Naturally occurring IgE-specific IgG autoantibodies have been identified in patients with asthma and other diseases, but their spectrum of functions is poorly understood.Address the hypothesis that: (i) IgG anti-IgE autoantibodies are detectable in the serum of all subjects but elevated in asthmatic patients regardless of atopic status as compared with controls; (ii) some activate IgE-sensitized basophils; and (iii) some inhibit allergen-induced basophil activation.IgE-specific IgG autoantibodies were detected and quantified in sera using ELISA. Sera were examined for their ability to activate IgE-sensitized human blood basophils in the presence and absence of allergen using a basophil activation test, and to inhibit allergen binding to specific IgE on a rat basophilic cell line stably expressing human FcεRI.IgG autoantibodies binding to both free and FcεRI-bound IgE were detected in patients with atopic and non-atopic asthma, as well as controls. While some were able to activate IgE-sensitised basophils, others inhibited allergen-induced basophil activation, at least partly by inhibiting binding of IgE to specific allergen.Naturally occurring IgG anti-IgE autoantibodies may inhibit, as well as induce, basophil activation. They act in a manner distinct from therapeutic IgG anti-IgE antibodies such as omalizumab. They may at least partly explain why atopic subjects who make allergen-specific IgE never develop clinical symptoms, and why omalizumab therapy is of variable clinical benefit in severe atopic asthma. IgE is thought to participate in host defense, but it also has a central role in the pathogenesis of allergy and asthma.1 or IgG4 isotype1 and IgG3 isotypes have been described predominantly in patients with chronic urticaria, while IgG2 and IgG4 isotypes have been described in other autoimmune disorders.14Previous studies in humans have identified the production of autoantibodies of the IgG or IgM class that bind specifically to IgE or FcεRI. Some are able to activate basophils and mast cells independently of antigens.Two general, striking features of these studies stand out. First, not all of these autoantibodies show proinflammatory activity,19Consequently, we set out to examine the possibility that IgG anti-IgE autoantibodies may in some individuals exert an anti-inflammatory, rather than a proinflammatory, effect. We elected to focus on asthma as an archetypal disease involving IgE-mediated mechanisms, in which exogenous IgG anti-IgE  has a proven therapeutic role at least in some individuals, and on anti-IgE rather than anti-FcεRI autoantibodies, for the same reason. We hypothesized that: (i) IgG anti-IgE autoantibodies are detectable in the serum of all subjects but elevated in asthmatic subjects regardless of atopic status as compared with controls; (ii) some of these antibodies can activate IgE-sensitized basophils; (iii) some of these antibodies do not activate IgE-sensitized basophils and can, furthermore, inhibit allergen-induced activation.in vitro assay to detect and quantify IgG anti-IgE autoantibodies in the serum of asthmatic subjects and controls. We then tested the ability of these sera to activate or inhibit IgE-sensitized blood basophils from a single atopic donor in the presence and absence of allergen and finally utilized a rat basophilic cell line stably expressing human FcεRI bound to in-house manufactured monoclonal IgE directed against the Phl p 7 component of timothy grass allergenTo address these hypotheses, we developed and calibrated an 1 or PEFR in response to inhaled bronchodilators ; ii) documented ≥8% variability of PEFR during a 24-hour period or ≥20% variability over a period of 1 to 2 weeks; or iii) a positive mannitol bronchial challenge test . Non-asthma was defined as absence of relevant symptoms, with FEV1 in the normal range. Atopy was defined as a positive in vitro IgE test to 1 or more of the following local aeroallergens: mixed grass, mixed tree, mixed mould, house dust mite (HDM), and cat and dog dander. Non-atopy was defined as negative in vitro IgE tests  (Grade 0 or ≤0.35 kU/L) to the same aeroallergens. The median and range % predicted FEV1 of the AA and NAA subjects and the NAC at the time of the study were 97 (64-143), 73 (40-119) and 106 (94-128), respectively. All patient sera were screened for anti-nuclear autoantibodies (ANA) using commercially available ANA-ELISA kits  according to the manufacturer's instructions.Atopic asthmatic (AA), non-atopic asthmatic (NAA), and non-atopic non-asthmatic control (NAC) subjects were recruited from the departmental asthma clinic at Guy's Hospital, London and databases or through advertisements. All participants gave written informed consent to participate in the study, which was approved by a local research ethics committee. A diagnosis of asthma was accepted based on relevant symptoms and 1 of the following criteria: i) documented ≥12% reversibility of FEV2SO4 and absorbance read at 450 nm using a Multiskan EX plate reader (Thermo Fisher Scientific). The assay was calibrated using a commercial IgG anti-IgE monoclonal antibody  or human IgG (Sigma-Aldrich). All samples were measured at least in duplicate. To further allow for any non-specific IgG binding to the plates and determine the threshold of sensitivity of the anti-IgE ELISA (0.27 ng/mL), commercial human IgG (Sigma-Aldrich) was used at a relatively high concentration (2 μg/mL) in place of sera as a negative control and the threshold defined as 3 standard deviations above the mean resulting absorbance.Maxisorp plates (Thermo Fisher Scientific) were coated either with 0.5 μg/mL of recombinant IgE  to measure IgG anti-IgE or with anti-human IgG  to measure total IgG, in 50 mM carbonate buffer pH 9.6 at 4°C overnight. Non-specific binding was blocked with SuperBlock blocking buffer (Thermo Fisher Scientific) or 3% milk powder (MP) in PBS-T at room temperature (RT) for 2 hours, and the plates then washed 3 times with PBS-T. Test sera were added at 1:5 to 1:20 dilutions in 1% MP-PBS-T and the plates incubated at RT for 1 hour then washed 3 times with PBS-T and incubated with anti-human IgG-HRP  for 1 hour at RT. The plates were washed again 3 times and the color reaction developed using TMB solution . The reaction was stopped by addition of 1.8 M HThe binding of IgG anti-IgE autoantibodies to FcεRI-bound IgE was determined by pre-coating the ELISA plate with recombinant FcεRIα  in carbonate buffer at 4°C overnight. After washing 3 times with PBS-T and blocking with 3% MP in PBS-T for 2 hours at RT, the plates were incubated overnight at 4°C with recombinant IgE (0.5 μg/mL in 1% MP in PBS-T). Plates were washed 3 times with PBS-T before the addition of the test sera and the assay developed as described above. Each sample was assayed at least in duplicate.7 cells/mL.PBMC from a single atopic non-asthmatic donor with total serum IgE >150 IU/mL and sensitized to HDM as determined by skin prick testing were isolated using Ficoll-Paque PLUS  and re-suspended in HBSS (Sigma-Aldrich) to a final concentration of 2 x 106 PBMC (50 μL of suspension) were incubated with sera diluted 1:2 in BAT buffer to 50 μL (HBSS with 2 mM CaCl2) for 30 minutes at 37°C and the reaction stopped by adding 2 mL of FACS buffer containing 2 mM EDTA on ice. The donor's own serum was used as a baseline control. As a positive control, instead of sera, PBMC were activated using polyclonal anti-IgE  in the concentration range 6 ng/mL to 6 μg/mL in BAT buffer.To test the activity of subject sera in the BAT assay, 1 × 10+ basophils expressing the activation marker CD63 compared with the baseline control.Flow cytometric analysis of basophil activation was adapted from a previously published protocol.6 PBMCs in 50 μL of BAT buffer from a single atopic donor sensitized to HDM with the sera (diluted 1:2 in BAT buffer to 50 μL), purified anti-IgE, or the donor's own serum as a baseline control for 30 minutes at 37°C. The cells were then washed and resuspended in 50 μL BAT buffer prior to the addition of serial dilutions of the major HDM antigen Der p 2 from 300 to 3 ng/mL for 30 minutes at 37°C. The reactions were stopped by adding 2 mL of FACS buffer containing 2 mM EDTA on ice, and basophil activation was determined as described above.To determine the effect of subjects' sera on allergen-induced basophil activation, we pre-incubated 10To deplete total IgG from sera, 500 μL samples were incubated with an equal volume of protein G Sepharose (Sigma-Aldrich) at 4°C overnight in a mini Bio-Spin chromatography column . The flow-through was recovered and re-incubated with fresh protein G Sepharose at 4°C overnight. IgG was measured in the final recovered flow-through by ELISA.3 buffer containing 0.5 M NaCl) at 4°C overnight in a mini Bio-Spin chromatography column, then washed with coupling buffer. Unconjugated sites were blocked with 0.2 M glycine buffer pH 8.0 for 2 hours at RT. After extensive washing with 5 cycles of the coupling buffer and 0.1 M acetate buffer pH 4, the IgE-coupled Sepharose was ready to be used. IgE-coupled Sepharose (50 μL) was mixed with subjects' sera (3 mL) at 4°C overnight. The eluate containing non-IgE binding antibodies was stored at −20°C for later analysis. Proteins bound to the IgE-coupled Sepharose were then eluted with 100 μL of 0.2 M glycine pH 2.5 into an equal volume of 100 mM Tris buffer pH 8.0. The eluted solutions were dialyzed in PBS at 4°C overnight, then mixed with protein G Sepharose (1:1) at 4°C overnight to capture IgG antibodies. The IgG anti-IgE antibodies were eluted off the protein G Sepharose with glycine, dialyzed with PBS as above, then quantified by ELISA and run on 10% SDS-PAGE under non-reducing conditions compared with recombinant IgE and IgG (Sigma-Aldrich). No contaminating IgE was found in purified antibodies, as determined by total IgE ELISA .To deplete IgE-binding proteins, IgE was cross-linked to cyanogen bromide (CNBr)–activated Sepharose 4B (Sigma-Aldrich) according to the manufacturer's instructions. Briefly, 100 μg of recombinant IgE anti-NP-BSA (manufactured in-house) were mixed with 100 μL of 1 mM HCl-swollen CNBr-activated Sepharose in coupling buffer  for 30 minutes at 37°C. After further washing with HBSS, the cells were resuspended and incubated with 50 μL of 1 μg/mL of in-house produced, freshly biotinylated Phl p 7 diluted in BAT buffer for 30 minutes at 37°C. Surface-bound IgE was detected using anti-IgE-FITC  and Phl p 7 binding to this IgE using streptavidin-APC (BioLegend), with analysis by flow cytometry.To determine the effects of our test sera on allergen binding to IgE on the surface of basophils, we used pure, recombinant P value of < .05 was considered significant .Statistical analysis was performed using 2-way ANOVA with Bonferroni correction or as otherwise stated. A A).Serum samples were screened for IgG-class antibodies specific for IgE using an in-house, custom ELISA as described in the A). A “normal” range for these measurements has yet to be defined, but some serum samples from the asthmatic subjects clearly showed concentrations of IgG anti-IgE in excess of the 95% confidence limit of the measurements in the normal controls (A). Nevertheless, many did not, and overall, there was no significant variation in the concentrations of these antibodies between the 3 groups. There were no systematic differences in binding of any of the IgE-specific autoantibodies to IgE alone and FcεRI-bound IgE, although in 2 of the samples from the non-atopic asthmatic subjects (NAA9 and NAA17), binding of the autoantibodies to FcεRI-bound IgE was below the lower limit of detection of the assay and therefore technically unquantifiable (see B). Purified IgG anti-IgE autoantibody from a single patient's serum was used as a standard to calibrate the ELISA for measuring FcεRI-bound IgE, because omalizumab does not bind to FcεRI-bound IgE. Gel electrophoresis confirmed that this antibody, as well as antibodies from the remainder of the study subjects, was pure and, in particular, free of contaminating IgE (see C and D).Omalizumab was used as a standard to calibrate the ELISA see , A. A “ncontrols , A. Neveable see , B. Puri IgE see , C and DA and B, in this article's Online Repository at www.jacionline.org). No correlation was observed in either case.Concentrations of IgG anti-IgE antibodies were compared with those of total IgE and anti-nuclear autoantibodies in all of the study subjects . To examine autoantibody activity, these same donor basophils were incubated with subjects' serum samples. Basophil activation was defined as >2.67% CD63+ cells, or 3 standard deviations above the mean percentage of basophils expressing CD63 following incubation with the donor's own serum under identical conditions as a baseline. According to these criteria, sera from 2 non-atopic asthmatic subjects  and 3 atopic asthmatic subjects  were able to activate basophils directly, whereas the others were not (C). This activity did not equate with elevated IgG anti-IgE autoantibody concentrations: not all basophil-activating sera contained high concentrations of IgG anti-IgE . None of the 5 basophil-activating sera, when pre-incubated with the same donor basophils under identical conditions, significantly reduced the degree of basophil activation induced by a concentration of Der p 2 optimal for this donor (B) and a range of higher and lower concentrations . In contrast, sera from 2 subjects (NAA16 and AA12), which did not directly increase basophil activation, significantly inhibited allergen-induced activation, while serum from a third subject (NAA2) exhibited an inhibitory effect of borderline significance .Considering the above data, we examined the ability of sera from all of the donors to modify allergen-induced activation of blood basophils from the same single atopic donor who was sensitized to the major dust mite allergen tivation , A. Noneis donor , B and aA) or IgE-binding proteins (B). Both maneuvers essentially abolished the ability of the sera to inhibit allergen-induced basophil activation, whereas purified IgE-binding proteins retained this activity in full (C and D). As a negative control to demonstrate that depletion of IgG or isolation of IgE-binding proteins did not generate basophil modifying activity per se, we showed that similar extracts from the non-inhibitory serum NAA7 showed no significant effects on allergen-induced basophil activation (C).To further characterize the observed inhibitory activity of the sera from subjects NAA16 and AA12 on allergen-induced basophil activation, we depleted either IgG , A or Igproteins , B. Both in full , C and Dtivation , C.Fig 3Phl p 7.Phl p 7 to the FcεRI expressed on the surface of the RBL-SX38 cells (A). Similarly, subsequent addition of fluorochrome-tagged, recombinant Phl p 7 to the IgE-coated cells resulted in its binding to this specific antibody (B). Pre-incubation of IgE-bound RBL-SX38 cells with 4 of the sera that activated basophils directly ex vivo but did not modify allergen-induced basophil activation ex vivo , or 1 serum sample from a donor in each group selected to contain anti-IgE at the threshold of the mean concentration observed in the controls with no basophil activating activity  prior to the addition of Phl p 7 did not significantly alter surface IgE or Phl p 7 binding (C and D). In contrast, 2 of the sera that inhibited allergen-induced basophil activation ex vivo  significantly reduced binding of recombinant allergen but not allergen-specific IgE to the RBL-SX38 cells (C and D).To begin to address the mechanisms of inhibition of allergen-induced basophil activation by the inhibitory sera NAA16 and AA12, we used the rat basophilic cell line RBL-SX38, which expresses surface human FcεRI, and recombinant IgE specific for the timothy grass allergen 38 cells , A. Simiantibody , B. Pre- binding , C and D38 cells , C and DWe describe a novel and quantitative technique for the measurement of serum IgG anti-IgE autoantibodies. Hitherto these have been identified indirectly using histamine release assays,Using this assay, we show that there is a subset of asthmatic patients who, regardless of conventional atopic status, have circulating IgG anti-IgE autoantibodies in excess of the range observed in a group of non-asthmatic control subjects, although this is not a consistent finding in the asthmatic subjects and does not permit a clear statistical distinction between the groups. Nevertheless, these observations are of great interest to us from the point of view of a possible role for IgE-mediated mechanisms in asthma, since we have previously reported data suggesting that IgE is expressed to excess in the bronchial mucosa of asthmatic patients regardless of conventional atopic status,B). Similarly, and in contrast to certain earlier studies,A). This may reflect low subject numbers, interference by IgE-specific autoantibodies with the determination of total IgE using different techniques,It is not clear what regulates the production of IgG anti-IgE autoantibody in individual subjects. We observed no correlation between the production of IgG anti-IgE antibodies and the production of other potentially pathogenic IgG autoantibodies such as anti-nuclear autoantibodies see , B. Simiin vivo. This is in contrast to omalizumab, which binds only free IgE. Our findings are congruent with previous studiesex vivo (C) excludes the possibility that it is an artefact of the separation procedure. Nevertheless, we cannot completely exclude the possibility that some basophil-modifying activity may have reflected the alternative or additional presence of IgG anti-FcεRI autoantibodies.in vitro assays, and further exploration of the properties of these antibodies is the subject of an ongoing further study. The unprecedented potential protective effect of some of these antibodies, however, is of great interest regardless of their precise specificities.Although we studied the properties of IgG anti-IgE autoantibodies from considerable numbers of non-atopic controls and non-atopic and atopic asthmatic subjects in this study, it is clearly arguable that, as in all similar studies, we may not have defined all of the properties of these antibodies or their relative incidence in the entire source populations in an unbiased manner. Having said this, we observed that in no case was their binding inhibited by prior binding of the IgE to its high-affinity receptor FcεRI, suggesting that these IgG species are able to bind to IgE already attached to its high-affinity receptor sex vivo , C excluWe undertook some preliminary investigations as to possible mechanisms by which inhibitory IgG anti-IgE autoantibodies could reduce allergen-induced basophil activation in our patients. Those we studied did not appear to dissociate IgE from its receptor, a property suggested in previous reports,4 response that could, at least in theory, include IgG anti-IgE autoantibodies. Indeed, 1 of these studies on wasp venom immunotherapyIt is finally apposite to note that production of IgG anti-IgE autoantibodies has been documented in murine “models” of allergen sensitization and tolerance•IgE-specific autoantibodies are detectable in atopic and non-atopic asthmatic subjects and controls. They bind to IgE regardless of whether or not it is bound to its high-affinity receptor.•Some of these autoantibodies activate basophils, whereas others inhibit allergen-induced basophil activation.In summary, inhibitory IgG anti-IgE autoantibodies may contribute to a natural regulatory mechanism that could conceivably influence the severity and presence or absence of diseases with IgE-mediated mechanisms, including asthma and allergic rhinitis, and the outcomes of therapeutic processes such as allergen immunotherapy and therapy with exogenous IgG anti-IgE. This demands further, potentially extremely exciting, research.


This paper discusses the first phase of a longitudinal study underway in Australia to ascertain the broad health benefits of specific types of biophilic design for workers in a building site office. A bespoke site design was formulated to include open plan workspace, natural lighting, ventilation, significant plants, prospect and views, recycled materials and use of non-synthetic materials. Initial data in the first three months was gathered from a series of demographic questions and from interviews and observations of site workers. Preliminary data indicates a strong positive effect from incorporating aspects of biophilic design to boost productivity, ameliorate stress, enhance well-being, foster a collaborative work environment and promote workplace satisfaction, thus contributing towards a high performance workspace. The longitudinal study spanning over two years will track human-plant interactions in a biophilic influenced space, whilst also assessing the concomitant cognitive, social, psychological and physical health benefits for workers. It will never fail you”.—Frank Lloyd WrightThe positive effects of nature, especially plants, upon human health and well-being, has been extensively researched and documented ,10,11,12According to Harvard biologist, E. O. Wilson ,30, we aStephen Kellert  as a leaet al. revealed that just one plant within the workspace can significantly enhance staff morale and simultaneously promote well-being and improve performance [Nature writer Wendell Berry  coined tformance .2 from occupants’ breathing. Cleaner air has also been found to have a causal relationship with better cardiovascular health and mental acuity [Their seminal research focused on the benefits of potted plants in reducing air pollution indoors. Plants played a central role in ameliorating volatile organic compound (VOCs) emitted from plastic or synthetic materials , and COl acuity ,43.Compelling evidence is mounting to encourage the incorporation of green spaces in work sites; this proposed two-year collaborative project between University of Western Sydney (UWS) and Brookfield Multiplex (BM), one of Australia’s largest construction companies, will examine if selected variables such as: a bespoke open plan site shed space; natural lighting and ventilation; introduction of plants; and collaborative work spaces have an impact on health and well-being.On each BM building site, the most essential component is the site office, adjoining the building site and serving as a temporary workplace for site managers who oversee the project. Although temporary, site managers move from one site office to another; thus the buildings constitute a long-term setting for their professional lives. In other words, “temporary” becomes “permanent” across different work sites.“Our goal with this project is to improve our site office environments which is an element of our broader business agenda of creating high performance site offices... because evidence tells us they make our people happier, healthier and more productive”.BM is a partner of 202020 Vision  and suppIn the first month of the study, the case study commenced with a “voluntary Saturday working bee” involving workers from a wide cross section of employees from apprentices through to upper management. This was a novel approach to work assignment, with the goal to empower workers and break down everyday hierarchy in the company to increase collaboration and solidarity.The bespoke workplace design of the biophilic site office see  was initFour specific purpose spaces, including: (1) collaboration hub; (2) design hub; (3) enclosed collaboration area; and (4) open plan area.Skylights to allow for natural light.Recycled and sustainable carpet and furniture.Blinds and operable windows with views of trees.Recycled timber decking.Doors to improve lighting and ventilation.Extended kitchen with a breakout area for recreation and informal team meetings.12 plant boxes at eye height between large rows of decks.2 floor-to-ceiling plant walls.Herb tower for the verandah.The following characteristics defined the site office see :Four spIt can be seen that the design outlined here has incorporated certain biophilic design principles as articulated by both Kellert ,31 and Ain situ for 24 months renovating a major suburban shopping mall in western Sydney, both quantitative and qualitative data will be obtained. The long-term research project incorporates a mixed-methods research design. Quantitative data for the longer-term project will be obtained from on-line survey results Connection to Nature Scale (CNS) [The aim of the two-year longitudinal study is to examine the impact of purposely introduced plants into a retrofitted site office shed which incorporates elements of biophilic design. Against this backdrop, our paper provides the initial findings from the first phase of a longitudinal two-year study (2014–2016). The site office workers are le (CNS) , employeThe preliminary data collection points during the first three months of this study are displayed in Peace/Madonna Lily (Spathiphyllum species)Mother in Laws Tongue/ Snake Plant (Sansevieria species)Zanzibar Gem (Zamioculcas zamiifolia)Cast Iron Plant (Aspidistra)Grey Star (Ctenanthe setosa)Several planning meetings were held with overall site manager and foreman of the site, 202020 Vision staff and Nursery and Garden Industry of Australia (NGIA) representative Matt Carroll, who were providing the plants for the retrofit. Discussions concerned the type of plants themselves as suitable for this environment . The final plant selection included:Decisions concerning what materials the plants would be seated in and exactly where in the site office they were to be located were important considerations. A biophilic approach features recycling natural materials wherever possible, so construction used wood pallets see , readilyVertical pallets had to be easy to assemble on the designated working bee day with staff, friends and family invited to the site to participate in constructing the pallets, choosing and planting the plants and placing them in the designated spots in the site office. An underlying premise was that a model of easy-to-construct, cheap and attractive pallets could be adopted in a home context; thus, not only does the workplace or company model the use of recycled materials and greening of the workplace, but also encourages greening of the workers’ homes as a flow-on effect. This pattern of modeling and transferable techniques develops in a new direction the overall Sustainability Plan for BM, while incorporating all six elements of biophilic features .The study commenced with a Saturday working bee organised for site office workers (n = 17), their partners (n = 6) and children (n = 3). The primary intent of the exercise was to foster collaborative and group ownership of the project. Qualitative interviews, observation, photographs and video footage were obtained during the working bee proceedings.Using elements of social capacity building and cohesive team building, two foremen were tasked with six apprentices and shown novel ways to up-cycle materials found on their worksite which would normally go into waste material. Workers and their families were actively engaged in transforming their site offices using innovative recycled office furniture and planter boxes see .The use of the “foreman-apprenticeship” relationship reinforced team building and increased social engagement. One of the managers remarked on the day, “obviously there is a huge team building exercise going on”. Another site-manager commented: “they kind of jumped in and the community collaborative spirit… the tapping into a connectedness with the plants and some of the people and you could see a different side of it coming in”. In sum, the day was heralded as an exemplary team building exercise although this was a secondary objective see .Once the site workers were up-skilled by the foreman, they were responsible for the construction of their personalized recycled planter box, choice of greenery and general maintenance for their plants. Workers were interviewed whilst building vertical pallet trolleys and vegetable gardens from recycled materials. The artifacts produced are shown in the following collages see .Three weeks after the working bee day, the researchers conducted onsite individual interviews (15 min per subject) with randomly selected workers across all levels of management (n = 12) to ascertain the initial impact of the biophilic intervention upon their workspace. Researchers actively probed workers to encourage them to reflect deeply on their responses. All workers were receptive to this approach and forthcoming with measured and thoughtful dialogue.Demographics: (i) Gender and Age, (ii) Role or Position and (iii) Time spent in Site OfficeRate this site office against previous site offices you have worked in (score out of 10).Does this office layout help or hinder the work environment at BM? Why/why not?Do you think it is beneficial in terms of health and well-being? Why/why not?Does this office do anything for you in terms of fatigue or stress? Why/why not?What about creativity and mental acuity—any changes by being in this office? Why/why not?In terms of collaboration and co-operation, have there been any noticeable differences? Why/why not?Did you come to the working bee© If not, what was your initial impression when you walked into the office space the following Monday?Name three qualities that best describe “the vibe” of this site.Interview questions:The narratives were transcribed and emergent themes coded. Collated results are outlined in the following section.Due to the nature of data collected during the interviews, observations and video footage, reviewing all of the explicate outcomes obtained in this phase is clearly beyond the scope of the paper. In addition, some emergent themes need to be confirmed during the ongoing longitudinal follow-up before they can be reported. However, our preliminary analysis of the results has revealed intriguing trends and insights. With regard to interview Question 1, a breakdown in the demographics of the 12 workers and their workplace information is provided in What stands out from this table is the relatively young age of people working in the site office, that the workers are predominantly male, and the variable amount of time that workers utilize this site office space. The site office space occupies the vast amount of working time for many of the staff on this building site.Individuals were asked during interview Question 2 to rate  previous site offices they had worked in against the biophilic office. This data makes obvious that the transformed biophilic site office was considered by its workers to be superior to previous site(s), in some cases by a very wide margin. It must be acknowledged that a limitation of our study lies here in the lack of a pre-and post- intervention design. In asking workers to make a retrospective evaluation of previous site sheds as against the present modified site shed, results may indicate bias.Data collected from interviews identified several emerging themes: the unique nature of this site office (compared to other site offices); sustainable workplaces and the transfer of learning from workplace to home (sustainable practices and ownership); high performance workplaces; impact of external surroundings of working site office; and the role and impact of “green space” (specific plants) in the workplace. For the purposes of this paper, two themes will be addressed: high performance workplaces and the role of plants in the workplace. The justification for the choice to focus on these elements is as follows: it was felt that since the initial interviews had been conducted so soon after the Working Bee (three weeks), the impact of that day may still have remained as a strong influencing factor on the workers’ responses. In addition, the researchers deduced that a longer time interval was required in order to tease out the nature of sustainable workplaces and if there was a transfer of learning from workplace to home. The role of the transformed green working space and the re-design in terms of open space collaborative spaces, seemed to suggest an immediacy of impact that should be reflected in the interviews.“For me this is the best office that I’ve ever worked in, so an 8 or a 9 (rating out of 10)”.“…and my last site, I didn’t want to go to the office. So out of 10, it would probably be 3”.The site office, although by its very nature transitory and temporary, is in fact the type of workplace setting that these workers and managers inhabit serially over their working lives in the construction industry. Although the site may be temporary, the workers are permanently in similar structures. Most workers noted an overwhelming positive difference between previous site offices and the present one:“And if you look at most site offices, they’re fairly cold, harsh. Walled sort of environments, and that’s not the sort of environment that’s conducive to really collaborating well and just creating an atmosphere in which you can present your ideas strongly. But it doesn’t have to be an absolute sort of contest till the death to get those ideas across”.Our workers described previous site offices as “aggressive”, “stark surroundings”, “sterile environment” and “stale kind of environment”. As one participant elaborated:“Sometimes I get stuck on certain things on site, and I come back to the office and you know, I’m looking for an answer first from someone. And I find that this office kind of gives me an opportunity to speak to different people. I’ll just walk past someone, and I’ll think to myself, maybe I can ask this person. You know, because you get an opportunity to see people in their open area. You’re not, you know,… sometimes if you’re in an office, you’re kind of restricted. Like people might not come up to you as much. But if you’re in an open area, you know, you kind of feel as though everyone’s on the same level in that sense, where you can walk past and just have a conversation and ask questions”.In contrast, many viewed the biophilic site office as increasing social capability and improving workplace relations. Not only had the extra spaciousness of the working environment been noticed, but also the more “functional light and airy spaces” (Almusaed and Asaad ) had cha“…Between the site team and the design team. So sometimes, because they are based on site, and 85% of their time is spent on site, they don’t get that opportunity to maybe look at drawings and at that same time, collaborate with the design team who has spent, you know, a couple of weeks looking into certain issues such as the way things are built. And you know, to have that opportunity here in the office where they can grab someone, ask a question, … the problem can be solved in five min rather than let the problem be ongoing for a couple of days before they bring it to the attention of the design team”.According to workers, spontaneous collaborations occurred that previously would not have taken place in another site. For example, informal collaborations expedited problem solving across teams:“There was more room—that was definitely a bonus. I came from a previous site where our site shed was quite limited, and there wasn’t really a lot of room to kind of set up and have an area to work with. It was, yeah, very different. We also didn’t have a lot of meeting rooms, so a lot of the conversations where we didn’t want to make a lot of noise, we would walk outside and talk on the phone. Other areas would be, I think for me a kitchen, especially. It was just more a lunch area. We didn’t have that prior; it was just a small kitchen, and you had your lunch at your desk. So it gives a kind of opportunity for a bit of balance, without working from your desk, because normally you just go to your desk and just check your emails while you’re eating your lunch… So yeah, and because we’re in between site and office, sometimes when we do come back to the office, we kind of just need, I don’t know… Sometimes it’s nice to walk into the kitchen and sit down and just recollect your thoughts and think about what you need to do and make some calls. …Another thing was, I guess, just interaction with other people. There weren’t kind of shelves in front of you, so you could see the person next to you and talk to them. And yeah, I guess it was just a very different layout for a site office”.The newly designed open plan arrangement suited this type of increased social interaction, as well as opportunities afforded by the range of smaller meeting places:“Personally, I haven’t got the best attention span anyway, so with everyone talking, especially, I’m in the design hub so there’s a constant flow of people coming in and asking questions. Sometimes, I find it very hard to concentrate, as in focus on reading the emails. A lot of what I do is reading reports and trying to knuckle down and get design done…I just literally was losing my temper a couple of seconds ago because I’m trying to read an email which is many pages of A4 long. I’ve printed it off. I can’t focus because there’s seven different conversations, and everyone asks you, because you’re sitting there, and it’s so open plan…”.Some workers found those increased interactions distracting, but these responses were in the minority:“I think collaboration in this job has been good, and a lot of that’s been because of the way the office is set up. A lot of it’s been from the initiatives that certain people in the office have driven: with every Friday at 2 o’clock, we review something in the meeting room... Yeah, I think it has been good for collaboration”.Still, the collaborative nature of the space was recognized by the same person:From natural lighting, furniture made with natural materials, white painted walls, and recycled carpet, to open windows and hearing bird sounds, all of the workers delineated different positive attributes of the unusual biophilic workspace. The workplace was enhanced from various perspectives. Early accounts from the qualitative interviews also suggest that this space increases social capacity and collaboration, and may lead to gains in productivity that we are exploring in the longer research project.“I guess as soon as you walk into our office from the front gate, you notice something different because it’s got that vibe of you know, you’re kind of secluded away. And you know, you don’t really know what to expect, and then you walk in, and I think it looks a lot more, I don’t know, modern, relaxing. I’m not sure what words to use, but it’s different in a very positive way”.The dominant effect of the redesign of the site office seemed to be a change in the social dynamics of the space. However, the researchers were keen to tease out the specific impact of the plants, especially how workers perceived green elements in the design:“Yeah, when I walk to my desk, and you see plants, and you’re just, you know, it’s different. You don’t feel as though you’re indoors the whole day, if that makes sense”.This user does not speak about the “green” of the site office directly here, but the plants were mentioned by this subject subsequently:But I do enjoy having them (the plants) now; I think it’s really nice. I’m not super-stressed at the moment. I’m in one of my calmer cycles, so I don’t know if that’s down to the plants, or whether that’s down to just where the job’s at. I like them.Another respondent who commented on his high stress levels in his last job, referred to the site office plants in this context:“I think natural light is really important; it’s one of my favourite aspects whenever we’re working on a job and designing a job. I think it’s important. Like I say, I really like the plants as well. I didn’t like the fertiliser smell for the first couple of days when they came in, but no, I like it, I do. I actually like the deck. I like there’s just a bit of a garden bit out the front; when you’re out there on the front, it’s nice. It’s nicer than what I’m used to. Last time I would sit outside the office on a roundabout pretty much making phone calls”.This same worker identified his own need for natural surroundings in the workplace:Look, I’m a bit of a sceptic to be honest with you, when it all sort of comes to this sort of stuff. But I was actually surprised when we did all the plants, because it actually, it is good. And it does make, it does create a better vibe within the office.Even the office worker who was least receptive of the biophilic intervention in the group acknowledged that the plants in some way contributed to the transformed working environment:“I’d say “energetic” would be one word: relaxed, calm, enjoyable. You know, this is probably the best, yeah, probably the best office I’ve worked in”.When questioned about the meaning of the word “vibe” this worker explained:“What did I think? I thought, wow there’s plenty of plants in here—very different for a site office. I thought it looked good. I had no issues, no issues at all with it. I think it is good. You know, our subbies (subcontractors) come in and have a meeting with us, and they go, geez, where did you get all the plants? I don’t think it’s going to have a negative impact on anyone who works here, that’s for sure”.The extent to which the plants contribute to the ‘vibe’ is unclear in this respondent’s interview. From other interviews, the plants were clearly having an impact:“Look, it may reduce stress and fatigue and stuff like that, but I think not knowing, it probably does. But it definitely takes away that sort of sterile environment that you sort of get in a lot of offices and that. So I may not directly know that it’s making me feel a lot better, but you walk in, and it doesn’t feel like your standard office”.And another states:“I suppose when you look up and see a bit of greenery around, it kind of reminds you you’re in a kind of living environment… We’re working long hours though; I’m sure it does help though, just not being such a stale kind of environment… Anything that’s natural, anything natural that’s I suppose growing and changing every day”.At this preliminary stage in the research, we note that green workplaces matter to workers who inhabit them in explicit ways, perhaps more so to those who inhabit temporary spaces such as site offices. Despite issues with responsibility for watering and maintaining the plants, we propose that the greenery itself contributes to a changed working environment:Lastly, the researchers caution that generalisations to other populations at this point in time could be problematic due to the interviews being conducted within a few weeks of the “working bee” intervention. Clearly, issues such as “recency effects” and “honeymoon periods” could have favourably skewed the results. The future six-month interval data collection over the two years of the project will enable the researchers to gauge the enduring impacts and better understand the processes and mechanisms underpinning these claims. Notwithstanding these acknowledged limitations, the researchers posit that their preliminary results act as a benchmark for future comparisons.Historically, site workers have been housed in temporary, “match-box-sized” offices for the duration of construction projects, extending up to multiple years. These offices become the “norm” for their office space as workers transition from one construction site location to another. In this Brookfield Multiplex Australian study, aspects of open plan design and green interior spaces were purposefully infused into the newly devised bespoke office site.Initial responses from occupants were clearly supportive of the newly introduced biophilic design elements to their workspace. Some benefits perceived by the workers using them included: enhanced collaboration amongst staff, including across teams, improved morale, and mitigation against stress. Even though this longitudinal study is ongoing for two years, early trends in responses from the first few weeks of occupancy suggested resounding approval by office workers. In short, the research is pointing towards a potential outcome that biophilic-designed site offices are linked to perceived social benefits and increased employee functioning including cooperation and mentoring, and to positive psychological effects, such as improved work satisfaction and higher morale. These are all characteristics of a high performance workplace and reinforce the findings of earlier research. What remains to be explored as the project continues are the effects of these design elements on the specific performance of workers and managers in their work spaces and the overall impact of plants in the workplace environment, including on non-subjective factors, such as employee retention and productivity. In addition, it remains to be seen to what extent the results are due to the biophilic elements or the impact of the Working Bee. Data collection currently ongoing until early 2016 will address a number of measures of workplace effectiveness and other quantifiable benefits, such as decreased absenteeism and improved mental acuity.


The Ron receptor tyrosine kinase (RTK) has been implicated in the progression of a number of carcinomas, thus understanding the regulatory mechanisms governing its activity is of potential therapeutic significance. A critical role for the juxtamembrane domain in regulating RTK activity is emerging, however the mechanism by which this regulation occurs varies considerably from receptor to receptor.1198 in the kinase domain promotes Ron activation, likely by relieving the inhibitory constraints imposed by the juxtamembrane domain.Unlike other RTKs described to date, tyrosines in the juxtamembrane domain of Ron are inconsequential for receptor activation. Rather, we have identified an acidic region in the juxtamembrane domain of Ron that plays a central role in promoting receptor autoinhibition. Furthermore, our studies demonstrate that phosphorylation of YTaken together, our experimental data and molecular modeling provide a better understanding of the mechanisms governing Ron activation, which will lay the groundwork for the development of novel therapeutic approaches for targeting Ron in human malignancies. Thereby, understanding the complex mechanisms by which RTKs are regulated is of clinical significance due to potential applications for therapeutic intervention against cancers ,2. Overeescribed . Consequescribed ,17,18.1238 and Y1239) in the C-lobe activation loop is required for receptor activation. Subsequent phosphorylation of two docking tyrosines in the c-terminal tail transduces a variety of signaling pathways mediated by Ron [Recepteur d'originenantais (Ron)  belongs to the Met proto-oncogene family ,20. Origd by Ron ,24  , a hallmark of kinase activation, and induction of a major signaling cascade downstream of Ron, the Ras-Map kinase pathway, were examined. Ron and the variants were also transiently transfected with an AP-1 luciferase reporter, and the induced AP-1 transcriptional response was measured. Equivalent levels of protein expression and cell surface expression of Ron and its variants were demonstrated by probing for HA and by the flow cytometry analysis . However, the triple mutation (EDE-AAA) dramatically upregulated receptor activity and downstream signaling to a level comparable to that induced by Ron∆1039–1047  interspersed with serines in the Ron JM-C region Figure A are rel) Figure A. HA-tag Figure 138/9, a h Figure 138/9, a h Figure 138/9, a h Figure 138/9, a h1039,S1041,S1043,S1047) are major components of the acidic JM-C region, the first three of which are predicted to be potential phosphorylation sites (NetPhos 2.0 Server) (data not shown) , absent in murine Ron, resulted in elevated receptor activity, as observed in murine Ron [∆JM-B possessed higher intrinsic catalytic capability than the wild type receptor, as demonstrated by a dramatic increase in phosphorylation of the activation loop tyrosines and a docking tyrosine, and in expression of an AP-1 reporter in both the presence and the absence of ligand  in the JM-B region, Ron∆JM-B-S, which did not result in deletion of the tyrosines in the JM-B region, was constructed  in a sample from the non small cell lung cancer , suggest1044-D1045-E1046) have the potential to interact with H1092 in the P-loop, Y1238 and Q1243 in the activation loop, and the side-chains of S1117 and R1118 between the β3 strand and the αC helix  -51  [1198 and N1139 is disrupted, potentially due to the phosphorylation of Y1198. Consequently, the regional stability of the inactive kinase domain collapses, and the αC helix reorients significantly compared to its position in the auto-inhibited model. This large scale movement could promote the displacement of the JM-C constraint, resulting in reorientation of the P-loop and activation loop, and realignment of the active site residues for recruitment of ATP and substrates. Therefore, when Y1198 is mutated to phenylalanine, the absence of phosphorylation at this residue would be predicted to fix the αC helix in the kinked non-productive orientation, stabilizing the kinase in the inactive state. However, when the inhibitory JM-C region is released by alterations in the juxtamembrane domain that disrupt the autoinhibition confered by the juxtamembrane domain, phosphorylation of Y1198 would not be required for reorientation of the kinase segments and receptor activation.Located at the interface between the αE and the αC helix, Yand FGFR ,52-55. Pand FGFR ,39,40. Iand FGFR , the nont (3Q6U)   retroviral vector. Mutants were generated from pcDNA-RON-HA or MSCV-RON-HA, with the Quik Change mutagenesis kit (Stratagene) according to the manufacturer’s instructions. The following PCR protocol was used and primers for the mutants are listed in Additional file Human embryonic kidney (HEK) 293 cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The TransIT-293 transfection reagents were purchased from Mirus Bio, LLC . The dual-luciferase reporter assay system was purchased from Promega Corporation . Antibodies against Ron 1238/9phospho-tyrosine, phospho-Erk, Erk and the HA epitope (262 K) were purchased from Cell Signaling. Goat anti-rabbit IgG-HRP was purchased from Santa Cruz Biotechnology, Inc . Recombinant MSP was purchased from R&D Systems. The AP-1 luciferase reporter cDNA was kindly provided by Dr. Avery August (Cornell University). All PCR primers were ordered from Eurofins MWG Operon, Inc. . Pfu Turbo DNA polymerase was purchased from Agilent Tech. ECL Plus Western blotting detection reagents were purchased from GE Healthcare .4 HEK 293 cells/well were seeded into 24-well plates. Twenty four hours later, a designated mixture of 60 ng of wild type or mutant forms of RON, 60 ng of AP-1 luciferase reporter plasmid, and 0.5 ng renilla were used for transient transfection with the Mirus-293 transfection reagent according to the manufacturer’s protocol. MSCV-neo or PCDNA3.1-neo plasmid were used as control vectors for each transfection. Cells were stimulated with 50 ng/ml (50 ug/ml stock) MSP 24 h following transfection. Luciferase assays were performed at 24 h later according to the manufacturer’s instructions (Promega). The fold increase is calculated as the firefly luciferase activity divided by the renilla luciferase activity.For the luciferase assay, 5 × 105HEK 293 cells/well were plated into six-well plates. Twenty four hours later, the cells were transiently transfected with 300 ng RON or its derivatives per well. Cells were stimulated with 200 ng/ml MSP for 48 h following transfection. 10 minutes later, cells were suspended in 400 ul ice-cold cell lysis buffer containing 150 mM NaCl, 20 mM Tris–HCl (pH 7.5), 5 mM EDTA, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 10 mM NaF. Cell lysates were centrifuged at 12,000 rpm for 20 min, and the supernatant lysates were transferred to prechilled tubes. Cell lysates were mixed with 4X denaturing SDS loading buffer and heated at 100°C for 8 min. Samples were resolved on an SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes. Membranes were then blocked with 5% nonfat milk or Bovine Serum Albumin in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h and probed with primary antibody at 4°C overnight. Membranes were washed three times in TBST and incubated with secondary goat anti-rabbit IgG-HRP for another hour. Membranes were washed three times in TBST before ECL plus Western blotting detection reagents were applied for visualization. For reprobing, membranes were stripped with 62.5 mM Tris–HCl (pH 6.8), 2% SDS, and 0.7% β-mercaptoethanol at 55°C for 30 min.A total of 2.5 × 105 HEK 293 cells/well were plated into 12-well plate. Twenty four hours later, the cells were transiently transfected with 200 ng RON or its derivatives per dish. PCDNA3.1-neo plasmid was used as control vectors for each transfection. Flow cytometry analysis was performed at 48 h later. 106 cells were incubated per tube in 50 ul FACS buffer (ice cold PBS + 2% FBS). Non-specific binding was blocked by the addition of 50 μL HAB for 2 minutes at room temperature. Cells were incubated on ice with 5 ug/ml anti-Ronβ (DX07) or IgG (control) for 30 min. Cells were washed twice with 1 ml FACS buffer and resuspended once more in 50 μL of FACS buffer. 50 μL HAB per tube was added again for blocking of non-specific binding. Cells were incubated with 5 ug/ml PE anti-mouse IgG1 as a secondary antibody to Ron for 30 min on ice. Cells were washed twice, resuspended in 1 mL of FACS buffer and analyzed for FLOW on a Beckman-Coulter FC500.A total of 1.2 × 10A comparison of 150 receptor kinase domain sequences was mapped onto the crystal structure of the Ron kinase domain (PDB code 3pls) using the CONSURF web server. Surface conservation and surface potential of the Ron kinase domain were illustrated. Potential JM-C binding site was proposed based on residue/sequence characteristics and relative distance.Modeling of the autoinhibited and the activated conformation of Ron was based on the crystal structure of the kinase domains of Ron (PDB code 3PLS) and Met (PDB code 3Q6U) respectively. The modeling was done manually using the COOT software . HydrogeSeven algorithms, BPS , D_R 5757, DSC , GGR 5959, GOR , G_G 6161, H_K , K_S 6363, L_G , and Q_SThe authors declare that they have no competing interests.XW performed all the experiments and wrote the manuscript, NY performed all of the molecular modeling, PAH designed the experiments and helped write the manuscript. All authors read and approved the final manuscript.A) Schematic diagram of mutants. Y1012 and Y1017 in the JM-B region are highlighted in bold. B) 293 cells were transiently transfected with HA-tagged wild-type or mutant Ron in the presence and absence of MSP. Lysates were blotted for p-Ron1238/9, HA, p-Erk and total Erk. C) Relative luciferase activity in 293 cells co-transfected with wild-type or mutant Ron, and an AP-1 luciferase reporter in the presence and absence of MSP.Tyrosines in the juxtamembrane domain of Ron are dispensable for the regulation of receptor activity. Click here for fileCell surface expression of wild type Ron and the Ron variants discussed herein. 293 cells were transiently transfected with wild-type or mutated Ron as indicated. Cells were harvested 48 h after transfection and analyzed by flow cytometry for protein membrane expression.Click here for fileA) In the inactive Ron model, the JM-C region (magenta) is predicted to pack against and interact with the activation loop (yellow) and P-loop (orange) near the active site of the kinase domain. Potential bonds among residues in the JM-C region (1044EDE) (magenta), the P loop (H1092) (orange), the active site  (green) and the activation loop  (yellow) are labeled with black dashed lines. Y1198 (cyan stick) in the kinase C-lobe hydrogen bonds to the backbone amide of N1139 at the c-terminus of the αC helix. B) In the active Ron model, phosphorylation of Y1198 results in reorientation of the αC helix and JM-D region. Consequently, the JM-C region is released from the active site, which enables ATP binding and reorientation of the activation loop. The orange and cyan stick represents AMP-PNP. Figure was generated using the software Pymol.Predicted structure of the autoinhibited and active form of Ron. Click here for file1198-N1139 hydrogen bond promotes the non productive orientation of the αC helix. Crystal structure of the autoinhibited Ron kinase domain (3PLS) is shown as cartoon. The αC helix and αE helix are colored in cyan and green respectively. Residues involved in the hydrogen bonds (yellow dash) linking the two helices are labeled in white.YClick here for filePCR primers for the mutant construction.Click here for file


Here we report about in vitro transphosphorylation of the only NAD+-dependent deacetylase (mDAC) present in the genome of Mycobacterium tuberculosis by eukaryotic-type Ser/Thr kinases, particularly PknA. The phosphorylated mDAC displayed decreased deacetylase activity compared to its unphosphorylated counterpart. Mass-spectrometric study identified seven phosphosites in mDAC; however, mutational analysis highlighted major contribution of Thr-214 for phosphorylation of the protein. In concordance to this observation, variants of mDAC substituting Thr-214 with either Ala (phospho-ablated) or Glu (phosphomimic) exhibited significantly reduced deacetylase activity suggesting phosphorylation mediated control of enzymatic activity. To assess the role of phosphorylation towards functionality of mDAC, we opted for a sirtuin knock-out strain of Escherichia coli (Δdac), where interference of endogenous mycobacterial kinases could be excluded. The Δdac strain in nutrient deprived acetate medium exhibited compromised growth and complementation with mDAC reversed this phenotype. The phospho-ablated or phosphomimic variant, on the other hand, was unable to restore the functionality of mDAC indicating the role of phosphorylation per se in the process. We further over-expressed mDAC or mDAC-T214A as His-tagged protein in M. smegmatis, where endogenous eukaryotic-type Ser/Thr kinases are present. Anti-phosphothreonine antibody recognized both mDAC and mDAC-T214A proteins in western blotting. However, the extent of phosphorylation as adjudged by scanning the band intensity, was significantly low in the mutant protein (mDAC-T214A) compared to that of the wild-type (mDAC). Furthermore, expression of PknA in the mDAC complemented Δdac strain was able to phosphorylate M. tuberculosis sirtuin. The growth profile of this culture in acetate medium was slow compared to that transformed with only vector. On the other hand, use of a kinase dead variant, PknA-K42N instead of PknA, did not display such behavior, which again supported phosphorylation mediated control of mDAC protein. Thus, our results ostensibly render evidence for cross-talk between two distinct post-translational modifications, phosphorylation and deacetylation, in any bacteria. Bioinformatic analysis further indicated conservation of Thr-214 among different mDAC orthologs, thereby arguing the event as mycobacteria specific.Sirtuins are NAD In fact, M. tuberculosis shows remarkable adaptability to the changing environment within the host to become a successful pathogen. Furthermore, within macrophage this bacterium survives on the fatty acids/cholesterol mainly derived from the host  was utilized for PCR amplification of m-dac (Rv1151c) and pknB (Rv0014c). These genes exhibited 100% identity in their nucleotide sequences compared to that of M. tuberculosis H37Rv (virulent strain). For this, primers  were designed incorporating restriction sites. All PCR reactions were carried out using Herculase fusion DNA polymerase as per standardized protocol . The PCR amplicons following restriction digestion(s) and subsequent purification were ligated at corresponding sites of vectors (NdeI/HindIII of pET28c/pVV2 or BamHI/HindIII of pMAL-c2X/p19kproHA) to obtain pET-mDAC or its variants or pVV-mDAC/-mDAC-T214A or pMAL-mDAC or pMAL-PknB or p19kproHA-PknB constructs. For use in some experiments, m-dac was PCR amplified using primers incorporating BamHI/HindIII sites and cloned in corresponding sites (in MCS1) of pETDuet-1 having pknA at NdeI/KpnI sites (in MCS2). Besides these, pMAL-PknA, p19kpro-PknA (Rv0015c either in pMAL or p19kpro), and pMAL-PPP (Rv0018c in pMAL) used in this study were described elsewhere  cells (except those in pMAL which were in TB1/DH5α cells only) for the expression as well as purification of recombinant proteins. Authenticity of all gene sequences was ensured through sequencing using an automated DNA sequencer (Applied Biosystems).To generate point mutants of mDAC (S38A/T39A/S179A/T197A/S212A/T214A/T214E/S222A) or PPP (G117D), two external and two internal primers (incorporating the desired mutations) were designed. This was followed by two sets of primary PCR reactions using pET-mDAC/pMAL-PPP as the template and single set of secondary PCR (using 1:1 mixture of primary PCR reaction products as template) by overlap extension method . All conE. coli BL21(DE3) or TB1 cells harboring different constructs were re-inoculated (1% inoculum) in fresh media (LB broth) supplemented with appropriate antibiotics (100 μg/ml ampicillin for constructs in pMAL and 50 μg/ml kanamycin for pET28c vectors), grown till OD600 of 0.6 and then induced with 0.4 mM IPTG (5 h at 23–25°C for His-tagged constructs or 3 h at 37°C for MBP-tagged constructs). Cells were harvested, re-suspended in lysis buffer  and sonicated for 10 min  at 4°C. For His-tagged proteins, supernatant fraction was loaded onto a Ni-NTA column, washed with 20 ml of lysis buffer containing 20 mM imidazole. The resin bound protein was then eluted in elution buffer (1 ml of lysis buffer containing 50 or 100 or 150 mM imidazole). Eluted protein fractions were pooled, diluted five times in lysis buffer and reloaded onto the column to remove contamination. Column was further washed with 20 mM imidazole and 1 M NaCl followed by elution as mentioned above . Imidazole from the protein preparations was usually not removed since its presence did not affect the enzyme activity. MBP-tagged protein, on the other hand, was purified using an amylose column and eluted with 10 mM maltose following manufacturer’s  recommended protocol. Protein concentrations of eluted samples were estimated by Bradford method (E. coli strain BL21(DE3) cells harboring pETDuet-mDAC/PknA, was utilized for carrying out kinetics of deacetylase activity or mass spectrometric experiments. Culture of M. smegmatis strain mc2155, transformation of pVV-mDAC and purification of recombinant protein is described elsewhere (Overnight cultures (14 h at 37°C) of d method  and storlsewhere .in vitro kinase assay described elsewhere  and 2 μCi of [γ- 32P]-ATP  was incubated in the presence of mDAC (1.79 μM/reaction) at 25°C for 30 min . The reaction was stopped by adding 5x SDS buffer. Samples were resolved in 12% SDS PAGE and gels were stained with Coomassie Brilliant Blue. Finally, gels were analyzed in a phosphoimaging device (Fuji Film model FLA 9000) and also exposed to Kodak X-Omat/AR film for autoradiography. The dephosphorylation activity of PPP was monitored following its (5 μM/reaction) incubation (25°C for 1 h) with samples undergone kinase reaction. Unless mentioned otherwise, all experiments were carried out for at least three times.Ability of PknA/PknB to transphosphorylate mDAC was determined in an lsewhere . BrieflyColor de Lys assay system,’ a microtiter plate (96 well) based colorimetric assay kit of Enzo Life Sciences. Briefly, mDAC protein (2.9 μM) and acetylated ‘Color de Lys’ substrate peptide  in assay buffer  were incubated with varying concentrations of NAD+ (0–16 mM) at 37°C for 120 min . This was followed by addition of equal volume (50 μl) of ‘Color de Lys’ developer solution to terminate the reaction (incubation at 37°C for 10 min) and the absorbance was measured at 405 nm in a micro-plate reader (Molecular Devices Spectramax plus 384 equipped with Softmax Pro 5.2 software). The amount of product formed was calculated by subtracting the blank  readings and the specific enzyme activity (μM deacetylated peptide produced/min/mg of protein) was assessed from standard curves prepared with ‘Color de Lys’ deacetylated standard (0–200 μM). Km and Vmax values were obtained from non-linear fit plots and the molecular mass of mDAC protein was considered as 28 kDa for calculating kcat. Unless mentioned otherwise, the experiments in the present study were done three times and data is represented as mean ± SD.The deacetylase activity of mDAC was determined using ‘Escherichia coli strain BW25113 (hereafter designated as wild-type) and deacetylase gene knockout strain JW1106 (cobB deleted variant hereafter referred as Δdac) were obtained from Coli Genetic Stock Center, Yale University, USA. The strains were revived in LB broth (37°C at 200 rpm) and streaked on LB (wild-type) or LB supplemented with 50 μg/ml kanamycin (Δdac) plates. Growth profile of these strains using nutrient rich conditions (LB medium) was monitored for 12 h following re-inoculation of overnight cultures . Growth of these strains in nutrient deprived conditions was assessed using acetate medium . For this, overnight cultures (LB medium) were re-inoculated in acetate medium and grown following similar conditions with extended duration of the culture . In studies monitoring the ability of mDAC to complement E. coli CobB function, Δdac strain transformed with pMAL or pMAL-mDAC or pMAL-mDAC-T214A or pMAL-mDAC-T214E was selected on LB-ampicillin (100 μg/ml) plates. One such colony was grown overnight in LB-ampicillin medium, either inoculated in acetate containing ampicillin (100 μg/ml) for growth curve or made competent by CaCl2 method  recommended protocol. Scanning of blots was carried out using Biorad-GS-800 Calibrated Densitometer equipped with Umax Magic Scan V5.2.Purified fusion proteins or whole cell lysates were resolved in SDS PAGE (12%) and transferred to nitrocellulose membrane (0.45 μ) at 120 V for 1 h using a mini-transblot apparatus . Following transfer, incubation with different primary antibodies  was carried out. For secondary antibody incubation, 1:5000 dilution of HRP conjugated anti-rabbit (for anti-pThr) or anti-mouse (for anti-His) IgG was used. The blots were finally developed for the signal through Luminata forteTM software1.Phosphorylated and unphosphorylated His-tagged mDAC proteins were subjected to trypsin digestion (in-solution) in 100 mM ammonium bicarbonate buffer, pH 8.5 at 37°C for 16 h. Following digestion, 0.5% formic acid  was added to the peptides before running them on UHPLC system  with C8 column coupled to 6550 iFunnel QTOF LC-MS (Agilent Technologies). Electron spray ionization (ESI) was operated in positive ion mode. The instrument acquired peptide ions over the m/z range of 100–3200 with an MS and MS/MS acquisitions rates of 2 and 4 spectra/second, respectively. Peptide sequences were identified utilizing ProteinPilotCD spectra of wild-type mDAC and different mutant (mDAC-T214A and mDAC-T214E) proteins were compared using a Jasco J-815 spectropolarimeter. Protein solutions (0.2–0.35 mg/ml) employing a cell with path length of 0.1 cm at 25°C was used for measurements in the far ultraviolet region (250–198 nm). Each spectrum reported is an average of 3 scans and the mean residue ellipticity (𝜃) was calculated considering 110 Da as the mean of amino acid residue molecular mass.Archaeoglobus fulgidus (PDB ID: 1ICI) which has been complexed with NAD+ , both PknA and PknB were able to transphosphorylate mDAC. However, magnitude of PknB mediated phosphorylation, as adjudged by scanning the band intensity, was considerably low compared to that of PknA (compare lanes 3 and 5). To ensure phosphorylation is not an experimental artifact, we used boiled mDAC in the incubation mixture with kinase, which led to a significant decrease in the level of its transphosphorylation . The use of PknA-K42N protein (a kinase-dead mutant of PknA) in the reaction mixture did not show any phosphorylation of mDAC . Furthermore, we used myelin basic protein, a common substrate for both PknA and PknB, as a positive control to monitor its transphosphorylation by these kinases. As expected, myelin basic protein was efficiently phosphorylated by both the kinases, which ensured that the difference in the level of phosphorylation of mDAC by PknA or PknB was not because of any discrepancy in their transphosphorylation abilities .Sirtuin activity in eukaryotes is reported to be influenced by the phosphorylation state of the protein . To gainin vivo, we co-expressed p19kpro-PknA or p19kproHA-PknB and pET-mDAC in E. coli strain BL21(DE3) and the transformants were selected over hygromycin (200 μg/ml) and kanamycin (25 μg/ml). Cell extracts from such transformants expressing both the proteins (PknA+mDAC or PknB+mDAC) were purified through Ni-NTA and assessed for recognition by anti-pThr or anti-His antibodies in western blotting. As depicted in Figure 1C (upper), anti-pThr antibody recognized mDAC protein when co-expressed with kinases, such as PknA (lanes 2 and 5) and PknB (lane 4). PknA/PknB mediated phosphorylation of mDAC was further evident by the fact that anti-pThr antibody did not recognize mDAC, when co-expressed with either vector alone (p19kpro/p19kproHA) or kinase-dead variant (PknA-K42N) of PknA . However, the intensity of phosphorylated mDAC band was significantly increased when co-expressed with PknA but not with PknB . The loading of samples was ensured in western blotting with anti-His antibody . Similarly, expression of PknA or PknA-K42N was ascertained by western blotting of lysate using anti-PknA antibody .To elucidate, whether mDAC is phosphorylated by PknA or PknB M. tuberculosis genome is known to reverse auto- or transphosphorylation activity of mycobacterial kinases . The reversible phosphorylating ability of PPP was further supported by the action of phosphatase-dead mutant PPP-G117D, which did not remove phosphate from phosphorylated mDAC .Since the only Ser/Thr phosphatase present in the  kinases , we moniE. coli strain BL21(DE3) transformed with pETDuet-mDAC/PknA. Assessment of the enzymatic activity as the function of protein concentrations indicated that about twofold more p-mDAC enzyme was necessary to obtain activity at equivalent range exhibited by mDAC . In accordance with this observation, even at substrate saturated conditions (10 mM NAD+ with 2.9 μM protein), p-mDAC exhibited ∼45% activity compared to unphosphorylated mDAC . Thus, eukaryotic-type Ser/Thr kinase, particularly PknA mediated transphosphorylation of mDAC modulated its catalytic activity.To monitor the effect of phosphorylation on the deacetylase activity, phosphorylated His-tagged protein (p-mDAC) was prepared from cultures of M. tuberculosis sirtuin (p-mDAC) was prepared from cultures of E. coli strain BL21(DE3) transformed with pETDuet-mDAC/PknA. Mass spectrometric analysis (LC-MS/MS) of p-mDAC protein after trypsin digestion yielded peptides . In fact, compared to mDAC, mDAC-T214A variant exhibited decrease in PknA-mediated transphosphorylation ability in a dose-dependent manner ; indicating leading role of Thr-214 in PknA mediated transphosphorylation of mDAC.Phosphorylated His-tagged + (0–16 mM) exhibited typical Michaelis–Menten curve  with Km and kcat/Km values of 2.5 ± 0.6 mM and 25.3 ± 0.6 mM–1.s–1, respectively. Contrastingly, both the mutant proteins (mDAC-T214A and mDAC-T214E) hardly showed any activity  so that their catalytic parameters could be detected. CD analysis of these mutant proteins compared to the wild-type did not depict any significant alteration in the secondary structures . Since both mDAC-T214A and mDAC-T214E proteins behaved similarly , it is indicative of the contribution of phosphorylation in regulating mDAC activity irrespective of the charge at Thr-214.Phosphorylation often provides negative charge to a protein, thereby modulating its activity . To gain+-dependent deacetylases are fairly conserved among bacteria and are involved in regulating carbon metabolism  in this study. In our preliminary experiments, we compared the growth of wild-type and Δdac in LB (nutrient rich) or in acetate (nutrient deprived) medium. As anticipated, there was no difference in growth pattern between E. coli strains in LB medium, while it was compromised in Δdac in nutrient deprived conditions . Furthermore, to assess whether CobB function could be complemented by mDAC, we transformed pMAL-mDAC in Δdac strain and monitored its growth in LB as well as acetate media. Since there was leaky expression of mDAC, no IPTG was used to induce expression. Interestingly, the complemented strain (Δdac strain transformed with pMAL-mDAC) exhibited enhanced growth in acetate medium compared to that of the knock out variant transformed with vector alone . As expected, the difference in growth was not observed in LB medium . The expression of mDAC in cell lysates was authenticated using anti-MBP antibody . We further transformed pMAL-mDAC-T214A or pMAL-mDAC-T214E in E. coli Δdac strain and monitored their growth in acetate medium. The growth in acetate medium of both the mutants (mDAC-T214A and mDAC-T214E), as assessed by monitoring OD600, were nearly identical and they exhibited an intermediate growth profile between the cobB knock out strain (Δdac) either transformed with vector or complemented with mDAC . The expression of different constructs (pMAL-mDAC or pMAL-mDAC-T214A/T214E or vector) in cell lysates was confirmed in western blotting using anti-MBP antibody . Thus, our findings insinuate the role of PknA mediated phosphorylation of mDAC at Thr-214 in regulating its functionality.NADtabolism . It is wnditions . Thus, tM. tuberculosis eukaryotic-type Ser/Thr kinases, in M. smegmatis is well known . The blot developed using anti-His antibody ruled out any discrepancy in amount of sample loading . The mDAC (phosphorylated and unphosphorylated) protein purified from E. coli strain BL21(DE3) was used as internal controls for the experiment to rule out any possibility of non-specificity of anti-pThr antibody. Collectively, these data strongly argue in favor of endogenous eukaryotic-type Ser/Thr kinase (like PknA) mediated phosphorylation of mDAC, when over-expressed in M. smegmatis.The presence of homologs of ll known . We furtin vivo functional interaction between eukaryotic-type Ser/Thr kinase like PknA and M. tuberculosis sirtuin (mDAC), we further utilized E. coli based expression system since it does not possess such endogenous kinases or phosphatase. For this, p19kpro (vector) or p19kpro-PknA (kinase) or p19kpro-PknA-K42N (kinase-dead mutant) was transformed in mDAC complemented Δdac strain and grown in nutrient deprived condition (acetate medium). Interestingly, growth was affected in the presence of PknA compared to that of the vector control (p19kpro). This effect was kinase specific since there was no E. coli growth inhibition in the presence of the kinase-dead variant, PknA-K42N . The expression of mDAC in cell lysates  and phosphorylation of protein(s) from cultures co-expressing p19kpro-PknA and MBP-mDAC following purification through amylose resin  was confirmed by western blotting using appropriate (anti-MBP/anti-pThr) antibodies. Thus, our findings strongly suggest that PknA mediated phosphorylation of mDAC affected functionality of this protein.To elucidate +-dependent deacetylases from different mycobacterial species. Protein sequences for mycobacterial deacetylases along with those belonging to different taxa were procured using NCBI2. Multiple sequence alignment by Muscle and Clustal omega revealed that mDAC is very much conserved in almost all mycobacterial species  but far distinct when compared with deacetylase sequences from other taxa representing its unique phylogenetic distribution. Of note, the deacetylase sequence from E. coli was taken as an out-group for the analysis. Furthermore, when closely examined Thr-214 remains conserved in all mycobacterial deacetylases except for M. chelonae which bears Asp at this position . Thus, it seems that phosphorylation at this threonine residue is conserved in all mycobacterial deacetylases.We used MycoRRdb database , NCBI BLM. tuberculosis genome that displays NAD+-dependent deacetylase activity. Since regulatory interplay between different signaling events is critical for adaptation of any micro-organism in a particular environment, such cross-talk is definitely important to elucidate. In fact, enzymes associated with these events are considered as vital targets for screening or designing of new anti-mycobacterials . Our results also indicated that co-expression of PknA and mDAC in E. coli yielded p-mDAC protein, which was recognized by anti-pThr antibody in western blotting. The phosphorylated protein (p-mDAC) exhibited decreased deacetylase activity compared to its unphosphorylated counterpart . Mass spectrometric studies with p-mDAC identified four serines  and three threonines  as phosphorylating residues (Table 1). Mutation (one at a time) of these residues to Ala followed by kinase assay with these proteins in the presence of PknA, however, indicated the role of Ser-179, Thr-197, and Thr-214 in PknA mediated phosphorylation of mDAC . Amongst these residues, we noticed major contribution of Thr-214 for transphosphorylation of mDAC .We observed that mDAC was transphosphorylated by PknA, a representative of mycobacterial eukaryotic-type Ser/Thr kinases . To establish functional implications of this finding, we opted for a phenotypic assay, where expression of NAD+-dependent deacetylase, CobB is necessary for optimal growth of E. coli in nutrient deprived acetate medium  in its knock out Δdac strain . Unlike mDAC, neither its phospho-ablated nor phosphomimic variant complemented growth phenotype, indicating the role of phosphorylation at Thr-214 in controlling the enzymatic activity as well as functionality of mDAC .To evaluate the effect of phosphorylation on the catalytic activity of the protein, we compared mDAC along with its phospho-ablated (mDAC-T214A) or phosphomimic (mDAC-T214E) variants. To our surprise, both the mutant proteins displayed hardly any enzymatic activities indicating direct role of phosphorylation in the process . These results, however, do not pin point in vivo functional interaction of a specific kinase in the process. Given the fact that there are several eukaryotic-type Ser/Thr kinases and a cognate phosphatase are present in all mycobacterial genomes sequenced to-date and some of them are essential in nature, it would be an arduous task to resolve this issue using M. tuberculosis as such. In this scenario, we again utilized mDAC complemented E. coli Δdac strain and transformed it with either PknA or its kinase dead variant, PknA-K42N, and monitored growth profile of the cultures in acetate medium. The outcome of this study indicated that at least PknA mediated phosphorylation of mDAC altered the growth phenotype. Thus, our results ostensibly established Thr-214 as the major phosphosite in mDAC and its phosphorylation is a eukaryotic-type Ser/Thr kinase mediated event.rculosis . We furt+ binding sites are fairly conserved. Interestingly, the presence of a charged residue (D/E/R) corresponding to Thr-214 of mDAC is evident, except for Cys in Sirt4 and Ala in Sirt7 . In fact, sequence alignment of mDAC with gram positive bacterial deacetylases also showed the presence of charged residue matching Thr-214 . Considering these results with mDAC activity assays , it seems that the generation of a charged environment corresponding to Thr-214 in other deacetylases is important in modulating their enzymatic activity. Nonetheless, it is distinct and unique in mycobacterial sirtuins where phosphorylation at Thr-214 per se is a molecular switch that governs NAD+-dependent protein acetylation/deacetylation trajectory. Although mechanistic detail of such a regulation is very remote to postulate in the absence of crystal structure of mDAC, phosphorylation induced conformational change of proteins are well known. The positioning of Thr-214 is in the flexible coiled region . Furthermore, its predicted structure based on the same protein from thermostable Archaeoglobus fulgidus (46% identity and 62% homology) through I-TASSER server indicated Thr-214 to be in unstructured region . Taken together with our results of enzymatic activity, it is very likely an indication of ‘action at distance’ control of the active site of mDAC by phosphorylation at Thr-214. Further studies in this direction, especially with mDAC crystal structure, would unravel such mystery.Most notably, we also obtained and analyzed 1:1 sequence alignment of mDAC with each of the human sirtuins and observed NADM. tuberculosis is fatty acids/cholesterol in nutrient restricted conditions as evident when bacteria reside within host macrophages (Several studies indicated that primary carbon source for rophages . The degrophages . In thisrophages , it woulrophages , in bactOur results established that eukaryotic-type Ser/Thr kinase mediated phosphorylation of mycobacterial sirtuin modulates its enzymatic activity as well as its functionality. Furthermore, we for the first time provide evidence for cross-talk between two distinct post-translational events, phosphorylation and deacetylation, thereby regulating bacterial signaling. Further work in this direction is necessary to unravel the mechanistic details.PC and GY conceived the idea; planned experiments, analyzed the results and wrote the manuscript; GY, SR, and NM carried out experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Transgelin2, one of cytoskeletal actin binding proteins has recently been suggested to be involved in the formation of immune synapses. Although detailed function of transgelin2 is largely unknown, interactions between transgelin2 and actin appear to be important in regulating cellular functions of transgelin2. Because protein phosphorylation can change ability to interact with other proteins, comprehensive phosphorylation analysis of transgelin2 will be helpful in understanding its functional mechanisms.Here, a specific protein label-free quantitative phosphorylation analysis method combining immuno-precipitation, IMAC phosphopeptide enrichment technique and label-free relative quantification analysis was used to monitor the phosphorylation changes of transgelin2 overexpressed in Jurkat T cells under protein kinase C (PKC) and protein kinase A (PKA) activation conditions, two representative intracellular signalling pathways of immune cell activation and homeostasis. A total of six serine/threonine phosphorylation sites were identified including threonine-84, a novel phosphorylation site. Notably, distinct phosphorylation patterns of transgelin2 under the two kinase activation conditions were observed. Most phosphorylation sites showing specific kinase-dependent phosphorylation changes were discretely located in two previously characterized actin-binding regions: actin-binding site (ABS) and calponin repeat domain (CNR). PKC activation increased phosphorylation of threonine-180 and serine-185 in the CNR, and PKA activation increased phosphorylation of serine-163 in the ABS.Multiple actin-binding regions of transgelin2 participate to accomplish its full actin-binding capability, and the actin-binding affinity of each actin-binding region appears to be modulated by specific kinase-dependent phosphorylation changes. Accordingly, different actin-binding properties or cellular functions of transgelin2 may result from distinct intracellular signalling events under immune response activation or homeostasis conditions.The online version of this article (doi:10.1186/s12953-015-0070-9) contains supplementary material, which is available to authorized users. Transgelin2 was characterized as a smooth muscle cytoskeletal protein and subsequently classified into the calponin family. Although the detailed function of transgelin2 is largely unknown, co-localization with F-actin cytoskeleton in the cytoplasm has been recently reported . Close hIn this study, a specific protein label free quantitative phosphorylation analysis method combining immuno-precipitation, IMAC phosphopeptide enrichment technique and mass spectrometric label-free relative quantitation was used to achieve comprehensive phosphorylation analysis of transgelin2 under any given conditions. While only two phosphorylation sites of transgelin2 were investigated in large-scale quantitative phosphorylation analyses of immune cells, five phosphorylation sites of transgelin2 were quantitatively analysed in this study. Reproducibility of the entire sample preparation procedure was first systematically evaluated. Our method was then used to investigate the phosphorylation changes of transgelin2 overexpressed in Jurkat T cells under two different kinase activation conditions. Jurkat T cells are a well-characterized model for studying immune response signaling. Protein kinase A (PKA) and protein kinase C (PKC) activation conditions were chosen in this study as the two representative serine/threonine kinase cascades of immune response homeostasis and activation, respectively ,21. We hConventional phosphatase inhibitor treatments of the cell lysates such as phosphatase inhibitor cocktails did not effectively improve the identification of transgelin2 Ser/Thr phosphorylation sites. Only one or two phosphorylation sites were typically identified. To increase the number of transgelin2 phosphorylation sites identified in the quantitative phosphorylation analysis, various phosphatase inhibitors such as calyculin A and okadaic acid were tested. Among the phosphatase inhibitors tested in our laboratory, calyculin A treatment exhibited the most significant changes in terms of the total numbers of identified phosphorylation sites and phosphopeptides. To show the phosphatase inhibitory effects of calyculin A, Jurkat T cells overexpressing GFP-tagged transgelin2 were treated with calyculin A for 30 min, okadaic acid for 30 min or untreated prior to cell harvest. These three cell samples were lysed separately with lysis buffers containing conventional phosphatase inhibitors, followed by immuno-precipitations with anti-GFP antibodies. The immuno-purified GFP-tagged transgelin2 samples were then subjected to SDS-PAGE and the resulting SDS-PAGE gel was sequentially stained with Pro-Q Diamond and Coomassie. The Coomassie staining results showed one major protein band that matched the molecular weight of GFP-tagged transgelin2, and the intensities of this protein band were not noticeably different among the untreated and two phosphatase-treated conditions Figure A. In con2 values ranged from 0.9851 to 0.9982.To demonstrate our reproducible phosphopeptide sample preparation procedure, three aliquots of cell lysates containing 5 mg proteins were independently subjected to immuno-precipitation, SDS-PAGE, in-gel digestion, and the IMAC phosphopeptide-enrichment processes. Table 183GAS*QAGMoxTGYGMPR196 and 183GAS*QAGMTGYGMoxPR196, whose only difference consisted of the location of the oxidized methionine, were clearly separated in the RPLC separation and unambiguously identified  of smooth muscle calponin1 (CNN1) is more important with regard to the actin-binding ability than the previously reported actin-binding site (ABS: aa142-163) [4-203 of Our specific protein label-free phosphorylation quantitative analysis revealed distinct phosphorylation changes of transgelin2 in Jurkat T cell lines under the PKC and PKA activation conditions, which are comparable to immune cell activation and homeostasis conditions, respectively. Among the six transgelin2 phosphorylation sites identified in this study, four phosphorylation sites were located in three potential actin-binding regions: the ABS, the calponin homology domain (CH) and the CNR1. Notably, phosphorylation sites located in two actin-binding regions demonstrate specific kinase-dependent phosphorylation changes. Threonine-180 and serine-185, which are located in the CNR, exhibited significantly increased phosphorylation exclusively under the PKC activation conditions. Serine-163, which is located in the ABS, exhibited solely PKA-dependent phosphorylation changes. The direct involvement of these two regions during actin binding has been previously demonstrated by functional analyses of transgelin2 homologues. Because the CNR was more important than the ABS with regard to the actin binding, substantial phosphorylation changes of the CNR under PKC activation conditions could effectively attenuate any interactions between transgelin2 and cytoskeletal actin. These results suggest that different actin binding capabilities or two conflicting cellular functions of transgelin2 may be meticulously controlled by specific kinase cascades activated under immune response activation or immune response homeostasis conditions. Finally, our specific protein quantitative phosphorylation analysis method which can monitor the phosphorylation changes of individual phosphorylation sites under any specific kinase activation conditions is especially useful for functional validation of protein phosphorylation because more than one type of protein kinases is likely to be involved in regulating the functional state of a target protein.HPLC grade acetonitrile (ACN), methanol, water and BCA Protein assay kit were purchased from Fisher Scientific . Fetal bovine serum (FBS), Geneticin (G418) and RPMI medium 1640 were purchased from GIBCO . Pro-Q Diamond phosphoprotein gel staining kit was purchased from Molecular Probes . Rabbit polyclonal anti-GFP was developed in rabbit by using purified recombinant full-length GFP protein (AbFrontier). All other chemicals were obtained from Sigma .Jurkat T cells were transiently transfected with cDNAs encoding GFP-tagged transgelin2 by Nucleofector Kit V (Amaxa) according to manufacturer’s instructions and then selected with 1 mg/mL Geneticin. Jurkat T cells  transfected with GFP-tagged transgelin2 were grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C. Jurkat T cells expressing GFP-tagged transgelin2 were grown to confluence and then subjected to serum deprivation for 18 h. These cells were subjected to each of the following treatment conditions. No-activation condition samples were untreated or treated with 100 nM calyculin A (LC Laboratories), or 1 μM okadaic acid for 30 min prior to cell harvest. PKA activation condition was induced by 100 nM forskolin and PKC activation was induced by 200 nM phorbol 12-myristate 13-acetate (PMA) for 30 min. Two protein kinase activation condition samples were then treated with 100 nM calyculin A for 30 min prior to cell harvest.No activation and kinase activation (PKA or PKC) cells were washed and harvested with cold phosphate buffered saline (PBS). The pellets were resuspended in lysis buffer  with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). The lysates were centrifuged at 13000 rpm for 10 min. The supernatant was transferred to a new tube. For immune-precipitation experiments, the immune complexes containing 5 mg proteins and anti-GFP conjugated Sepharose 4B  were gently mixed for overnight at 4°C and then washed twice with lysis buffer. Eluted proteins by heating of the beads at 95°C for 10 min in Laemmli SDS-PAGE sample buffer were separated by 10% SDS-PAGE. For phospho-specific fluorescent staining, all gels were treated with fixation solution (3 × 30 min), washed with deionized water (3 × 10 min), and stained with Pro-Q Diamond staining solution for 90 min. After Pro-Q Diamond staining, gels were imaged using a FLA-7000 imaging analyzer (Fujifilm). Following scanning of Pro-Q Diamond-stained gels, the gels were also stained with Coomassie Brilliant Blue (CBB) R250. The gel pieces were subjected to in-gel digestion as described previously . For lin4H2PO4) and the resulting phosphopeptide samples were analyzed by micro RPLC-MS/MS. Micro RPLC-MS/MS analyses were carried out only once for each phosphopeptide-enriched sample to show reproducibility of our label-free quantitative phosphorylation analysis.A fused-silica capillary  was packed with 3 cm of 5 μm 120 Å ReproSil Pur Aqua C18 (Phenomenex) using a high-pressure Bomb for desalting purposes. The tryptic digest samples were loaded onto the column at a flow rate of 2 μL/min. The column was washed with 1 mL of 1% formic acid and 120 μL of IMAC binding buffer  was used to elute peptides. PHOS-Select iron affinity gel (15 μL of 50% beads slurry) was incubated with the desalted peptides for 1 hr at room temperature. After beads were washed with 1 mL of the IMAC binding buffer, bound phosphopeptides were eluted using 200 μL of IMAC elution buffer  pump (Agilent Technologies) coupled to a linear ion trap mass spectrometer  with an in-house manufactured nano-ESI interface. For micro RPLC-MS/MS analysis, samples were injected into a trap column  and separated with an analytical column . Buffer A (0.1% FA) and Buffer B  were used to elute bound peptides with a split flow system (flow rate: 250 nL/min) for 120 min linear gradient. In a positive ion mode, spectra were acquired with cycles of one full MS scan in the LTQ (m/z 400–2000) followed by 10 data-dependent MS/MS scans with normalized collision energy of 35% and dynamic exclusion time of 30 s.t test was used for statistical analysis between no-activation condition and two kinase activation condition samples.MS/MS spectra were searched against in-house database containing various transgelin2 homologues, GFP, IgG sequences and bovine alpha casein sequences using SEQUEST algorithm (Bioworks 3.2). Methionine oxidation and phosphorylation of serine, threonine, and tyrosine as variable modifications and carbamidomethylation of cysteine as a fixed modification were applied to the search. DTAselect (v.1.9) was used to filter the search results with the following criteria: fully tryptic digest ends, Xcorr > 1.8 for charge state 1+, Xcorr > 2.5 for charge state 2+ and Xcorr > 3.5 for charge state 3+. Assignments of the phosphopeptide sequences were further confirmed by manual validations on filtered MS/MS spectra. For the quantification of phosphorylation levels, selected ion chromatograms of identified phosphopeptides were constructed using Xcalibur 2.1.0 SP1 program (Thermo) and integrated peak areas were then calculated with a built-in feature of Xcalibur program for comparison purposes. Relative quantification of each phosphopeptide was performed by comparing peak-areas of no-activation condition and those of two kinase activation condition samples. Student’s