the values sought by customers when buying ice cream. Besides, customers will also consider promotional information and convenience when buying ice cream.As shown in Figs. Therefore, consumers of ice cream pay attention to the economical value of promotions, regardless of the consumption situation. In the dating and friends-gathering situations, consumers are more concerned with pleasure. For these consumers, the feeling of dating with their valentine or being with their friends is more important, and so is the safety of the food. For consumers visiting the store simply for ice cream, the sense of satisfaction resulting from eating ice cream is more important.Consumers in the on-a-date situation expect to receive relaxing services. In contrast, consumers who visit the store simply for ice cream expect special treatment from the store. As a result, they may pay more attention to the staff’s attitude and hope to receive adequate respect from them. For consumers in the on-a-date situation, brand word-of-mouth is one of the factors affecting their consumption. They wish to recommend safe food to their date and believe they can gain a sense of assurance from a store with positive word-of-mouth.Consumers visiting the store in a friends-gathering situation pay attention to the sense of belonging. This kind of situation gives them a sense of belonging to a specific group. Besides, since they usually have a large number of friends coming to join them, store location is important. They may have reduced intention to visit stores located in a more distant area.For consumers in a friend-gathering situation or craving-for-ice-cream situation, product diversity is important. These two groups of consumers expect to have new feeling or experiences in each purchase. A store with high product diversity allows them to try different items and gain the value of freshness in each visit.In this study, we used a means-end chain approach to examine the expected values underlying in ice cream consumers in three different consumption situations. Using one-on-one interviews and laddering, we collected value perceptions from 60 ice cream consumers. From their value perceptions, we identified the important links for them and analyzed differences in the attribute-consequence-value chain between consumption situations. We discussed the customer value expected by consumers of ice cream from domestic ice cream stores and also the customer value expected by customers in different consumption situations.We summed the attributes, consequences, and values provided by all the participants into an implication matrix. This matrix shows the numbers of direct and indirect links between factors on three levels. Based on this matrix, we further drew HVMs to more clearly illustrate the relations between factors. The HVM for the entire sample was created with a cutoff of 3. To identify important value chains, we used thick lines to indicate links in the top 50 % of links ranked by frequency (i.e. having 6 or more links). From these links, we obtained three major stable chains, including “promotion-good bargain-economy”, “taste-delicious-pleasure”, and “store location-convenience-efficiency”. These stable chains suggest “economy”, “pleasure”, and “efficiency” are key values emphasized by ice cream consumers. In addition to the pleasure of eating ice cream, consumers also pay attention to the cost-benefit ratio of the product and efficiency of the service when making a decision for ice cream consumption.The analysis of customer value in different consumption situations shows there is no direct and stable chain in the HVM for consumers in the on-a-date situation. Though below the level of strong relation (cutoff = 6), the number of links between “taste-delicious-pleasure” is very close to this standard, suggesting the existence of an underlying chain relation. The HVM for consumers in the gathering-with-friends situation shows “taste-delicious-pleasure” is a major chain, suggesting the attribute of “taste” leads to the consequence of eating “delicious” food and ultimately to the value of “pleasure”. From this chain, we can infer consumers who visit an ice cream for gathering with friends pay more attention to the “taste” attribute of ice cream and whether they can have a pleasant feeling after visiting the store. In other words, this type of consumers expects to have a pleasant and joyful feeling in this consumption situation. The HVM for consumers visiting the store simply for ice cream shows “taste-delicious-satisfaction” is a major chain, suggesting the attribute of “taste” leads to the consequence of eating “delicious” food and ultimately to the value of “satisfaction”. From this chain, we can infer consumers who visit an ice cream store due to a craving for ice cream pay more attention to the “taste” attribute of ice cream and whether they can feel a sense of satisfaction after visiting the store. In other words, consumers in this situation expect to achieve mental satisfaction by satisfying their craving for ice cream.Products Taste is one of the most important attributes for customers. Although Cold Stone uses its US origin as a key appeal to customers, we suggest this company introduce new products based on local customers’ flavor preferences obtained through a survey and develop new or seasonal products on a regular basis. Generally, customers expect to have a diversity of product choices and be able to find one that not only meets their preference but also gives a new experience. Besides, this company can offer free sampling of their ice cream. This service allows customers to directly taste the food before they buy, so it can also help promote buying behavior.Price Consumers expect to get the best product at the lowest cost. Therefore, price discount is an important factor affecting customers’ buying intention and behavior. Although Cold Stone launches promotional activities from time to time, many consumers do not know about these activities until the activities have expired or until they are told through word-of-mouth. Therefore, we suggest Cold Stone reinforce their marketing through social media to allow customers to access promotional information at anytime and anywhere. Besides, it can also distribute promotional leaflets in the streets near their stores to attract nearby consumers.Activities Our findings indicate that most customers expect to gain some pleasure from eating ice cream. Cold Stone emphasizes it is an ice cream specialty store full of passion and joy, but not all customers are clear about the brand image it claims. To increase customers’ knowledge of its brand image, Cold Stone can add joyful elements to the names of its ice cream products or use celebrity appearances in its advertisements to underscore the joyful feeling its products can offer. Besides, it can also use themed parties to create a venue full of joy and imagination for customers. This marketing approach may deepen customers’ impression of surprises and happiness offered by the company.Store location Ice cream is not a daily necessity. Customers usually have low intention to travel a long way to buy ice cream. Therefore, when setting up a new store, Cold Stone can consider places in an important business district or places with a convenient parking area nearby, such as department stores, wholesale stores, parks, and so on. Stores set in these kinds of places are more easily accessible to customers. Besides, stores located in places with more people coming and going can attract more consumers who have a craving for ice cream.The “on-a-date” situation and the “gathering-with-friends” situation For customers in either situation, “taste” leads to “pleasure”. This indicates customers in these situations expect to gain the value of pleasure in the consumption process. Therefore, in addition to incorporating joyful elements into their products and advertisements, Cold Stone can provide set meals for two (or more) and interactive games to amplify customers’ feeling of sharing and pleasure in these situations.The “craving for ice cream” situation For customers in this situation, “taste” leads to “satisfaction”. This indicates customers in this situation expect to gain the value of satisfaction in the consumption process. This type of customer is more concerned with satisfaction of their taste. Therefore, while increasing the diversity of products, Cold Stone can provide a description of each product item and offer the free sampling service to help customers understand the content  and flavor  of each item. These approaches are helpful for customers to pick the right item for themselves and then gain the value of satisfaction.The results show the underlying major link for the on-a-date situation is the same as the major link for the gathering-with-friends situation. Therefore, we view customers in these two consumption situations as in the same market segment. Below are our suggestions:The limitations of this study and suggestions for future researchers are as follows: (1) include multiple ice cream brands in the survey: due to time and human resource constraints, we only selected Cold Stone stores in Taichung City as our subjects. Future researchers are advised to conduct a cross-region study of several ice cream brands. This kind of study may produce more comprehensive findings about ice cream consumers’ value perceptions. (2) Include other variables in the analysis: in addition to a general analysis of customer value among customers of ice cream stores, we also analyzed customer value in different consumption situations. However, future researchers can include variables, such as demographic variables or family life cycle, in the analysis. (3) Use a large sample: we used soft laddering in the interviews with customers to well know the researched area. Future researchers can use hard laddering with more structured interview and data collection procedure, making it possible to collect more samples in minimizing a researcher’s influence.


Drosophila inactivation no afterpotential D (INAD) is a PDZ domain-containing scaffolding protein that tethers components of the phototransduction cascade to form a supramolecular signaling complex. Here, we report the identification of eight INAD phosphorylation sites using a mass spectrometry approach. PDZ1, PDZ2, and PDZ4 each harbor one phosphorylation site, three phosphorylation sites are located in the linker region between PDZ1 and 2, one site is located between PDZ2 and PDZ3, and one site is located in the N-terminal region. Using a phosphospecific antibody, we found that INAD phosphorylated at Thr170/Ser174 was located within the rhabdomeres of the photoreceptor cells, suggesting that INAD becomes phosphorylated in this cellular compartment. INAD phosphorylation at Thr170/Ser174 depends on light, the phototransduction cascade, and on eye-Protein kinase C that is attached to INAD via one of its PDZ domains. Drosophila phototransduction cascade are tethered together by the inactivation no afterpotential D (INAD) scaffolding protein to form a supramolecular complex that is referred to as the INAD signaling complex [2 hydrolysis in the light by PLCβ [in vitro kinase assay using protein kinase C only when Thr669 was substituted by Ala providing further evidence for a possible link between phosphorylation and formation of the disulfide bond within PDZ5. The authors propose the transient receptor potential (TRP) ion channel as the ligand of INAD PDZ5. They also provide evidence that TRP additionally binds to INAD PDZ3. According to their model, TRP is released from PDZ5 in the light and is eventually released from PDZ3 as well. A fly transgenically expressing an INAD protein in which Thr669 was substituted with Ala disrupting the hydrogen bond between PDZ4 and PDZ5, displayed prolonged activation kinetics of the macroscopic light response that was caused by an increase in quantum bump latency. Additionally, this fly displayed a prolonged refractory period being in line with the proposition that PDZ5 is locked in the oxidized state and thus is uncoupled from TRP.Components of the  complex –8. INAD  complex . INAD li complex –4,7, nei complex , and ret complex ,12. The  complex ,14. Late complex . Recentl complex . The aut complex . Acidifi by PLCβ , obstruc2+ levels was sufficient to trigger Thr170/Ser174 phosphorylation.The current evidence outlined above suggests that light-dependent phosphorylation of INAD might contribute to the dynamic molecular rearrangements of this scaffolding protein. However, the INAD phosphorylation sites that might regulate the distortion of the ligand binding pocket within PDZ5 are not known. Here, we report the identification of eight INAD phosphorylation sites in a mass spectrometry approach. We show that phosphorylation of two of these sites, Thr170/Ser174, depends on light, the phototransduction cascade, and eye-PKC. Light-independent elevation of intracellular CaImmunization of rabbits was conducted at the University of Karlsruhe according to the animal protection ordinance and was approved by the regional administrative council Karlsruhe (reference number 35-9158.82/977/99). All efforts were made to ensure animal welfare and minimize suffering.Drosophila melanogaster strains and mutants were used: w Oregon R, yw;inaD1 [trpP343 [trpP365 [ninaE17 [Gqα1 [norpAP24 [inaCP209 [trpD621G,trpP343 [The following w;inaD1 , yw;;trptrpP343 , w;;trptrpP365 , yw;;ninninaE17 , w;Gqα17 [Gqα1 , w,norpAorpAP24 , w;inaCtrpP343 . Flies wFor analysis of the INAD phosphorylation state, flies were illuminated with a white 18-watt fluorescent lamp, 2400 lux, or kept in the dark, for 12–18 h before they were subjected to protein extraction for 1D or 2D gel electrophoresis and subsequent Western blot analyses or to immunoprecipitation and subsequent mass spectrometry analysis. For 1D gel electrophoresis prior to Western blot analysis, flies were manually decapitated and extracts were prepared as described previously . For 2D To generate a phosphospecific antibody against INAD phosphorylated at pThr170 and pSer174, a phosphopeptide, NH2-T(pT)FTA(pS)MRQC-CONH2, comprising pThr170 and pSer174 was synthesized, coupled to keyhole limpet haemocyanin, and injected into rabbits. The first immunization was accomplished using Freund’s complete adjuvant and for concomitant boosts, Freund’s incomplete adjuvant was used. Peptide synthesis and immunization was done by Pineda Antibody Service. Affinity purification was carried out as described previously . For immin vitro dephosphorylation, head extract (600 μg of protein) was diluted into a total volume of 210 μl of the supplied buffer supplemented with 2 mM MnCl2, a cocktail of protease inhibitors , and 2 μl of lambda phosphatase (New England Biolabs). Samples were then incubated at 30°C for 60 min. 600 μg of protein were cup-loaded onto 18 cm IPG strips with a linear pH gradient of 6–11  and proteins were subjected to isoelectric focusing in an IPGphor3 system  using the following parameters: 2 h, 150 V, step; 2 h, 300 V, step; 8 h, 1000 V, gradient; 3 h, 8000 V, gradient; 5 h, 8000 V, step; 50 V, step until processing. The current was limited to 50 μA per strip. After isoelectric focusing, the IPG strips were equilibrated in equilibration buffer  glycerol, 2% SDS) containing 1% dithiothreitol and then in equilibration buffer containing 2.5% iodoacetamide 15 min each. The strips were applied to a 26 cm × 20 cm 12% SDS polyacrylamide gel and overlaid with 0.5% agarose containing 0.01% (w/v) bromphenol blue. A small piece of filter paper was soaked with Prestained protein marker (Bio-Rad) and was placed beside the strip. Gel electrophoresis was performed at an initial current of 10 mA per gel for 1 h and then at 12 mA per gel over night at 20°C. Gels were then subjected to Western blot analysis or were stained with colloidal Coomassie brilliant blue to recover protein for mass spectrometry.Approximately 300 fly heads were homogenized in 200 μl 2D lysis buffer  using a micropestle. Homogenates were extracted on ice for 30 min and were then centrifuged for 10 min at 12,000 × g at 4°C. The resulting supernatant was used for 2D gel electrophoresis. Protein concentrations were determined using the Rotiquant Bradford assay (Roth). For Drosophila head extracts was immunoprecipitated using 3 μg of α-INAD or α-TRP antibodies.Immunoprecipitations of INAD and TRP were performed as described previously . The INAPrior to nano-LC-ESI-MS analysis, INAD was in-gel digested with trypsin. Mass spectrometry was performed on a Thermo LTQ-Orbitrap instrument (Thermo) as described before , with moProtein transfer onto PVDF membranes (Bio-Rad) was carried out as described previously . PrimaryImmunocytochemistry was carried out as described previously , with monorpAP24 mutant flies lacking the central effector enzyme of the phototransduction cascade, phospholipase Cβ are similar , and eye-PKC is a member of the INAD signaling complex [Drosophila TRP protein , only spots in the more acidic range were detected whereas no spots were detected in extracts from dark-adapted wild type flies. In the inaCP209 mutant, no spots were detected using the α-pThr170/pSer174 antibody regardless of the light condition. The fact that more than two spots were detected with the α-pThr170/pSer174 antibody in extracts from light-adapted flies points to the existence of different INAD isoforms that are phosphorylated at Thr170/Ser174 and at several other sites.A likely candidate for the kinase phosphorylating INAD at Thr170/Ser174 is eye-PKC because eye-PKC is a Cain vitro ,30. Addipe flies . Thus, eantibody . Using t2+ concentrations by usage of different trp mutants resulted in altered phosphorylation levels of Thr170/Ser174. Elevated Ca2+ levels led to strong phosphorylation whereas lowered Ca2+ levels resulted in weak phosphorylation of T170/S174. We propose that INAD phosphorylation at Thr170/Ser174 is ultimately regulated by intracellular Ca2+ levels. Upon phosphatase treatment of fly head protein extracts, INAD spots on 2D blots became even more condensed than in dark-adapted untreated head extracts. This points to the existence of sites that are phosphorylated in the dark. Indeed, phosphorylated Ser40 was identified from dark-adapted flies in our mass spectrometry approach. However, we cannot rule out that there is also Ser40 phosphorylation or even enhanced Ser40 phosphorylation in the light. Generally, we were not able to detect INAD phosphorylation sites robustly enough for quantification by mass spectrometry.In this study, we report the identification of eight INAD phosphorylation sites by a nano-HPLC-MS approach. INAD phosphorylation was further investigated by 1D or 2D gel electrophoresis and concomitant immunoblotting using a pan-specific α-INAD antibody detecting INAD regardless of its phosphorylation state and a phosphospecific antibody that detected phosphorylation of INAD at T170/S174. Comparison of the intensity of spots on the 2D immunoblots suggests that a considerable amount of INAD becomes light-dependently phosphorylated supporting a physiological relevance of INAD phosphorylation. After light adaptation, about half of the INAD spots on 2D gels are detected by an α-T170/S174 antibody, suggesting that a significant number of the INAD molecules are phosphorylated at one of these two sites in the light. We found that the light-dependent isoelectric point shift observed in 2D blots depended on PLCβ indicating an involvement of the phototransduction cascade in INAD phosphorylation. Indeed, impairments in rhodopsin, Gqα, and PLCβ resulted in compromised INAD phosphorylation at Thr170/Ser174. Manipulation of intracellular Ca2+ levels and by DAG. Indeed, in the eye-PKC null mutant, inaCP209, phosphorylation of INAD at Thr170/Ser174 was drastically reduced.Thr170 and Ser174 are predicted PKC phosphorylation sites and PKC is activated by rising intracellular Cain vitro studies showing that INAD can be phosphorylated in purified INAD signaling complexes containing eye-PKC [This finding is in line with  eye-PKC ,30. The  eye-PKC .in vivo phosphorylation site that we detected in the PDZ4/PDZ5 region was T544. However, the available data do not allow us to estimate the phosphate occupancy of this site in light- and dark-adapted flies. It remains to be determined whether or not phosphorylation of T544 affects INAD structure and is physiologically relevant. Additionally, Ser163, Thr170, and Ser174 are located in the linker region connecting PDZ1 and PDZ2 that harbors a putative calmodulin binding site [in vivo may be modulated by phosphorylation of Ser163, Thr170, and Ser174. In this study we demonstrate that INAD is not phosphorylated at Thr170 and Ser174 in the dark while a significant amount of INAD molecules, represented by distinct spots on 2D gels, becomes phosphorylated at these sites in the light.Putative physiological effects of INAD phosphorylation might at least in part be conveyed by the regulation of the formation of the disulfide bond within PDZ5. As was shown, formation of the disulfide bond depends on eyePKC . Since ding site . More sping site . BindingS1 FigFigs. A to G show fragmentation spectra and tables derived from tryptic INAD peptides. The coverage of the peptide sequence by b and y ions and the calculated mass for each fragment ion are shown in the tables below the spectra, in which observed b and y ions are highlighted in red and blue, respectively.(DOCX)Click here for additional data file.S2 Fig2-CGRKK(pT)QKGD-CONH2 containing phosphorylated Thr849 of TRP, NH2-CARKN(pT)FASD-CONH2 containing phosphorylated Thr864 of TRP, NH2-CADEVpSLADD-CONH2 containing phosphorylated Ser936 of TRP, and NH2-T(pT)FTA(pS)MRQC-CONH2 containing phosphorylated Thr170 and Ser174 of INAD. The dephosphopeptides were similar to the respective phosphopeptides except for the lack of the phosphoryl groups. B and C, To check linearity of the signal intensities obtained with the antibodies, different amounts of protein extracts from wild type heads were supplemented with protein extracts from inaD1 null mutant heads to ensure equal overall protein content. Three head equivalents were loaded onto a gel and subjected to Western blot analysis using α-INAD and α-pThr170/pSer174 antibodies. B shows representative Western blots and C shows results from three independent experiments. Error bars show SEM.A, 2.5 μg of phospho- and dephosphopeptides were spotted onto a nitrocellulose membrane and the membrane was then blocked and incubated with the α-pThr170/pSer174 antibody and a secondary α-rabbit IgG conjugated to horse radish peroxidase. Enhanced chemiluminescence signals were recorded. Phosphopeptides were NH(DOCX)Click here for additional data file.


Nitric oxide is involved in many physiologic processes. There are efforts, described elsewhere in this volume, to deliver nitric oxide to tissues as a therapy. Nitric oxide also contributes to pathophysiologic processes. Inhibiting nitric oxide or its production can thus also be of therapeutic benefit. This article addresses such inhibitory strategies. Nitric oxide is an important biological mediator of functional and metabolic processes in nearly every organ system. This gaseous free radical moderates endothelial function, neurotransmission, immunity and cell death by activating guanylate cyclase (GC), which increases intracellular cGMP levels and cGMP-dependent protein kinase . Elevatil-arginine analogs, which fail to reduce mortality in septic shock  oxadiazolo-quinoxalin-1-one (ODQ) is a selective sGC inhibitor in rats, shown to restore basal ganglia function and improve motor symptoms in Parkinson's disease .All hemoglobins are potential scavengers of NO. The chemically modified human-derived, hemoglobin conjugate, pyridoxalated hemoglobin polyoxyethylene such a scavenger. It has the potential to increase systemic blood pressure while reducing vasopressor and ventilation needs in shock. A Phase III clinical trial on its use in septic shock with systemic inflammatory response syndrome is pending publication .Several herbal agents have demonstrated possible use as nitric oxide inhibitors. Notably, (-)-epigallocatechin-3-gallate (EGCG) inhibits migration of mammary cancer cell, while grape seed proanthocyanidins inhibits migration of nonsmall cell lung cancer cell, by inhibiting NOS and GC .Arginase has emerged as another regulator of nitric oxide homeostasis. Arginase is an enzyme in the urea cycle that hydrolyzes L-arginine to urea and L-ornithine. It suppresses nitric oxide production through numerous mechanisms. It may contribute to endothelial dysfunction in hypertension, aging, ischemia-reperfusion, psoriasis, erectile dysfunction, arthritis, diabetes and pathological wound healing ,39.DDAH inhibition may provide an alternative means to inhibit nitric oxide . HomocysNitric oxide also activates potassium, big potassium (BK) and calcium channels, most of which can be blocked pharmacologically by specific agents. It might be predicted that signaling molecules and enzyme cofactors in nitric oxide synthesis may serve as promising targets. For instance, nitric oxide activity is regulated by tetrahydrobiopterin and calmodulin. The synthesis of nitric oxide is calcium-dependent, therefore calcium mobilizing agonists could provide therapeutic targets in the future.Nitric oxide is involved in a wide spectrum of human physiology and pathophysiology. Pharmacological agents to inhibit nitric oxide may modify or arrest the course of some of these NO-mediated pathologies. There are numerous limitations to our current knowledge on nitric oxide inhibition. We must keep in mind that most of the pharmacotherapy studies on nitric oxide inhibition were performed in animals or on small groups of patients, with a short course of treatment. The use of nitric oxide inhibitors in patients with poly-pharmacy remains a challenge, as there is inadequate understanding of interactions between nitric oxide inhibitors and other drugs. Known interactions have already been reported with statins, fibrates, thiazolidinediones, metformin, antioxidant vitamins, aspirin, n-3 polyunsaturated fatty acids and plant flavonoids. Nitric oxide provokes many cellular responses and modulates physiological functions differently depending on the organ system. Systemic nitric oxide inhibition may be limited by the widespread involvement of nitric oxide in most body systems. To further complicate matters, depending on the disease studied, changes in nitric oxide may either ameliorate or exacerbate the pathophysiology of the disease. This proves to be a particular challenge in patients with co-morbidities. Finally, there is a paucity of studies that survey the chronic use of NOS inhibitors. One animal study shows that chronic NOS inhibition may also produce long-term biological effects by enhancing early atherogenesis in animals . The potElevation of nitric oxide has been implicated in many pathophysiological processes.Nitric oxide inhibitors are promising agents for treating various cardiovascular, dermatological, neurological and oncological conditions.At present, potential pharmacological inhibition of nitric oxide is achieved via inhibition of nitric oxide synthase, inhibition of downstream mediators and nitric oxide inhibition/scavenging.Nitric oxide inhibition should be performed with caution in patients with renal or cardiovascular disorders.New approaches to inhibit nitric oxide are being studied.Our understanding of nitric oxide inhibition would benefit from large scale, long-term animal and human studies.


Interestingly, mitotic phosphorylation inactivates TAZ oncogenic activity, as the non-phosphorylatable mutant  possesses higher activity in epithelial-mesenchymal transition, anchorage-independent growth, cell migration, and invasion when compared to the TAZ-S89A mutant. Accordingly, TAZ-5A has higher transcriptional activity compared to the TAZ-S89A mutant. Finally, we show that TAZ-S89A or TAZ-5A (to a greater extent) was sufficient to induce spindle and centrosome defects, and chromosome misalignment/missegregation in immortalized epithelial cells. Together, our results reveal a previously unrecognized connection between TAZ oncogenicity and mitotic phospho-regulation.The transcriptional co-activator with PDZ-binding motif (TAZ) is a downstream effector of the Hippo tumor suppressor pathway, which plays important roles in cancer and stem cell biology. Hippo signaling inactivates TAZ through phosphorylation (mainly at S89). In the current study, we define a new layer of regulation of TAZ activity that is critical for its oncogenic function. We found that TAZ is phosphorylated  TAZ  is a transcriptional co-activator that is involved in human cancer and stem cell function . TAZ proDrosophila [TAZ activity/function is regulated largely through the Hippo tumor suppressor pathway, which was originally discovered in osophila  and is hosophila –24. The osophila , 26. Manosophila , 28. TAZosophila . Furtherosophila  or throuosophila . BesidesWe recently showed that some members of the Hippo pathway are phosphorylated by mitotic kinases Aurora and CDK1 during mitosis , 33. We We and others showed that TAZ protein is upshifted on SDS-polyacrylamide gels during mitotic arrest induced by Taxol or nocodazole (both agents arrest cells in G2/M by binding to microtubules) , 35. As Since TAZ is a paralog of YAP and mitotic phosphorylation of YAP is mediated by the mitotic kinase CDK1 , we testin vitro with His-tagged TAZ as the substrate. Figure in vitro  and transforms MCF10A cells , 25, 40,MCF10A cells expressing TAZ-S89A/4A formed colonies in soft agar; however, all our other transfectants failed to produce any obvious colonies when 5,000 cells were seeded Figure . Again, Several studies showed that TAZ/TAZ-S89A also promotes cell migration, invasion and metastasis in animal , 20, 41.TAZ is a transcriptional co-activator, and functions mainly through the TEAD1–4 transcription factors in the Hippo pathway , 42, 43.We next examined whether TAZ or its phosphorylation mutants are able to trigger mitotic defects. MCF10A cells stably expressing vector, TAZ-S89A, or TAZ-5A (TAZ-S89A/4A) were used for this purpose. Consistent with our recent studies, immunofluoresence staining with α-tubulin and γ-tubulin antibodies showed normal microtubule/spindle formation and centrosome number during mitosis in most control cells Figure . In contAlthough recent studies have demonstrated important roles for TAZ in promoting tumorigenesis, the underlying mechanisms are largely unclear. The current study identified novel phosphorylation of TAZ during mitosis and importantly, the mitotic phosphorylation regulates TAZ's oncogenic activity Figures , 5. InteOur data not only reveal a new layer of regulation for TAZ's oncogenic activity, but also highlight a previously unrecognized mechanism through which TAZ exerts its oncogenic function. Intriguingly, recent studies have shown that most of the Hippo core tumor suppressor proteins, such as Mst1/2, Lats1/2, WW45, Mob1 are involved in regulating mitosis . FurtherUpon treatment with anti-mitotic agents (including Taxol), YAP , 35 and We recently found that YAP  is required for the spindle checkpoint activation induced by Taxol . YAP regAdditionally, studies from Yang's group showed that there is also a significant difference between TAZ and YAP phosphorylation induced by Taxol treatment. For example, TAZ is degraded after phosphorylation while YAP protein stability is not affected during Taxol treatment , 36. We HA-TAZ was a gift from Kun-Liang Guan (Addgene plasmid #32839) . To makeHEK293T, HeLa, and MCF10A cell lines were purchased from American Type Culture Collection (ATCC). The cell lines were authenticated at ATCC and were used at low (<20) passages. MCF-10A cells were cultured as described , 58. TheLuciferase reporter assays were performed in 24-well plates, using HEK293T cells. 8XGTIIC-Luciferase , SV40-RTo make His-tagged human TAZ, a full-length TAZ cDNA was subcloned into the pET-21c vector . The His-tagged proteins were expressed in E. coli and purified on HisPur™ Cobalt spin columns (Pierce) following the manufacturer's instructions.32P-ATP . The samples were resolved by SDS-PAGE, transferred onto PVDF (Millipore) and visualized by autoradiography followed by Western blotting or detected by phospho-specific antibodies.About 1 μg of His-TAZ was incubated with 100 ng recombinant CDK1/cyclin B complex  or HeLa cell total lysates in kinase buffer  in the pThe TAZ (V386) antibody from Cell Signaling Technology was used for Western blotting throughout the study. Rabbit polyclonal phospho-specific antibodies against TAZ S90, S105, T326, and T346 were generated and purified by AbMart. Anti-β-actin, anti-GFP, and anti-cyclin B antibodies were from Santa Cruz Biotechnology. Anti-glutathione S-transferase (GST) and anti-His antibodies were from Bethyl Laboratories. Anti-phospho-S10 H3 and anti-vimentin antibodies were from Cell Signaling Technology. Anti-E-cadherin antibody was from BD Biosciences. Anti-β-tubulin (Sigma), anti-α-tubulin (Abcam), and anti-γ-tubulin (Biolegend) antibodies were used for immunofluorescence staining.Immunoprecipitation, Western blotting, and lambda phosphatase treatment were done as previously described . MetabolCell fixation, permeabilization, fluorescence staining, and microscopy were done as previously described . For pepIn vitro analysis of invasion and migration was assessed using the BioCoat invasion system (BD Biosciences) and Transwell system (Corning), respectively, according to the manufacturer's instructions and as we previously described [Colony formation assays in soft agar were performed as described . In vitrescribed , 61.t-test.Statistical significance was performed using a two-tailed, unpaired Student's


The Hippo pathway plays a central role in tissue homoeostasis, and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include a kinase cascade of MST1/2 and LATS1/2 and the transcription co-activators YAP/TAZ. In response to stimulation, LATS1/2 phosphorylate and inhibit YAP/TAZ, the main effectors of the Hippo pathway. Accumulating evidence suggests that MST1/2 are not required for the regulation of YAP/TAZ. Here we show that deletion of LATS1/2 but not MST1/2 abolishes YAP/TAZ phosphorylation. We have identified MAP4K family members—Drosophila Happyhour homologues MAP4K1/2/3 and Misshapen homologues MAP4K4/6/7—as direct LATS1/2-activating kinases. Combined deletion of MAP4Ks and MST1/2, but neither alone, suppresses phosphorylation of LATS1/2 and YAP/TAZ in response to a wide range of signals. Our results demonstrate that MAP4Ks act in parallel to and are partially redundant with MST1/2 in the regulation of LATS1/2 and YAP/TAZ, and establish MAP4Ks as components of the expanded Hippo pathway. A variety of signals have been reported to either activate or inhibit the Hippo kinase cascade. Here, Meng et al. show that mitogen activated protein kinase kinase kinase kinase (MAP4K) family members function in parallel to and are partially redundant with MST1/2 in regulating LATS in response to upstream signals. Drosophila to mammals, has been recognized as a master regulator of cell fate, tissue homoeostasis and organ size12346Drosophila: Ste20-like kinase Hippo (Hpo) with its adaptor protein Salvador (Sav), the NDR family kinase Warts (Wts) with its adaptor Mats, and the transcriptional effector Yokie (Yki). The Hpo–Sav complex phosphorylates and activates Wts–Mats, which in turn phosphorylates and inhibits Yki. Dysregulation of this Hpo/Wts kinase cascade leads to aberrant activation of Yki and uncontrolled growth in the Drosophila eye and wing8910111213Tissue homoeostasis is maintained through the precise regulation of cell proliferation, apoptosis and differentiation; dysregulation of any of these processes can result in aberrant tissue growth and carcinogenesis. The Hippo pathway, which is conserved from The core components of the Hippo pathway in mammals consist of Mammalian Ste20-like kinases 1/2  and their adaptor protein Sav family WW domain-containing protein 1 , Large tumour suppressor 1/2  and their adaptor proteins MOB1A/1B (homologues of Mats), and the two Yki homologues Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). The physiological importance of the Hippo pathway in mammals has been revealed in many different genetically engineered mouse models. For example, deleting MST1/2 or overexpressing YAP in the liver leads to hepatomegaly and hepatocellular carcinogenesis in mice1215A variety of signals have been reported to either activate or inhibit the Hippo kinase cascade. In epithelial cells, apical–basal polarity regulates activities of Wts/LATS through interactions between the upstream components and intercellular junction-associated proteins19202119242526in vitro kinase assay with LATS1 as the substrate and identified several mitogen-activated protein kinase kinase kinase kinase (MAP4K) family members, including Hematopoietic progenitor kinase 1 (HPK1/MAP4K1), Germinal centre kinase (GCK/MAP4K2), Germinal centre kinase-like kinase (GLK/MAP4K3), HPK/GCK-like kinase (HGK/MAP4K4), Misshapen-like kinase 1 (MINK1/MAP4K6) and TRAF2 and NCK interacting kinase (TNIK/MAP4K7), as potent LATS1/2-activating kinases. We further demonstrate that MAP4Ks are physiological regulators of LATS1/2 and YAP/TAZ in response to a number of upstream signals. Our results indicate that MAP4Ks are components of the Hippo pathway by directly phosphorylating and activating the LATS1/2 kinases.Although the role of MST1/2 in the Hippo pathway has been firmly established, accumulating evidence indicates that MST1/2 are not essential for LATS1/2 activation under various conditions192324To test the dependency of YAP/TAZ phosphorylation on MST1/2 or LATS1/2, we generated MST1/2-dKO and LATS1/2-dKO HEK293A cells using CRISPR /Cas9 technologyWe tested the roles of LATS1/2 and MST1/2 in YAP/TAZ phosphorylation in response to a variety of signals. High cell density induced YAP phosphorylation in wild-type (WT) HEK293A cells, as shown by YAP-S127 phosphorylation and mobility shift in phos-tag gels . TAZ conWe and others have recently observed that energy stress caused by glucose starvation or 2-deoxyglucose  promotes YAP phosphorylation2021MST1/2 phosphorylate the hydrophobic motif of LATS1/2 (LATS-HM), resulting in LATS1/2 activationin vitro kinase assays to identify candidates that can directly phosphorylate the LATS hydrophobic motif. A truncated human LATS1  was expressed and purified from Eschericia coli, and used as a substrate for the in vitro kinase assays. We screened a human kinome libraryConsidering the indispensable role of LATS1/2 in YAP/TAZ regulation, identification of the MST1/2-independent LATS1/2 kinases is crucial for understanding the Hippo pathway signal transduction. We, therefore, screened the human kinome using MAP4K2/4/6 and MST1/2 both belong to the STE20-like kinase family, and their kinase domains are highly homologous to one anotherTo verify that MAP4K4, but not an associated kinase in the MAP4K4 immunoprecipitate, was responsible for LATS1-HM phosphorylation, we generated a MAP4K4 kinase-inactive mutant (MAP4K4-K54R). This mutation abolished MAP4K4's ability to phosphorylate LATS1 and LATS2 . We nextin vitro. Both MST2 and MAP4K4 enhanced LATS2 autophosphorylation, as determined by LATS activation loop phosphorylation (pLATS-AL), as well as the ability of LATS2 to phosphorylate YAP  were also significantly decreased by MAP4K4 overexpression  transcription factors to promote gene transcription. Phosphorylation of YAP leads to its cytoplasmic localization and reduces its interaction with TEAD. As expected, MAP4K4 overexpression decreased YAP-TEAD4 association . Consistpression . In contKO cells . Collectin vivo functions of MAP4K4, we deleted MAP4K4 by CRISPR in HEK293A cells (Drosophila Misshapen (Msn) and also expressed in HEK293A cells  reporterhpo null mutant clones, marked by lack of GFP, grew large and displayed increased ex-Z expression compared with GFP-positive WT cells  knockdown of MST1/2 does not affect LATS or YAP phosphorylation induced by serum depletion or high cell confluence23 signals . It is nDrosophila Hpo gene (homologue of MST1/2), and the major signal output of the Hippo pathway is the inhibition of YAP/TAZ. This study, together with other reports, shows that the central regulatory molecule of the Hippo pathway in mammals is LATS because it is essential for YAP/TAZ regulation by virtually all signals tested. Surprisingly, the Hpo homologue MST1/2 are largely dispensable for YAP/TAZ regulation. We would like to suggest a broad and new definition of the ‘Hippo pathway'. Proteins that specifically influence LATS kinase activity and the functional output of YAP/TAZ should be considered as Hippo pathway components. Even though MAP4Ks act independently of MST1/2, we propose that MAP4Ks are components of the Hippo pathway based on their direct role in LATS1/2 phosphorylation and activation.The Hippo pathway was named after the Drosophila also supports Hpo-independent regulation of Wts. Loss of function of the Hpo kinase causes strong phenotypes in imaginal discs and in enterocytes89101441428hpo mutants, indicating the existence of another Wts-activating kinase in DrosophilaDrosophila has two MAP4K homologues, Msn (corresponding to MAP4K4/6/7) and Happyhour (corresponding to MAP4K1/2/3/5). Hpo is a master regulator of Drosophila midgut homoeostasis by controlling intestinal stem cell proliferation through suppressing Yki-induced Upd1/2/3 synthesis and secretion from enterocytes41424344et al. showed that Msn is also important for the midgut homoeostasis through controlling Upd3 secretion from another type of midgut cells, enteroblasts, by inactivating Yki through Wts independently of Hpoet al. proposed that Msn and Hpo activate Wts in a non-redundant mannermsn knockdown slightly enhanced the hpo loss of function phenotype in imaginal disc clones , indicating the existence of other LATS-activating kinases. Serum deprivation-induced YAP phosphorylation was blocked in the MM-5KO cells while high cell-density-induced YAP phosphorylation was still present, though much reduced, in the MM-8KO cells. On the other hand, the 2-DG-induced phosphorylation of LATS and YAP/TAZ was completely abolished in the MM-5KO cells. These data suggest that different LATS-activating kinases may preferentially participate in relaying different upstream signals. However, it is also apparent that there is no simple one-to-one linear relationship between upstream signals and the different subgroups of MSTs/MAP4Ks to LATS activation. Our kinome screen identified additional kinases that could phosphorylate LATS directly −1 puromycin selection for 3 days and then were sorted onto 96-well plates with only one cell in each well. The clones were screened by Western blot with gene-specific antibodies and at least two independent clones for each gene deletion were used for each experiment described. The details of guide RNA sequences are provided in CRISPR genomic editing technology was used for the deletion of genes of interest2, 2 mM MnCl2, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35) was used for most of the kinases except for CAMK family kinases, for which 1.2 μM calmodulin and 2.0 μM calcium were supplied. The immunoblot with an antibody detecting phosphorylated LATS hydrophobic motif was performed to detect LATS-HM phosphorylation signals. The detailed information of the tested kinases and the results are shown in The flag-tagged human kinome constructs were transfected into HEK293A cells individually. The kinases were purified from the transfected cells by immunoprecipitation with anti-flag antibodies and protein A/G magnetic beads with a high stringency buffer . The beads were subsequently washed with a buffer containing 50 mM Tris-HCl pH 7.5 and 150 mM NaCl, and then used for kinase assays with a truncated form of human LATS1  at 30 °C for 30 min. A standard kinase assay buffer . 30 min after kinase reaction, recombinant GST-tagged YAP purified from y w hsFlp/Y; ex697FRT42D ubi-GFP/FRT42D, hpo424344454647; hh-Gal4/msn-RNAi. The msn-RNAi line was the verified TRiP line BSC28791The genotype of the animal shown in 5 cells per well were seeded onto six-well plates. For high confluence, 6.0 × 105 or 8 × 105 cells were seeded per well. For all the other treatments, low confluence cells were treated with 2-DG (25 mM), serum deprivation, and LatB (0.2 μg ml−1).HEK293A and U2OS cells were maintained in DMEM containing 10% fetal bovine serum. For low confluence, 1.5 × 10−1 Doxycycline was used to induce the gene expression for 24 h.For tetracycline-inducible gene expression, pRetroX-Tet-on-advanced and pRetrox-tight-puro plasmids (Clontech) were used to generate the stable cells according to the manufacturer's instructions. 500 ng mlWestern blot was performed following standard methods. 7.5% phos-tag gel was used to resolve the phosphor-YAP proteins. The detailed information of the antibodies is provided in HEK293A cells were sparsely seeded on fibronectin-coated plates. 24 h later, culture medium was replaced with fresh medium for 90 min before the serum deprivation. An antibody targeting YAP/TAZ  was used for detection. The results were quantified in three randomly chosen fields for each sample.CTGF and CYR61: CTGF-Forward: 5′-CCAATGACAACGCCTCCTG-3′, CTGF-Reverse: 5′-TGGTGCAGCCAGAAAGCTC-3′; CYR61-Forward: 5′-AGCCTCGCATCCTATACAACC-3′, CYR61-Reverse: 5′-TTCTTTCACAAGGCGGCACTC-3′.RNAs were purified from the cells with tetracycline-inducible expression of MAP4K4, and subjected to reverse transcription  that contains random primers and oligo-dT. The real-time PCR was performed with the Applied Biosystems 7300 with primers targeting How to cite this article: Meng, Z. et al. MAP4K Family kinases act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway. Nat. Commun. 6:8357 doi: 10.1038/ncomms9357 (2015).Supplementary Figures 1-19 and Supplementary Tables 1-2


Polo-like kinase 1 (Plk1) is one of the major kinases controlling mitosis and cell division. Plk1 is first recruited to the centrosome in S phase, then appears on the kinetochores in late G2, and at the end of mitosis, it translocates to the central spindle. Activation of Plk1 requires phosphorylation of T210 by Aurora A, an event that critically depends on the co-factor Bora. However, conflicting reports exist as to where Plk1 is first activated. Phosphorylation of T210 is first observed at the centrosomes, but kinase activity seems to be restricted to the nucleus in the earlier phases of G2. Here, we demonstrate that Plk1 activity manifests itself first in the nucleus using a nuclear FRET-based biosensor for Plk1 activity. However, we find that Bora is restricted to the cytoplasm and that Plk1 is phosphorylated on T210 at the centrosomes. Our data demonstrate that while Plk1 activation occurs on centrosomes, downstream target phosphorylation by Plk1 first occurs in the nucleus. We discuss several explanations for this surprising separation of activation and function. Drosophila, where Bora was first identified as a co-factor for Aurora A, it was shown that Bora is located in the nucleus and translocates to the cytoplasm in early prophase (Polo-like kinase 1 (Plk1) is an important kinase during the cell cycle. It controls several key processes that drive cells into and through mitosis such as centrosome maturation, spindle assembly, sister chromatid cohesion, cytokinesis, and recovery from a DNA damage-induced arrest . This loprophase . Howeverprophase .Here we show, using a nuclear localized FRET-based biosensor, that initial substrate phosphorylation by Plk1 in the nucleus is not the result of a diffusing FRET-probe, but that substrate phosphorylation by Plk1 initially occurs in the nucleus. However, we find that sequestration of Plk1 in the nucleus prevents phosphorylation of T210. This is in contrast to sequestration of Plk1 to the centrosomes, where Plk1 can get phosphorylated on T210. Thus, our data show that Plk1 is phosphorylated and activated at the centrosome, but Plk1 activity is first seen to rise in the nucleus. We were unable to reconstitute Plk1 function by mutants of Plk1 that strictly localized at the centrosomes or in the nucleus, indicating that Plk1 needs to be able to diffuse from the centrosomes to the nucleus in order to be fully functional. Finally, we show that Bora localizes strictly in the cytoplasm in human cells and Bora degradation is induced approximately 2 h before cells enter mitosis. This degradation does not completely remove all Bora, as we have shown previously, and Bora/Aurora A continue to activate Plk1 also in mitosis , 17. Takl-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cell lines expressing LAP-Plk1, AKAP-LAP-Plk1, H2B-LAP-Plk1, and GFP-Bora under the control of tetracycline-inducible were cultured in DMEM containing Tet system approved fetal bovine serum (Lonza). Antibodies that were used were directed against Plk1  supplemented with 6% FCS (Lonza), 2 mM nst Plk1 , 19, Plknst Plk1 , Aurora HindIII (NEB). AKAP-LAP-Plk1 was generated in the following manner: the AKAP centrosomal binding doamina was amplified by PCR using the forward primer 5′-AAGCTTGCCACCATGGCCAACATTGAAGCC-3′ and the reverse primer 5′-CTTAAGCTTCTCATGCCAGCATGAAATTG-3′ and ligated into the LAP-Plk1 consruct using the restriction enzymes HindIII and EcoRI (NEB). The pTON-GFP-Bora construct has been described previously  for 24 h followed by a 6 h release and 1 h incubation with Adriamycin (0.5 μM). Afterwards, cells were kept for 16 h in nocodazole (250 ng/ml). Recovery was induced by adding caffeine (5 mM). Unperturbed mitotic entry was assayed by a 24 h thymidine block followed by a release into nocodazole. For reconstitution assays, expression was induced by addition of tetracycline (1 mg/ml) at the indicated times. For FACS analysis, cells were transfected were harvested by trypsinization and fixed with ice-cold 70% ethanol. Cells were stained using the anti-Histone H3-pS10 antibodies (Millipore) and Alexa488-conjugated secondary antibodies (Molecular Probes). DNA was stained using propidium iodide and samples were analyzed on a FACSCalibur flow cytometer (BD biosciences). Cell cycle distribution was determined by flow cytometry counting 10l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, and were imaged with DIC on a Zeiss Axiovert 200M using 20 × 0.75NA objectives or on a Deltavision imaging system using 20 × 0.75NA objectives. Images were taken every 20 min. GFP-Bora levels were quantified by measuring the integrated density of the GFP signal in cells. Background was subtracted using an area that contained no cells. The FRET-based biosensor for monitoring PLK1 activity has been described previously (http://rsb.info.nih.gov/ij/).For time-lapse microscopy, cells were grown on LabTek II chambered coverglasses in Leibovitz’s L-15 medium (Gibco) supplemented with 6% FCS (Lonza), 2 mM eviously , 20. Theg for 2 min. Soluble nuclear fraction was obtained by extraction of pelleted nuclei with an equal amount of buffer B  and spinning down at 2000 × g for 2 min. Insoluble chromatin was washed with buffer B and finally resuspended in SDS sample buffer. For immunoprecipitations, cells were lysed in with 1 ml lysis buffer  on ice for 10 min. The lysate was cleared by centrifugation and 10% of supernatant was used for whole cell lysate. Immunoprecipitations were performed using S-protein beads (Novagen) or with antibodies bound to Dynabeads Protein A (Life Technologies). Beads were washed with TBST and incubated with the rest of the lysate at 4°C for 24 h. Beads were washed extensively with lysis buffer, after which bound protein was eluted with Laemmli sample buffer.Chromatin fractionation was performed as described . SolublePlk1 localizes both at the centrosomes and kinetochores during G2. While most of the literature suggests that Plk1 is activated at the centrosomes, this is mainly based on immunofluoresence using anti-phospho-T210 antibodies , and theSince substrate phosphorylation by Plk1 is first observed in the nucleus during G2, we next asked if Plk1 needs to be on the centrosomes or in the nucleus in order to promote entry into mitosis during recovery from a DNA damage-induced arrest. To this end, we generated stable cell lines expressing tetracycline-inducible and RNAi-resistant variants of Plk1 that were either freely diffusible, or exclusively localized at centrosome or in the nucleus. For this purpose, we used EGFP-TEV-S (LAP)-tagged wild-type Plk1 , 24, an Drosophila, Bora has been shown to initially localize in the nucleus, then transfer to the cytoplasm in early prophase until nuclear envelope breakdown ..Drosophireakdown .Because we were unable to monitor endogenous Bora by immunofluorescence, we performed a fractionation assay to separate cytoplasmic proteins from the nuclear proteins . We syncPlk1 localization is highly dynamic during the cell cycle. Activation starts in G2, presumably at the centrosomes, but activity monitored by a FRET-based biosensor is first observed in the nucleus approximately 5 h before mitosis , 14. SimHere, we provide proof that stable phosphorylation of Plk1 targets first occurs in the nucleus. Since the H2B-tagged and diffusible probes showed similar profiles, we ruled out the possibility that detection of Plk1 activity was affected by diffusion or active import of the phosphorylated FRET-probe from the cytoplasm Figure .There are several possible explanations for the preferential target phosphorylation by Plk1 in the nucleus. Phosphorylation of the FRET-based biosensor can easily be reversed as Plk1 inhibition decreases the CFP/YFP-ratio to basal levels in approximately half an hour . This obWe find that a version of Plk1 that is tethered to the centrosome can be phosphorylated at T210, albeit less than the wild type version of Plk1. However, it is not possible to functionally rescue Plk1 function when Plk1 is tethered to the centrosome, something others have observed as well . In addiPhosphorylation of Plk1 at T210 by Aurora A requires Bora , 12. OurIn addition to the downstream regulation, upstream regulation of Aurora A activity during G2 and how this relates to specific timing of T210-phosphorylation on Plk1 is currently unclear. Aurora A relies on several co-factors to exert its functions and we have previously shown that the Aurora A co-factor TPX2 does not contribute to the activation of Plk1 during G2 and mitosis . HoweverThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.