Rosa damascena Mill.) is one of the most high-value essential oil-bearing plants in the world. However, the flower yield and quality of essential oil of R. damascena are largely influenced by the pruning practices and balanced supply of plant nutrition. The objective of this study was to test the hypothesis whether the pruning system and foliar fertilization of MgSO4 would influence the flower yield, growth and secondary metabolites profile of R. damascena. A field experiment of 10 treatment combinations comprising two pruning systems  and five levels of MgSO4  was conducted. The experiment was conducted in randomized block design with factorial arrangement. Overall, the flower yield ranged from 503.66 to 1114.47 g bush-1, while oil content varied from 0.039 to 0.046% of the fresh flower. Irrespective of foliar spray, partial pruning produced significantly (P ≤ 0.05) higher flower yield (893.02 and 503.66 g bush-1) compared with complete pruning system in both the years. Regardless of pruning system, the foliar application of MgSO4 @ 15.0g L-1 registered about 26–38% higher flower yield compared with water spray control. The major constituents of essential oil were citronellol (19.75–48.88%), E-geraniol (9.63–29.6%), Z-citral (0.07–5.97%), nonadecane (6.76–22.32%), and heneicosane (2.87–10.21%). The principal component analysis revealed that the major hydrocarbons such as nonadecene, nonadecane, and heptadecane are positively and highly correlated with each others. The results suggest that higher yield and quality of R. damascena can be achieved through partial pruning system in combination with foliar application MgSO4 under rainfed acidic conditions.Damask rose ( Rosa damascena Mill.), a perennial shrub of the Rosaceae family, is widely known for its high-value essential oil content in the flower. Though R. damascena is being commercially cultivated in different parts of the world , the rainfall is erratic, and 70–80% of the total rain is received during monsoon (June–September); but the gentle winter rain is useful for crop productions. Now there is a pressing need for enhancing crop productivity under rainfed conditions. However, the efficiency of plant nutrients applied in the soil is low under rainfed conditions. Moreover, unpredicted rainfall also makes difficult to determine the level and timing of fertilizer application.R damascena. Among the essential plant nutrients, magnesium (Mg2+) is one of the important secondary nutrients, which occurs in the center of the chlorophyll molecule and therefore plays a major role in plant photosynthesis . Thus, nutrient use efficiency (NUE) is very low under this situation. Furthermore, under rainfed conditions, application of nutrient in soil cannot meet the internal demand during critical stages. The foliar application of plant nutrients is an alternative approach to increase the NUE and to meet the internal demand under these conditions. Under nutrient deficiency condition, the foliar application technique ensures instant translocation of nutrients to various plant parts through leaf tissues . The soil of experimental plot was silty clay in texture, and the reaction of the soil was acidic. The physico-chemical properties of the experimental soil are presented in Table 1.The field experiment was conducted at the experimental farm of CSIR-Institute of Himalayan Bioresource Technology , Palampur, India, during the cropping seasons of 2012–2013 and 2013–2014. According to the USDA soil taxonomy classification system, the soil of experimental area belongs to Alfisols . The expR. damascena  field was used, and the planting geometry was 1.5 m between rows and 0.75 m within rows. A basal dose of 100 kg nitrogen (N), 21.85 kg phosphorus (P), and 41.50 kg potassium (K) was applied by urea (46% N), single super phosphate (16% P2O5), and muriate of potash (60% K2O), respectively, during both the cropping seasons. Irrigation was not given during the course of study, since the crop was grown under rain-fed conditions. However, other recommended agronomic practices for R. damascena were adopted as per requirement for better growth and development. The experiment was laid out in randomized block design (RBD) with two-factors factorial arrangement and three replications. Ten treatment combinations consisting two different types of pruning [complete pruning (C) and partial pruning (P)] and five different concentrations of MgSO4  were tested. The pruning operation was done during 50th meteorological standard week (MSW) at 90 cm height from the ground level in 2012–2013 and 2013–2014. In case of partial pruning system, five new shoots were left without pruning in each bush, and the remaining shoots were pruned at 90 cm height from the ground level. The MgSO4 solutions for different treatments were diluted with water (about 800 L ha-1), and sprayed twice; the first foliar spray was applied at axillary bud development stage, and the second spray was done at flower bud appearance stage.In this study, 5-year-old plantation of -1) was recorded. New shoot initiation rate (No. old shoot-1) was also recorded. The data on number of flowers (No. shoot-1), flower weight (g flower-1), flower yield (g new shoot-1 and g bush-1) were recorded day-to-day basis from initial date of harvesting to end of the flowering. The flowers were harvested by manual picking in the morning (6:00–9:00 AM) to prevent the loss of volatile compounds from the flower.Two plants were randomly selected for each treatment from each replication, and the selected plants were tagged for growth and yield observation. After pruning, number of old shoots (No. bush2 SO4 (Merck) and collected in a glass vial. The sealed oil samples were stored in a dark place at 4°C until analysis.The essential oil was extracted from fresh flowers harvested separately from each plot. The oil was extracted by hydro-distillation for 4 h on a Clevenger-type apparatus using a 5.0 L distillation system. The flower and water ratio was 1:2 (w/v). The essential oil from each plot sample was measured, and the oil content (w/w) in flower was expressed as percentage on a fresh weight basis. The extracted essential oil was dehydrated by anhydrous NaThe compounds of oil samples were identified by using a Shimadzu QP2010 GC-MS system  attached with an AOC-5000 auto injector and a ZB-5  fused silica capillary column . The conditions for analysis were identical to those previously described . The ret-1. Then the individual compounds were quantified based on peak area percentage of the chromatogram.All the GC analyses of rose oil samples were carried out by a Shimadzu GC-2010 gas chromatograph  equipped with flame ionization detector (FID) and a ZB-5 capillary column . The operating conditions for analysis were identical to those previously described . The nit-1 tissue) was calculated based on the absorbance values as per standard equations . The absorbance of the extracts at 645 and 663 nm was recorded with a spectrophotometer . Finally, the total Chl content . Then, Kel Plus nitrogen analyzer unit was used for estimation of total N content in the leaves. In case of P and K estimation, a mixture of concentrated H2SO4 and perchloric acid (5:1) was used for digestion. Then, a spectrophotometer  and a flame photometer  were used for the estimation of total P and K, respectively, as per standard procedure  to test the sole effect of pruning system and foliar application of MgSO4, and pruning system × MgSO4 interaction by using Statistica 7 software . In this experiment, a two-factor factorial RBD was used with three replications. Differences among the treatment means were assessed by the least significant difference (LSD) value at P = 0.05. Correlation matrix was conducted by using Statistica 7 software to investigate relationships between the yield and yield attributes. The regression equation between yield and MgSO4 doses was also developed using same software. However, the heat maps of chemical profiling of essential oil were prepared with the help of R data analysis software (version 3.1.3). Principal component analysis (PCA) was performed to evaluate the influences of treatment combinations on chemical profiling of essential oil as a bi-plot, and nature of variations among the treatment combinations was also projected. The factor loading values represent the correlations of each variable with the principal components (PCs).The growth and yield data obtained from R. damascena, new shoot initiation rate (No. old shoot-1) and number of flowers (No. bush-1), were significantly (P ≤ 0.05) influenced by the system of pruning during both the years (Table 2). In this study, partial pruning system registered significantly (P ≤ 0.05) higher new shoot initiation rate (4.63 and 10.80 No. old shoot-1) and number of flowers (350.47 and 266.80 No. bush-1) compared with the complete system, irrespective of foliar application of MgSO4. However, number of petals (No. flower-1) and flower weight (g flower-1) were not influenced by the system of pruning during both the years, and these two parameters remained inconsistent over the years.The analyzed data revealed that two main yield attributes of 4 on new shoot initiation rate (No. old shoot-1), number of petals (No. flower-1), and flower weight (g flower-1) were not significant (P ≥ 0.05). However, number of flowers (no. bush-1), the important yield component of R. damascena, was significantly (P ≤ 0.05) influenced by the foliar application of MgSO4; and maximum numbers of flowers (357.50 and 250.67 no. bush-1) were recorded with MgSO4 @ 15.0g L-1 (Table 2). Irrespective of system of pruning, the application of MgSO4 @ 15.0g L-1 registered about 30 and 36% higher number of flowers compared with water spray control during 2012–2013 and 2013–2014, respectively. The percentage of blind shoots was also significantly (P ≤ 0.05) affected by the system of pruning during 2013–2014 cropping season; however, the effect of foliar application of MgSO4 on blind shoot (%) was insignificant, least percentages of blind shoot (14.15 and 20.71%) were recorded with the foliar application of MgSO4 @ 10.0g L-1 during both the years. The interaction effects of pruning system and foliar application of MgSO4 on blind shoot were significant (P ≤ 0.05) during 2012–2013 (Table 2).The effects of foliar application of MgSO4 on the flower yield (g bush-1) of R. damascena were significant (P ≤ 0.05) in both the years (Table 2). Regardless of foliar application of MgSO4, the partial pruning system increased flower yield by about 23 and 75% compared with complete pruning system during 2012–2013 and 2013–2014, respectively. Among the foliar treatments of MgSO4, the application of MgSO4 @ 15.0g L-1 recorded significantly (P ≤ 0.05) higher flower yield (1114.47 and 830.69 g bush-1) compared with water spray control during both the years (Table 2). Though the effects of the foliar application of MgSO4 @ 5.0g L-1, MgSO4 @ 10.0g L-1, and MgSO4 @ 15.0g L-1 on flower yield were statistically at par (P ≤ 0.05) in 2012–2013; significantly (P ≤ 0.05) higher flower yield was recorded with MgSO4 @ 15.0g L-1 compared with MgSO4 @ 5.0g L-1 and MgSO4 @ 10.0g L-1 in 2013–2014. Regardless pruning system, the foliar application of MgSO4 @ 15.0g L-1 registered about 26 and 38% higher flower yield compared with water spray control in 2012–2013 and 2013–2014, respectively. The interaction effect between pruning system and foliar application of MgSO4 on the flower yield (g bush-1) was significant (P ≤ 0.05) in 2012–2013 cropping season.The analyzed data revealed that the effects of pruning system and foliar application of MgSO4 doses as independent variable and flower yield and number of flowers as dependent variable . In this experiment, the flower yield was increased with the corresponding increasing concentration of MgSO4 doses up to 15.0 g L-1, thereafter the yield was declined. Thus, the second degree polynomial relationship  was found between flower yield and level of MgSO4 doses . The similar relation was found between number of flower and concentration of MgSO4 doses with an equation of y = 223.41 + 8.123 x - 0.275 x2, and R2 = 0.774. The correlation matrix among the flower yield, yield attributes, and growth parameter was also established, and the data revealed that flower yield (g bush-1) was significantly and positively correlated with the number of flower  and new shoot initiation rate  . However, a negative correlation was observed with the percentage of blind shoot . The negative correlation  was also found between the percentage of blind shoot and new shoot initiation rate. A significant (P ≤ 0.01) and a positive correlation (r = 0.78) was found between the flower weight (g flower-1) and number of petal .The regression equations were established considering MgSOP ≥ 0.05) of pruning system and foliar application of MgSO4 on the essential oil content (%) in the flowers of R. damascena during both the cropping seasons . However, regardless of foliar spray of MgSO4, complete pruning system registered slightly higher oil content (0.046 and 0.042%) compared with the partial pruning system. Among the foliar treatments, the applications of MgSO4@ 5.0g L-1, @ 10.0g L-1, and @ 15.0g L-1 registered highest oil content (0.046%) in 2012–2013 cropping seasons irrespective of pruning system . The lowest oil content (0.040 and 0.044%) was recorded with water spray control and higher doses of MgSO4 (@ 20.0g L-1) in both the years.In our study there was no significant effect . The lowest numbers (26 and 23) of compounds were identified with the interaction effects of partial pruning system and water spray control treatment during both the years. However, the maximum contribution (97.25 and 97.54%) by the identified compounds in total volume of essential oil was observed with interactive effects of complete pruning system and foliar application of MgSO4 @ 20.0g L-1 and water spray.In this experiment, we have identified a total of 33 compounds in 2012–2013, which contributed about 93–98% of the total volume; whereas 30 compounds were identified in 2013–2014 . The heat maps containing 33 and 30 compounds which depict the changes of chemical profiling of rose essential oil were observed due to pruning system and foliar application of MgSO4. The data in the heat map showed that the accumulation patterns of two major monoterpenoids, citronellol, and E-geraniol, were inconsistent over the years . However, the maximum shearing of citronellol (39.82%) and E-geraniol (29.68%) was observed with PM0  and PM3 , respectively, in 2012–2013 cropping seasons . In 2013–2014, CM4 (complete pruning with foliar application of MgSO4 @ 20.0g L-1) and PM3  registered maximum quantity of citronellol (48.88%) and E-geraniol (24.84%), respectively . Flowers obtained from the PM2  treatment registered highest concentration (1.27 and 1.45%) of linalool during both the years. In the present investigation, the two major hydrocarbons, nonadecane, and heneicosane, also show diverse accumulation patterns under different treatment combinations in both the years . The minimal level of nonadecane accumulation was recorded with CM1 (complete pruning with foliar application of MgSO4 @ 5.0g L-1) followed by PM2  in 2012–2013 season, whereas in second cropping season, the minimum shearing of nonadecane was recorded with higher concentration of MgSO4 @ 20.0g L-1) under both types of pruning system .The chemical profiles of rose essential oil under different treatment combinations are presented by heat maps  during 2012–2013; however, partial pruning system registered significantly (P ≤ 0.05) higher Chl content (3.58 mg g-1) in 2013–2014. Although the effect of foliar application of MgSO4 on total Chl content in leaves was insignificant (P ≥ 0.05) in both the years, the Chl concentration was gradually increased with corresponding increasing concentration of MgSO4 and the utmost value (3.07 and 3.45 mg g-1) was attained with MgSO4 @ 15.0g L-1 during both the years. We also observed that the accumulation of N, P, and K in the leaf was significantly (P ≤ 0.05) influenced by pruning system, and the maximum values were registered with the partial pruning system during 2013–2014 cropping season. On the other hand, irrespective of pruning system, the effects of foliar application of MgSO4 on the accumulation of N and P were insignificant (P ≥ 0.05) in both the years. However, K concentration in leaf was significantly (P ≤ 0.05) influenced by the foliar application of MgSO4 in 2012–2013 cropping season, and the maximum value was recorded with water spray control. The Mg concentration in leaves was not influenced by the system of pruning during both the years. However, the effect of foliar application of MgSO4 on Mg accumulation in leaves was significant (P ≤ 0.05) during 2012–2013 cropping season and the maximum value was observed with MgSO4 @ 15.0g L-1. In case of interaction effect between pruning system and foliar application of MgSO4, the insignificant (P ≥ 0.05) results were found in both the cropping seasons (Table 3).The results presented in the 1) and second component (PC2) jointly explained 83.25 and 68.24% of the total variations for 2012–2013 and 2013–2014 cropping seasons, respectively . The relationships among the variables in the space of the first two components (PC1 and PC2) with factor loadings are presented in Figures 6A,C, and indicate how each variable contributes to the PCs. In this experiment, PC1 has positive coefficients with α-pinene, β-pinene, myrcene, linalool, phenyl ethyl acetate, citronellol, E-geraniol, eugenol, geranyl, acetate, methyl eugenol, and α-guaiene in first cropping season . However, only four compounds  have positive coefficients with PC1 in 2013–2014 . On the other hand, highly negative loading values were found with heptadecane (-0.97 and -0.82), octadecane (-0.98 and -0.84), nonadecene (-0.92 and -0.89), nonadecane (-0.97 and -0.97), heneicosane (-0.95 and (-0.94), and tricosane (-0.92 and -0.71) in both the cropping seasons .The principal component analyses (PCA) were performed by using the sets of 20 and 17 compounds of essential oil for 2012–2013 and 2013–2014, respectively. The components, which are quantified in all the treatments, are used for PCA. The results from PCA revealed that the first component  and PM1  were separated by the PC1 from the rest of the treatments and placed in the negative end of the PC1 in 2012–2013 . These two treatments were separated from each other by PC2. In 2013–2014, the treatments PM2 , PM3 , and CM4 (complete pruning with foliar application of MgSO4 @ 20.0g L-1) were separated from rest of the treatments, and they formed individual group .The PCA bi-plots also indicated that there were three and four distinct groups among the treatment combinations during 2012–2013 and 2013–2014, respectively. The treatment PM-1) was significantly (P ≤ 0.05) higher with partial pruning system compared with the complete system. This result might be due to larger number of dormant vegetative buds present in partially pruned bushes. Moreover, partial pruning maintains a sequence of axes leading from leaves to stem and root system for allocation of photosynthates  was not significantly (P ≥ 0.05) affected by pruning system, the partial pruning system produced significantly (P ≤ 0.05) higher flower yield compared with complete pruning system, regardless of foliar spray. This result might be attributed to the cumulative effects on higher new shoot initiation rate and number of flower (No. bush-1) which ultimately enhanced flower yield. The flower yield is positively correlated with new shoot initiation rate  and number of flower  . It had been reported that partial pruning increased relative water content (RWC) and maintained higher photosynthesis process and nutrient supply through root profile (Table 2). These results could be due to the fact that the environmental conditions, particularly temperature and rainfall were not favorable during vegetative growth phase and reproductive stage . The maximum and minimum temperature (°C) at flower bud formation and flowering stages in 2013–2014 were quite low compared with 2013–2014 cropping season . However, the effect of partial pruning system to increase flower yield was more pronounced in second cropping season (Table 2). The higher flower yield with partial pruning system might be attributed to the higher level of photosynthetic pigment and N content in leaves (Table 3). Though the overall yield in first cropping season was higher, the yield increased by the foliar application of MgSO4 @ 15.0g L-1 was more consistent over the years.Though the flower weight . In this study, regardless of pruning system, the maximum number of flower (357.50 and 250.67 no. bush-1) and flower yield (1114.47 and 830.69 g bush-1) were recorded with the foliar application of MgSO4 @ 15.0g L-1 during both the cropping seasons. This result may be due to the fact that the foliar application of MgSO4 under rainfed conditions increases the availability of Mg for formation of photosynthetic pigment and hastens physiological activities for flower buds formation. The effect of Mg on the various chloroplast enzymes has been reported by 2; R2 = 0.752) between flower yield and level of MgSO4 doses . In our experiment, total Chl concentration and nitrogen content in the leaves were higher in all MgSO4 treated plants (Table 3). This result may be a cause to increase the number of flowers and flower yield in MgSO4 treated plots in the present study.On the other hand, Mgt health . Neverth4 also considerably increased the total Chl content in leaves in our study, regardless of pruning system. These results may be due to the fact that the Mg is the central atom of the chlorophyll molecule, hence plays a major role in plant photosynthesis  with partial pruning system (Table 3). These results could be due to the fact that partial pruning increased the root proliferation, in which root uptakes more nutrients from larger area of greater depth. The results are in conformity with the findings of 4 treated plants compared with water spray control. These results could be due to the fact that the Mg deficiency prevents uptake of mineral nutrients under rainfed acidic soil conditions. Thus, foliar application of Mg is the effective measure to increase the nutrient uptake pattern in these situations.The concentrations of N and K in the leaves were not influenced by pruning system in 2012–2013 cropping season. These results could be due to the dilution effect of nutrient content. However, in 2013–2014, the N, P, and K accumulations in leaves were significantly increased . Among the foliar spray, moderate level of MgSO4 registered little bit higher oil content. This result may be due to the fact that Mg and its counter ion sulfur influence various biochemical activities. The effects of sulfur to increase the essential oil content have been reported in dragonhead plants , the shearing percentage in total volume was quite high. Thus, the numbers of compounds are not responsible for contributing in total volume of rose essential oil.The essential oil yield of d plants  and basid plants . It had  vulgare . The maj vulgare . Though,4 in both the years . The accumulation patterns of different volatiles were very dynamics, with citronellol varying from 19.75 to 48.88%, E-geraniol from 9.63 to 29.68%, Z-citral from 0.07 to 5.97%, nonadecane from 6.76 to 22.32, and heneicosane from 2.87 to 10.21%. Two major monoterpenoids citronellol and E-geraniol, attained its maximum level of accumulation with PM0 and PM3, respectively, in 2012–2013 . However, the minimal accumulation of nonadecane was recorded with CM1 followed by PM2. These results may be due to the fact that the pruning system coupled with foliar application of MgSO4 influences the biosynthesis of various compounds. The influences of foliar application of plant nutrients to change the percentages of major components of essential oils have been reported in many medicinal and aromatic plants such as oregano . The analyzed data indicates that the compounds viz., α-pinene, β-pinene, myrcene, linalool, phenyl ethyl acetate, and E-geraniol have highly positive coefficient with PC1 in first cropping season. Thus, these compounds are influenced by the similar factors. On the other hand major hydrocarbons such as nonadecene, nonadecane, and heptadecane are positively and highly correlated with each others. Thus the inverse relationship was found between hydrocarbons and monoterpene.In this study, the major components were considerably influenced by the interactive effect of pruning system and foliar application of MgSO oregano , French  oregano , and lem oregano . The mul4 alter the flowering behavior, flower and essential oil yield, and profiling of secondary metabolites of R. damascena under rainfed acidic conditions. The partial pruning system produced significantly (P ≤ 0.05) higher flower yield compared with complete pruning system, regardless of foliar spray. However, the effect of pruning system on flower yield was not consistent over the years. On the other hand, the foliar application of MgSO4 @ 15.0g L-1 registered about 26–38% higher flower yield compared with water spray control. The effects of partial pruning system and foliar application of MgSO4 to increase flower yield were more pronounced in second cropping season. Substantial variations in major compounds  of essential oil were also observed in this experiment. Thus, it can be concluded that the partial pruning system and foliar application of MgSO4 @ 15.0g L-1 may be adopted to increase the flower and oil yield with desired quality. However, further studies are required to understand the role of other factors particularly plant nutritions and environmental factors on enzymatic activities.The results reveal that the pruning system and foliar application of MgSOPP: Develop the concept, design the experiment, data analysis, and manuscript writing. MM: Data collection and chemical analysis.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.


There are no core outcome domain or measurement sets for Total Joint Replacement (TJR) clinical trials. Our objective was to achieve an International consensus by orthopaedic surgeons on the OMERACT core domain/area set for TJR clinical trials.We conducted surveys of two orthopaedic surgeon cohorts, which included (1) the leadership of international orthopaedic societies and surgeons , and (2) the members of the American Academy of Orthopaedic Surgeons’ Outcome Special Interest Group (AAOS-Outcome SIG), and/or the Outcome Research Interest Group of the Orthopaedic Research Society . Participants rated OMERACT-endorsed preliminary core area set for TJR clinical trials on a 1 to 9 scale, indicating 1–3 as domain of limited importance, 4–6 being important, but not critical, and 7–9 being critical.Eighteen survey participants from the IOS group and 69 participants from the AAOS-Outcome SIG/ORS groups completed the survey questionnaire. The median (interquartile range [IQR]) scores were seven or higher for all six proposed preliminary core areas/domains across both groups, IOS and AAOS-Outcome SIG/ORS, respectively: pain, 8  and 8 ; function, 8  and 8 ; patient satisfaction, 8  and 8 ; revision surgery, 7  and 8 ; adverse events, 7  and 7 ; and death, 7  and 8 . Respective median scores were lower for two additional optional domains: patient participation, 6.5  and 6 ; and cost, 6  and 6 .This study showed that two independent surveys dervied from three groups of orthopaedic surgeons with international representation endorsed a preliminary/draft OMERACT core domain/area set for Joint Replacement clinical trials. Total Joint Replacement (TJR) is one of the most common elective procedures performed in the US and worldwide. The rate of utilization of this highly cost effective and clinically successful procedure has, in part, been attributed to the increasing prevalence of osteoarthritis, obesity, and an aging population –3. DespiThe method of development of TJR trial core domain set was a multi-step, data-driven process that required a consensus by key stakeholders, including but not limited to orthopaedic surgeons and patients; step-by-step details are available in a previous publication . ConsensThe objective of the current study was to assess whether a broader orthopaedic community will engage in further development of the draft TJR trial core domain set, endorse or propose modification. We performed two separate surveys with three groups of orthopaedic surgeons to obtain a consensus on the draft TJR trial core domain set. We surveyed: (1) the present/past leadership of international orthopaedic societies (IOS) (survey 1); and (2) the members of the American Academy of Orthopaedic Surgeons’ Outcome Special Interest Group (AAOS-Outcome SIG) and the Outcome Research Interest Group of the Orthopaedic Research Society (ORS) at the annual meetings (survey 2), using each groups’ email list.We surveyed three groups of orthopaedic surgeons, key stakeholders for this consensus, by performing two separate surveys. These surgeons included not only clinicians, but also orthopaedic clinical trialists and leading total joint registry investigators. We met orthopaedic surgeons face to face at the AAOS meeting and subsequently obtained email lists for three groups of orthopaedic surgeons, categorized as follows for two independent surveys: (1) the present/past IOS leadership (survey 1); and (2) the members of the AAOS-Outcomes SIG and the Outcome Research Interest Group of the ORS (survey 2), who attended the respective meetings at the AAOS-2015.We pre-tested a survey questionnaire that queried the importance of six core domains/areas  and two optional areas  for reporting in joint replacement clinical trials. Ratings were on a 1–9 scale, 1–3 indicating domain of limited importance, 4–6 being important domain, and 7–9 being critically important domain. If both groups of orthopaedic surgeons were to rate each core domain “7–9”, i.e., critical, it would indicate complete consensus, and the TJR core domain set would be considered endorsed by orthopaedic surgeons at this stage. If a majority of the six core domains, but not all, were rated as critical by both orthopaedic surgeon groups, it would indicate incomplete consensus and would lead to discussion with surgeons regarding  whether we needed modification of the draft TJR trial core domain set. In case of lack of consensus, modification of core domain set would be done. A similar approach was planned for the future, in case of lack of complete consensus between surgeon and patient surveys for the draft TJR trial core domain set, leading either to its endorsement or modification (separate surveys of the same core domain set in patients)  Total Joint Replacement (TJR) Clinical Trial Draft Core Domain Set, submitted).Summary statistics are calculated for the two cohorts of survey participants. We calculated the median (interquartile range) ratings for each of the preliminary core domain/area within each group, the orthopaedic surgeon/registry leadership group and the AAOS-Outcome SIG/ORS orthopaedic surgeon groups.There were 18 survey participants from the IOS group and 69 participants from the AAOS-Outcomes SIG/ORS outcome groups who completed the questionnaire. Of the survey respondents, respectively, 100 and 78% were male, >90% in both were arthroplasty researcher/surgeons, and 67 and 64% were 55 years and older , and Functional Outcomes in Total Joint Replacement (FORCE-TJR). In the last few meetings, the OMERACT TJR WG has made significant progress in assembling a multi-stakeholder group and completed the literature reviews examining the frequency of reporting of various domains  in TJR clinical trials .At OMERACT 2014 TJR WG, a preliminary core domain set for TJR clinical trials was developed based on systematic literature reviews, the OMERACT filter 2.0, and two separate surveys at orthopaedic surgery meetings and the OMERACT pre-meeting . This prIn the current study, both groups of orthopaedic surgeons achieved consensus on the six TJR clinical trial core domains (scores of 7 or above) and did not propose any new core domains. These are very important findings, since these Delphi involved two groups of orthopaedic surgeons independent of those who had participated in the development of the preliminary TJR clinical trial core domain set. This is not surprising since this TJR core domain set was developed over several years after multiple systematic reviews and with multi-stakeholder input consisting of orthopaedic surgeons, patients who had undergone arthroplasty and methodologists who have an extensive experience with developing and validating core domain and measurement sets. The OMERACT’s philosophy of a multi-stakeholder involvement from the beginning of the process, and a data driven approach, are key to successfully achieving a broad stakeholder consensus on this draft TJR clinical trial core domain set. This evidence indicates that preliminary TJR core domain set initially developed with multi-stakeholder input and endorsed at OMERACT 2014 meeting was endorsed independently by orthopaedic surgeons.Core domain sets are those domains that should be  reported in every clinical trial for the condition, regardless of the intervention, as they are critical to the conceptualization of the condition/disease. One must not confuse a core domain with the primary outcome for each clinical trial, which is always specific to each research question. In some studies, core domain measures will be primary outcome, since they are the focus of the research question; in other studies, they may be secondary study outcomes. Our work indicates that this draft TJR core domain set is now ready for the next phase, namely, the identification and validation of measures that accurately reflect each core domain, and further dissemination of this TJR core domain set to key stakeholders.We received qualitative feedback from the surveys, which has helped us with further specification of two core domains. One suggestion was to consider any reoperation, not just revision, due to its relevance to patients- this indicates that the core domain would be revision/reoperation. Another suggestion was to specify whether death was procedure-related vs. not procedure-related. With further refinement of the core domains, these two proposals will be the subject of future WG discussions. It is interesting and reassuring that several of domains brought up one participant each  were considered previously. Based on Delphi and detailed discussions with surgeons, patients and other stakeholders, they were recommended not for inclusion in the core domain set, since they were not considered to represent an independent core domain, above and beyond the six domains already included.We noted that both groups independently rated the additional non-core domains  as important but not critical; the median scores were 6 to 6.5, appropriately lower than the core domains, since these are not core domains. Non-core domains are those domains that may be additionally reported in a TJR clinical trial, depending on the clinical question being asked.Our study findings must be interpreted with caution, since they may not be generalizable to other groups. Our orthopaedic surgeon samples were 78 and 100% male, which may limit the generalizability of the findings to all surgeons. The gender distribution among orthopaedic surgeons is not balanced in general. We developed this draft TJR core domain set for clinical trials; it is possible that with validation and future work, this set or a similar set may be endorsed for observational studies as well.Our study has several strengths, including use of the same survey in two groups of stakeholders, inclusion of both leaders of orthopaedic societies as well as the group of orthopaedic surgeons and researchers from community and academic practices.In conclusion, the current study shows further independent endorsement of the draft TJR core domain set for reporting of TJR clinical trials. The next steps in this process, are a wider endorsement of these by OMERACT and/or orthopaedic organizations that we have been collaborating with. This will be  followed by the development of a valid, data- and consensus-driven TJR clinical trial core outcome measurement set. Our group will aim to complete this step over the next 2 years. The process will be completed with multi-stakeholder input with a particular emphasis of involvement of patients and TJR surgeons.


The Tec kinases, closely related to Src family kinases, are essential for lymphocyte function in the adaptive immune system. Whilst the Src and Abl kinases are regulated by tail phosphorylation and N-terminal myristoylation respectively, the Tec kinases are notable for the absence of either regulatory element. We have found that the inactive conformations of the Tec kinase Itk and Src preferentially bind ADP over ATP, stabilising both proteins. We demonstrate that Itk adopts the same conformation as Src and that the autoinhibited conformation of Src is independent of its C-terminal tail. Allosteric activation of both Itk and Src depends critically on the disruption of a conserved hydrophobic stack that accompanies regulatory domain displacement. We show that a conformational switch permits the exchange of ADP for ATP, leading to efficient autophosphorylation and full activation. In summary, we propose a universal mechanism for the activation and autoinhibition of the Src and Tec kinases. The phosphorylated ITAMs recruit the tandem SH2 domain tyrosine kinases Syk and ZAP-70 in B- and T-cells respectively, which leads to activation of Tec family tyrosine kinases upon their PI3K-dependent recruitment4. Activated Btk and Itk phosphorylate phospholipase C-γ6, which triggers calcium flux in the cell by producing the second messenger inositol-1,4,5-trisphosphate (IP3). Dysregulation of Btk in humans causes the heritable disorder X-linked agammaglobulinemia7, characterised by a lack of circulating antibodies and a corresponding susceptibility to opportunistic infection. Itk dysfunction, on the other hand, specifically affects the development of T-cells and has been implicated in a number of lymphoproliferative disorders10, as well as playing a role in HIV replication and infectivity12.The Tec family kinases Btk and Itk are essential for B- and T-lymphocyte development in the adaptive immune system. Engagement of the B- or T-cell receptor triggers a cascade of intracellular phosphorylation events, initiated by phosphorylation of ITAM motifs in the cytoplasmic tails of activated receptors by Src family kinases13 and sharing in common a conserved SH3-SH2-kinase domain module. Src kinases are localised to the plasma membrane via lipid modification of their N-termini, whereas Tec kinases are regulated by the lipid second messenger PIP3, responding specifically via their N-terminal PH domain14. Src and Tec kinases are activated by engagement of intracellular ligands for their SH2 and SH3 domains, events that promote activation loop phosphorylation and downstream signalling17. Deletion of the PH domain or mutation of the phosphotyrosine-binding SH2 domain of Itk results in a loss of Itk activation18.Whilst the Tec and Src kinases are often referred to as separate families, they are in fact very closely related, belonging to the same branch of the tyrosine kinase phylogenetic tree21. While Src and Hck are proposed to be maintained in this conformation by intramolecular engagement of their phosphorylated C-terminal tail21 and N-terminal myristoylation of Abl stabilises a similar conformation19, the Tec kinases notably lack either feature, leading to proposals that the PH domain might fulfil this function23. However, the recent crystal structure of the SH3-SH2-kinase domains of Btk shows a similar domain arrangement to Src in the absence of both the PH domain and a C-terminal tail23. The interface between the regulatory and kinase domains is mediated by a variable linker between the SH2 and kinase domains that forms a network of interactions essential for maintaining the inactive state. Displacement of the SH3 domain of Hck activates the kinase by relieving these inhibitory constraints17 and promoting a conformational switch in the N-terminal end of the catalytic domain24.The structures of Src, Hck, and Abl kinases show a conserved intramolecular assembly of their regulatory SH3, SH2, and kinase domains that maintains the kinase in an autoinhibited conformation26. In the Tec kinases, activation loop phosphorylation in cells depends on the activity of upstream Src kinases30, presumably via recruitment and activation of the kinase by SH2 and SH3 domain-ligands. Like the Src kinases, the Tec kinases robustly autophosphorylate in vitro31. However, while the Src kinases have been extensively studied, the Tec kinases have been suggested to be mechanistically distinct, despite their evolutionary similarity.Activation loop autophosphorylation regulates the activity of Src tyrosine kinasesWe set out to determine the structure and mechanism of activation of the T-cell kinase Itk. To our surprise, we discovered that both Itk and Src bind ADP significantly stronger than the non-hydrolysable ATP analogue, AMPPNP. We found that activation loop phosphorylation or C-terminal tail deletion had no effect on the nucleotide binding preference or autoinhibited conformation of Src in solution. We describe the crystal structure of autoinhibited Src bound to ADP and use molecular dynamics to rationalise the binding preference for ADP. We show that Itk adopts a stable, globular conformation in the absence of its PH domain that is well described by the autoinhibited state of Src. We demonstrate the allosteric activation of both Itk and Src by mutation of a conserved hydrophobic stack between the regulatory and kinase domains, which converts the kinase from an ADP- to an ATP-binding conformation that undergoes robust auto-phosphorylation. In summary, we present a general mechanism for the allosteric activation and autoinhibition of both Src and Tec family tyrosine kinases.21. Subsequent structures of Src and Btk under different crystallisation conditions, however, have revealed an ordered conformation of the activation loop independent of nucleotide and Mg2+ binding32.Sequence and structural homology of Itk to Src Fig.  indicate32K, Src32K, and Src32KΔC, Fig. 32K binds ADP with a Kd of 23.7 ± 1.7 μM, while AMPPNP was estimated to bind with 15-fold lower affinity , in which the activation loop is fully ordered32  and a SH2 domain mutant of Src that is incapable of binding phosphotyrosine peptides  data on all four proteins in solution in the presence of ADP and magnesium .To address whether Itkium Fig. . The scable Fig. . The strpes Fig.  indicate32K , this protein adopts the same conformation as Src32K in solution  is reoriented to form a hydrogen bond between the amide nitrogen of its indole ring and the main chain carbonyl of Leu303, while Lys293 in the αC helix maintains a stacking interaction with Trp238 by reorienting its side chain; together, these interactions stabilise the active conformation of the αC helix.By contrast, active Lck, lacking its regulatory domains and SH2-kinase inter-domain linker, exhibits a collapsed hydrophobic stack  Figs , 3a. The32K as judged by a 10% increase in the radius of gyration and the maximum dimension of the particle . We could not fit the data with the structure of Src (χ2 = 35.29), so we turned to rigid body modelling of the individual domains to try to better fit the scattering curve. Allowing only the SH3 domain to move did not result in an acceptable fit, but by allowing both the SH2 and SH3 domains to move freely, we were able to fit the scattering with a reasonable chi-square  ItkADP Fig. . ActivatADP Fig. , perhaps32KΔC L255A). Due to limiting protein concentrations, we were unable to obtain a binding curve by fluorescence anisotropy, so we instead used thermal stability measurements to investigate its nucleotide-binding properties. Determination of accurate binding constants from thermal stability measurements requires knowledge of the enthalpy of unfolding. However, for a given protein, the enthalpy of unfolding is independent of the ligand being tested, so comparison of the concentrations of nucleotide required for half maximal stabilisation is valid43. In contrast to Src32K, Src32KΔC L255A binds ATP approximately three times better than ADP and is stabilised to a greater extent by ATP than ADP . However, disruption of the hydrophobic stack in Src results in an 11-fold activation of the kinase and very efficient autophosphorylation 38.Interestingly, Src lacking its C-terminal tail retains its binding preference for ADP, while solution X-ray scattering measurements indicate that Src53, though a more recent SAXS study showed that a point mutant in the pocket that prevents myristoyl binding does not lead to disassembly of the regulatory domains54. Furthermore, recent investigations of ancestral Src kinase in unicellular choanoflagellates and filastereans have revealed that, while the C-terminal tail is present, kinase activity is not suppressed by Csk56, suggesting that phosphorylation of the C-terminal tail was a later evolutionary adaptation.In the Abl kinases, which lack a C-terminal tail, docking of the myristoylated N-terminus to a hydrophobic pocket on the C-lobe of the kinase domain was proposed to stabilise the autoinhibited conformation61. Whilst the C-terminal tail of Src is of undoubted importance in the cell, we find that it has no effect on Src conformation and only a very modest effect on Src activity in vitro. Indeed, one can dispense with either the C-tail or the capacity to bind it, and Src still adopts the autoinhibited conformation. Therefore, although v-Src is transforming in cells, it is unlikely due to the intrinsic disassembly of its autoinhibited conformation. Indeed, the SH2 domain mutant of c-Src incapable of engaging its C-tail is not transforming63. Likewise, the Tec kinases Btk and Itk require a functional SH2 domain for their activation, despite lacking a C-tail64. Notably, while constitutively membrane localised Btk has transforming activity, mutation of its SH2 domain abrogates this activity64.All of these observations are contrasted by cellular studies demonstrating that deletion of the tail or mutation of its phosphorylation site leads to hyperactivation of Src and cellular transformation3 production. Consequently, Src kinases are, on average, exposed to a higher density of SH2 and SH3 domain ligands. In vitro, the phosphorylated C-terminal tail of Src inhibits engagement of the SH2 domain by exogenous ligands supplied in trans by a factor of ~20050. We therefore favour a mechanism in which the phosphorylated C-terminal tail acts as a competitive inhibitor of SH2 domain engagement with activating ligands in the cell, rather than a physical latch that maintains the closed conformation.A distinguishing feature of the Src kinases is the presence of a membrane anchor, in contrast to the Tec kinases, which are transiently recruited upon PIP65, its disruption does not appear to lead to a conformational switch in either the regulatory domains or the nucleotide binding site of the kinase domain, an observation consistent with a recent molecular dynamics study66.Another widely used proxy for Src activation is activation loop phosphorylation. We show, however, that activation loop phosphorylation of Src alone does not change its overall conformation or binding preference for ADP, indicating that the regulatory and kinase domains are still assembled in the autoinhibited conformation. Whilst the electrostatic network stabilising the inactive conformation of the activation loop is undoubtedly important67, mutation of the equivalent residue in Btk (Trp395) and Itk (Trp356) renders them inactive70. Whilst these apparently opposite observations have been used to argue that these kinases are mechanistically distinct, Trp395 of Btk is observed in the same, active conformation as Trp260 of Src and Trp238 of Lck, supporting the notion that they employ similar mechanisms to activate their respective kinase domains71. Recent molecular dynamics studies have also demonstrated the importance of the invariant tryptophan in stabilising the active conformation of both Src and Tec kinases72. The invariant tryptophan therefore fulfils a dual role in stabilising both the inactive and active conformations.The invariant tryptophan in the N-lobe of the kinase domain has previously been the focus of efforts to understand the allosteric activation of these kinases. While mutation of Trp260 in Src and Hck results in their hyperactivation73. Mutation of this residue has previously been shown to abrogate Csk mediated inhibition of Src in a yeast cell-based assay41. We observed a dramatic switch in affinity for ATP, which is accompanied by efficient activation loop autophosphorylation and attainment of full catalytic activity. That Src and Itk bind ATP in their active conformations is not surprising, but the selective binding of ADP by the inactive kinases has not been previously observed. We speculate that this may serve to prevent promiscuous kinase activity and restrict kinase activation exclusively to situations in which the regulatory domains, and specifically the SH3 domain, have been engaged by intracellular ligands. As such, one could regard the hydrophobic stack as the ultimate regulatory node that controls the activation of Src and Tec family kinases, irrespective of activation loop phosphorylation or C-terminal tail dephosphorylation.In order to avoid compromising the catalytic machinery in Src and Itk, we mutated a hydrophobic residue in the inter-domain linker that acts as a ‘molecular glue’ between the kinase and regulatory domains. Critically, this residue inserts its side chain into a hydrophobic stack on the back of the N-lobe of the kinase domain, which stabilises the inactive conformation of the kinase74. Whilst these structures are reminiscent of active Csk75, the SH2 domain actually adopts distinct positions and makes different contacts with the kinase N-lobe in each structure. It is therefore unclear how and to what extent the SH2 domain may modulate catalytic activity through the N-lobe in the various family members.Whilst mutation of the linker led to a dramatic increase in binding affinity for ATP, it resulted in a very modest conformational change in Itk, consistent with regulatory domain displacement, but not complete disassembly of the regulatory and kinase domains. A putative conformation of the mutant Itk shows the SH2 domain to be in close proximity to the N-lobe of the kinase domain, though we cannot be certain of the respective domain arrangement given the resolution limits of the SAXS data. Intriguingly, the SH2 domain of c-Abl was found to enhance kinase activity through an interaction with the kinase N-lobe that stabilises the active conformation of the αC-helix, a mechanism that was also demonstrated for Fes76 iterative cycles of substrate phospho-transfer will be ensured as long as the regulatory domains remain engaged with their cognate ligands. While the core catalytic machinery is conserved between Itk and Src, additional regulatory features restrict their activities. In the case of Itk, the N-terminal PH domain restricts activity to PIP3-containing membranes. In the case of Src, which has an N-terminal membrane anchor, we propose that its C-terminal tail prevents spurious activation in the context of a high density of potentially activating ligands at the membrane.Our data are consistent with the following model Fig. : In the In summary, we propose a general mechanism for the allosteric regulation of the Src and Tec kinases. We describe the basic unit of kinase control and show that the inactive state is bound and stabilised by ADP. Based on the strong sequence and structural homologies between the Src, Abl, Tec, Csk, and Fer kinases, we predict that the mechanisms of autoinhibition and kinase activation are also conserved. We propose that nature has evolved various sub-family specific adaptations, such as the C-terminal tail of Src or PH domain of Itk, that further refine the control and localisation of enzymatic activity.The following antibodies were used in this study: Src ; Src pY416 ; Itk ; Btk pY551 (Itk pY511) (BD Pharmingen clone 24a).171–620 (Itk32K), mouse Src85-535 (Src32K), and mouse Src85-523 (Src32KΔC) as well as their respective point mutants were expressed as GST fusions in baculovirus-infected Sf9 insect cells. For the generation of Tyr527-phosphorylated Src85-535, the protein was co-expressed with human Csk using pFastBac Dual (Invitrogen). Proteins were purified by sequential glutathione-sepharose affinity chromatography, TEV cleavage, high-resolution anion exchange, and size exclusion chromatography in a final buffer of 50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM TCEP, 1% (v/v) glycerol.Human Itk500 ng protein was dissolved in 20 mM Tris 7.6, 50 mM KCl, 1 mM TCEP and loaded on an Eksigent MicroLC column  using a Dionex Ultimate 3000 HPLC system (Thermo Scientific). The proteins were separated on a gradient from 5% to 90% acetonitrile in water and 0.1% formic acid for 30 min at a flow rate of 30 µL/min. Mass spectra were recorded on a Sciex TripleTOF 5600 instrument equipped with a Turbo V DuoSpray ion source in positive mode. Data was analysed in PeakView version 2.1 (Sciex) using the Bio Tool Kit to reconstruct the uncharged average protein mass. All other samples were diluted in 0.1% formic acid (FA) to a concentration of 10 ng/μL and 50 ng were loaded on an Aeris Widepore C4 column, 3.6 µm particle size, dimensions 2.1 × 150 mm (Phenomenex), using a Dionex Ultimate 3000 HPLC system (Thermo Scientific) with a working temperature of 55 °C, 0.1% FA as solvent A, 90% acetonitrile, 0.08% FA as solvent B. Proteins were separated in a 6 min gradient from 10 to 70% solvent B at a flow rate of 300 μL/min. Mass spectra were recorded on a Waters Synapt G2-Si equipped with a ZSpray ESI source. Data were analysed in MassLynx V 4.1 using the MaxEnt 1 process to reconstruct the uncharged average protein mass.2 with ADP, ATP, or AMPPNP concentrations ranging from 0–16 mM. Samples were measured in triplicate using a BioRad iQ™5 Multicolor Real-Time PCR Detection System. For detection of phosphorylated protein, samples were diluted and 1 ng and 15 ng were loaded to SDS-PAGE for Src and Itk proteins, respectively. Phosphorylation was measured by immunoblot.Thermal stabilities as a function of nucleotide concentration were measured by differential scanning fluorimetry (DSF). Samples contained 0.1 mg/ml protein in 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM TCEP, and 5 mM MgCl32K, ItkL351A or Src32K were incubated with 40 μM Mant-nucleotide in 30 mM Tris pH 7.5, 200 mM NaCl, 5 mM MgCl2, and 1 mM TCEP. Measurements were made with a Perkin Elmer LS50B fluorimeter  at 20 °C. For each data point 50–60 measurements, each with an integration time of 1 s, were averaged. For competition nucleotide displacement assays, 40–80 μM of protein was incubated with 40 μM Mant-ADP until equilibrium was established. Unlabelled nucleotide was titrated into the reaction and fluorescence anisotropy monitored. The equilibrium inhibition constant for the unlabelled nucleotide was calculated from the fitted curve.The binding constants for ADP and AMPPNP were determined for Src and Itk proteins by fluorescence anisotropy, using 2′/3′-Mant-labelled nucleotides (Jena Bioscience). Increasing concentrations of Itk2 on BioSAXS beamline BM29, ESRF. Data reduction and analysis was performed using the BsxCuBE data collection software and the ATSAS package77. Scattering data for Itk32K L351A and Src32K R175L were collected using an online size exclusion chromatography setup on BM29. The proteins were applied to a Superdex 200 column equilibrated in 20 mM Tris, pH 8.0, 100 mM NaCl, and 1 mM TCEP and images were acquired every second for the duration of the size exclusion run. Buffer subtraction was performed by averaging 50 frames either side of the peak. The program AutoGNOM78 was used to generate the pair distribution function (P(r)) for each isoform and to determine Dmax and Rg from the scattering curves (I(q) vs. q) in an automatic, unbiased manner. Ab initio molecular envelopes were computed by 20 iterative cycles of simulated annealing starting with a dummy atom model in DAMMIF79. The models were aligned, averaged, and filtered using DAMAVER80. The final models for Itk and Src were superimposed using SUPCOMB81. The theoretical scattering curve for Src was calculated from the PDB entry 2SRC using CRYSOL82. The agreement between the experimental scattering curves for Src32K, Itk32K, Src32KΔC and Src32K R175L was evaluated in terms of the chi-squared value using DATCMP. For Itk32K L351A, rigid body modelling was performed using CORAL77 with 2SRC.pdb Δ521–533 (C-tail) as the starting model. The rigid domains of Src were defined as follows: SH3 (84-139), SH2 (148–246), kinase (260-520). The linker residues were implemented in CORAL as dummy residues. Iterative runs of CORAL were performed in which either the SH3 domain alone or both the SH3 and SH2 domains were allowed to move, while the kinase domain was fixed.SAXS data were collected on 0.5–4 mg/ml samples of Itk or Src in 50 mM Tris, pH 7.6, 200 mM KCl, 1 mM TCEP, 1% (v/v) glycerol, 2 mM ADP, 2 mM MgCl32K at 2.6 mg/ml was co-crystallized with 2 mM ADP and 5 mM MgCl2 in 50 mM PIPES, pH 6.5, 1.2 M sodium tartrate, and 20 mM DTT. Crystals were cryoprotected in reservoir solution plus 22–24% glycerol and plunge frozen in liquid nitrogen. Data were collected at 100 K on ID30A3, ESRF, Grenoble. Crystals grew in space group P212121 as previously reported for the human apo-protein21, but with altered unit cell parameters in b and c . The structure was solved by molecular replacement using 2SRC as a search model. Residues 205–212 in a loop on the surface of the SH2 domain and residues 110–117 of the SH3 domain were slightly shifted from their position in 2SRC due to lattice contacts and were therefore rebuilt. The structure was refined by iterative cycles of simulated annealing all-atom refinement in refmac. ADP and a single magnesium ion were built into clear electron density in the nucleotide-binding site and the structure refined to a final Rfree = 26.8, R = 20.8 at 2.42 Å resolution.Src35, using the GROMOS 54A7 parameter set to describe molecular interactions36. Relative binding free energies between ADP and ATP were computed using extended thermodynamic integration37 from simulations of the ligands free in solution or bound to the protein. For a more detailed description of the simulation setup, the reader is referred to the supplementary material.Molecular dynamics simulations were performed using the GROMOS simulation package32K or Itk32K respectively in 50 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM TCEP, and 1 mM ATP and incubated at 30 °C. 5 μl samples were taken at each time point and loaded to SDS-PAGE. Phosphorylation was measured by immunoblot. Data were fitted in Origin using a sigmoidal function to model the slow first step (autophosphorylation by unphosphorylated kinase), the fast second step (trans-phosphorylation of unphosphorylated kinase by phosphorylated kinase), and the stationary phase in which substrate (the kinase itself) has been consumed (phosphorylated).Autophosphorylation reactions were performed in a total volume of 100 μl containing 67 nM or 1 μM Src2, and 0.05 mg/ml BSA. Reactions were incubated at RT for the specified time and luminescence was measured using a TECAN Infinite F500 plate reader.Kinase assays were performed using the ADP-Glo kit (Promega) according to the manufacturer’s instructions. Kinase reactions contained 500 nM purified kinase, 50 μM SRC substrate , and 1 mM ATP in 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM TCEP, 2 mM MgClCrystallographic coordinates have been deposited in the Protein Data Bank with accession code 6F3F.Supplementary Information


Here, we systematically seek to improve F49A-FTP by modifications in its three structural domains; we generated variants with (1) altered chemokine sequence , (2) shortened and elongated linker region, and (3) modified toxin domain. Only F49L-FTP displayed higher selectivity in its binding to US28 versus CX3CR1, the endogenous receptor for CX3CL1, but this was not matched by a more selective killing of US28-expressing cells. A longer linker and different toxin variants decreased US28 affinity and selective killing. Thereby, F49A-FTP represents the best candidate for HCMV treatment. Many viruses encode internalizing receptors suggesting that not only HCMV but also, for instance, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus may be targeted by FTPs. Immunotoxins as antiviral therapeutics are largely unexplored but have promising prospective due to their high selectivity potential and their unparalleled efficiency. One recent example targeted the virus-encoded G protein-coupled receptor US28 as a strategy for specific and efficient treatment of human cytomegalovirus (HCMV) infections. US28 is expressed on virus-infected cells and scavenge chemokines by rapid internalization. The chemokine-based fusion-toxin protein (FTP) consisted of a variant (F49A) of CX Pseudomonas exotoxin A (PE) is a highly toxic protein that has been used to generate several immunotoxins undergoing evaluation in clinical trials . In . In 3CL1x of 501 . Thus, Fthe FTPs , F49A-FT potency , Table 23CL1   . The FTP [41aa])  but had ]) ) between the chemokine domain of CXns of PE . The F49ns of PE ; Table 3 Figures . In summ3CR1-expressing cells compared to F49A-FTP  turned out to be the best FTP to control virus infections and superior selectivity in killing US28-expressing cells compared to all tested FTPs.As a final step of this study, we investigated if we could increase the antiviral activity of the FTP by fusion of the chemokine with the full catalytic active domains of PE  in variant 7, F49A-FTP-7 . This vaF49A-FTP  and onlyF49A-FTP . We furtF49A-FTP  and cons3CR1) of the chemokine part alone, which, after selection, was fused to the catalytic active domains of PE , in addition to an extra variant at position 49 (F49E) to create FTPs by fusion of the chemokine fragment with the Pseudomonas exotoxin fragments and compared their binding and killing properties to those of F49A-FTP. F49L-FTP had the highest selectivity index in terms of binding, yet due to an improved killing of CX3CR1 expressing cells and maintained high killing of US28-expressing cells, the selectivity in terms of killing was reduced. Thus, to create a highly US28-selective FTP as F49A-FTP, it is essential not only to determine its binding affinity to the target receptor but also to investigate its specific killing property. A change of the naturally occurring ligand-receptor complex can influence the molecular properties of the receptor, for example, the rate of receptor internalization or its intracellular trafficking, that is, important receptor features for toxin delivery.In this study, we generated novel FTPs with the attempt to improve the previously published F49A-FTP  in termsns of PE  7]. The. The3CR1: CX3CL1 . For CX3eceptors , 36. Impeceptors . For US2eceptors , 39. F493CL1 and also deletion of domain II of PE reduced the efficacy of the FTPs in killing US28-expressing cells compared to F49A-FTP. This is consistent with previous studies where eliminating the furin cleavage site by deletion or preventing cleavage with a point mutation in the sites reduced the cytotoxicity of a series of immunotoxins [To control HCMV infections, selective targeting of the infected cells is required, but also a high efficacy of the FTP to kill cells before the virus spreads is required. Therefore, we further investigated if we could increase the efficacy of F49A-FTP by modifying the linker region or the catalytic active domains of PE. Elongation of the linker region with parts of the mucin-like stalk of CXnotoxins .3CR1-expressing cells, the prototype F49A-FTP still presents the best candidate therapeutic to treat HCMV infections. To sum up, the presented rational strategy of modifying the chemokine domain, the linker region, and the cytotoxic domains did not improve the prototype FTP. However, as the general understanding of PE toxicity is incomplete, more knowledge is needed. Moreover, questions remain regarding the binding and action of F49A-FTP. So far, the binding of the CX3CL1 variants was tested in competition with CX3CL1, but it remains to be determined if the modified CX3CL1 domain of F49A-FTP can compete against the broad spectrum of CC-chemokines binding to US28, for instance, in an inflammatory situation. Moreover, it remains to be described how the CX3CL1-based FTPs act on cells expressing the membrane bound CX3CL1, as the soluble form of CX3CL1 has been shown to bind to transmembrane CX3CL1 with high affinity, that is, with the latter acting as a receptor that thereby influences the communication between cells [Besides changes in domain II, changes in the full catalytic domains of PE  increased the cytotoxicity and antiviral property of the FTP (F49A-FTP-7). However, the increased killing was observed in both US28- and CX3CR1-expressing cells, indicating a generally improved toxicity of the FTP, again consistent with previous studies . As seleen cells .Immunotoxins targeting virus-encoded receptors represent promising drugs, not only for HCMV therapy but also for other herpesviruses by targeting their virus-encoded GPCRs. EBV and KSHV infections can cause cancer and targeting of their GPCRs could be a novel anticancer treatment strategy. The broad-spectrum chemokine receptor ORF74 from KSHV thus seems highly suitable for immunotoxin targeting, as this receptor (1) can induce the onset of Kaposi's-like lesions , 43, (2)3CL1-based FTP targeting the HCMV-encoded GPCR US28 can be modified for highly effective killing of virus-infected cells by modifying the three structural domains of the FTP . By inserting single point mutations in the core domain of CX3CL1, the FTP loses affinity for CX3CR1, but not for the virus-encoded receptor US28. Changes in the linker region do not improve the activity of FTP, whereas changes in the catalytic active domains of PE increase the killing efficacy for US28-expressing cells and thereby the antiviral activity. Thus, CX3CL1-based FTP can be used as scaffold to create highly efficient and selective FTPs to control HCMV infections. As several other virus-exploited GPCRs have been described, the principle of antiviral therapy targeting these receptors may not be limited to US28 for the targeting of HCMV but may be expanded to the targeting of ORF74 for KSHV treatment and BILF1 and/or EBI2 for the treatment of EBV-mediated diseases.Immunotoxin based antiviral drugs offer a novel antiviral mechanism for combination therapy and for treating infections that have become resistant to the current first-line intervention. Here, we show that a CX


Coprinopsis cinerea, CcPDH, the founding member of the recently discovered auxiliary activity family AA12. CcPDH contains a C-terminal family 1 carbohydrate binding module (CBM1), an N-terminal family AA8 cytochrome domain, and a central AA12 dehydrogenase domain. We have studied the ability of full-length CcPDH and its truncated variants to drive catalysis by two Neurospora crassa LPMOs. The results show that CcPDH indeed can activate the C-1-oxidizing N. crassa LPMO 9F (NcLPMO9F) and the C-4-oxidizing Neurospora crassa LPMO 9C (NcLPMO9C), that this activation depends on the cytochrome domain, and that the dehydrogenase and the LPMO reactions are strongly coupled. The two tested CcPDH-LPMO systems showed quite different efficiencies, and this difference disappeared upon the addition of free PQQ acting as a diphenol/quinone redox mediator, showing that LPMOs differ when it comes to their direct interactions with the cytochrome domain. Surprisingly, removal of the CBM domain from CcPDH had a considerable negative impact on the efficiency of the CcPDH-LPMO systems, suggesting that electron transfer in the vicinity of the substrate is beneficial. CcPDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool for studying cellulose-oxidizing LPMOs.Fungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs catalyze the oxidative cleavage of glycosidic bonds after activation by an external electron donor. So far, only flavin-dependent oxidoreductases (from the auxiliary activity [AA] family AA3) have been shown to activate LPMOs. Here, we present LPMO activation by a pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase (PDH) from IMPORTANCE Lytic polysaccharide monooxygenases (LPMOs) are currently receiving increasing attention because of their importance in degrading recalcitrant polysaccharides and their potential roles in biological processes, such as bacterial virulence. LPMO action requires an external electron donor, and fungi growing on biomass secrete various so-called glucose-methanol-choline (GMC) oxidoreductases, including cellobiose dehydrogenase, which can donate electrons to LPMOs. This paper describes how an enzyme not belonging to the GMC oxidoreductase family, CcPDH, can activate LPMOs, and it provides new insights into the activation process by (i) describing the roles of individual CcPDH domains , (ii) showing that the PDH and LPMO enzyme reactions are strongly coupled, (iii) demonstrating that LPMOs differ in terms of their efficiencies of activation by the same activator, and (iv) providing indications that electron transferring close to the substrate surface is beneficial for the overall efficiency of the CcPDH-LPMO system. Plant biomass is a promising raw material for industrial use because of its abundance in nature. However, industrial utilization of biomass via a biochemical conversion route is limited due to its resistance to enzymatic degradation . The recLPMOs are copper-dependent enzymes that catalyze the oxidative cleavage of glycosidic bonds in various polysaccharides, such as cellulose –7, hemic–8–16–b-type heme-binding AA8 cytochrome domain and a C-terminal flavin adenine dinucleotide (FAD)-dependent AA3_1 dehydrogenase domain activation , 20, 21.24–e domain . Some CDe domain . The AA3e domain . Then, iransfers , 31. The an LPMO , 31. In  an LPMO .Coprinopsis cinerea (CcPDH), which is the first PQQ-dependent enzyme in eukaryotes , and a C-terminal CBM1. The PQQ cofactor binds with high affinity, and the binding constant, dK, is 1.1 nM (d-glucosone (2-keto–d-glucose), l-fucose, and some rare pyranoses but is inactive toward glucose and cellobiose -dependent pyranose dehydrogenase (PDH) from the plant saprophytic basidiomycete karyotes . This ens 1.1 nM . The PQQllobiose , 34. Of nd PcCDH , indicatrsus SHE ).CcPDH in plant cell wall degradation, such a role is suggested by the presence of the CBM1 and by the fact that the gene adjacent to the CcPDH-encoding gene encodes an LPMO . In the present study, we investigated the potential of CcPDH to drive catalysis by N. crassa LPMO 9C and N. crassa LPMO 9F , two well-characterized LPMOs from the ascomycete Neurospora crassa, and we compared CcPDH with ascorbic acid as an electron donor. Since, in contrast to CDH, CcPDH is inactive toward glucose and cello-oligosaccharides, reducing end-containing LPMO-generated products released from the cellulosic substrate are not oxidized by CcPDH. Thus, CcPDH is a good candidate to study the activity and product profiles of cellulose-active LPMOs. Furthermore, as shown here, product formation by CcPDH can be analyzed chromatographically, simultaneously with the analysis of LPMO-generated products, generating detailed insights into how the two enzymes work together.Although there is no known role for d-glucose dehydrogenase (2KGDH) from Pseudomonas aureofaciens, a bacterial homolog of CcPDH, oxidizes the aldehyde group of d-glucosone at the C-1 position, yielding 2-keto-d-gluconic acid, which is a precursor of erythorbic acid . In the present study, we used external standards to identify the products generated by CcPDH from d-glucosone and l-fucose. The retention times of the reaction products generated from d-glucosone and l-fucose were 16.6 min and 8.9 min, i.e., identical to the retention times of 2-keto-d-gluconic acid and l-fuconic acid, respectively . Thus, as expected, CcPDH oxidizes the C-1 position of these substrates. In order to exclude the possibility that LPMOs may be activated by these reaction products of CcPDH, we incubated LPMOs with 2-keto-d-gluconic acid, l-fuconic acid, or products generated by CcPDH from after enzyme inactivation by boiling. None of these compounds were able to activate the LPMOs . The control experiments depicted in Fig. S1 and S2 also showed that quantification of PDH action is possible in the case of d-glucosone as the substrate, since the oxidized product, 2-keto-d-gluconic acid, gives a clear sharp peak with a good response factor in the required concentration range . In the case of l-fucose, detection of l-fuconic acid was not sensitive enough in the required concentration range. In principle, the sharp l-fucose peak with a high response factor  would allow for the quantification of PDH action through measuring l-fucose consumption.In a previous study , we showCcPDH can activate NcLPMO9C (NCU02916), a C-4-oxidizing LPMO with a C-terminal CBM1 domain, and NcLPMO9F (NCU03328), a C-1-oxidizing single-domain LPMO  showed negligible (but larger than zero) amounts of LPMO products  did lead to significant product formation (t) = 0 , the AA8-AA12 two-domain enzyme, and the AA12 single-domain enzyme, and studied their ability to oxidize d-glucosone and activate the LPMOs in reactions similar to those described above.In order to investigate whether d-glucosone to a much lower extent than the full-length enzyme shows that, in the absence of the AA8 domain, the presence of an electron-accepting LPMO is not sufficient to (efficiently) reoxidize the active site of the AA12 domain. Accordingly, CcPDH.CcPDH is similar for the two LPMOs and thus seems independent of the presence (NcLPMO9C) or absence (NcLPMO9F) of a CBM1 domain in the LPMO itself. Of note, the low-level background activation of LPMOs by CcPDH in the absence of substrate is also reduced upon removal of the CBM , the two CcPDH-LPMO systems work approximately equally quickly, as judged from the observed CcPDH activity . Thus, LPMOs seem to differ considerably when it comes to their ability to interact with the AA8 cytochrome domain attached to AA3 and AA12 dehydrogenases.NcLPMO9F  (NcLPMO9b propionate of the AA8 domain in MtCDH (31.8% sequence identity with the AA8 domain of CcPDH) may have favorable interactions with the copper active site of NcLPMO9F , and these AA8 domains are highly similar to each other in terms of amino acid sequence and spectral, biochemical, and electrochemical features  LPMOs that are not bound to substrate suffer from oxidative self-inactivation  and in the presence of a reductant, the LPMO generates its own H2O2 from O2, which, notably, would require two electrons. Thus, no matter which LPMO mechanism one assumes, under the conditions used in this study, each LPMO catalysis consumes two electrons. This explains why the observed activities of CcPDH, which needs to get rid of two electrons in each cycle, and the LPMO, which needs to acquire two electrons in each cycle, are tightly coupled. A key remaining question for all scenarios without externally added H2O2 is how and when the second electron is transferred to the catalytic center, perhaps especially for scenarios where the LPMO is bound to substrate and has a less accessible copper site. It is worth noting that transfer of the second electron likely is rate limiting, since one-electron reduction of the copper in a free LPMO  is orders of magnitude faster than LPMO catalysis driven by, e.g., ascorbic acid  becomes rate limiting. Increasing the PDH concentration would then have a double effect, since more NcLPMO9C molecules will become catalytically competent whereas, at the same time, production of the cosubstrate H2O2 will increase.The experiments with varied enzyme ratios show large differences between the CcPDH, on the other hand, is inactive against the most common sugars in biomass  can be distinguished from oxidized cello-oligosaccharides in HPAEC-pulsed amperometric detection (HPAEC-PAD) analyses, which allow not only proper detection of oxidized cello-oligosaccharides but also simultaneous monitoring of dehydrogenase and LPMO activities. Thus, we believe that CcPDH will turn out to be a useful tool in future studies of LPMOs and associated redox enzyme systems involved in biomass degradation.Small molecular reductants, such as ascorbic acid, are regularly used as an electron donor for LPMOs, while such compounds are generally unstable, may lead to side reactions, and could lead to the inactivation of LPMOs at higher concentrations , 51. Stu-mannose ) and will-fucose were purchased from Wako Pure Chemical Industries . d-Glucosone (2-keto–d-glucose) was purchased from Santa Cruz Biotechnology . 2-Keto-d-gluconic acid hemicalcium salt hydrate, l-fucono-1,4-lactone, cytochrome c from bovine heart, and Avicel PH 101 were purchased from Sigma-Aldrich . Phosphoric acid-swollen cellulose (PASC) was prepared from Avicel as described by Wood (Pyrroloquinoline quinone (PQQ) disodium salt and  by Wood .CcPDH (AA8-AA12-CBM1), truncated two-domain CcPDH (AA8-AA12), and the single-domain PQQ-dependent dehydrogenase domain (AA12) were heterologously expressed in the methylotrophic yeast Pichia pastoris and purified as described previously  by incubating enzymes with an excess of CuSO4 (at a 3:1 molar ratio of copper/enzyme) for 30 min at room temperature, as described previously .Full-length eviously . AA8-AA1l et al. . Proteineviously . After s2+-saturated forms) of CcPDH and its truncated versions were prepared by incubating 20 μM enzyme with 200 μM PQQ and 2 mM CaCl2 for 30 min on ice, followed by removal of excess PQQ and CaCl2 using PD-10 desalting columns. PQQ saturation was confirmed by analyzing the enzyme activity of holo-CcPDH with and without the addition of extra (5 μM) PQQ and (50 μM) CaCl2. The enzyme activity of CcPDH was measured spectrophotometrically by monitoring the reduction of cytochrome c at 550 nm and 30°C  (PIPES)-NaOH (pH 7.0), 1 mM l-fucose, and 50 μM cytochrome c in a total volume of 500 μl. The addition of extra PQQ and CaCl2 to the putatively saturated enzyme had no effect on enzyme activity, indicating that the holo-enzyme was fully saturated .Holo-forms  with bovine serum albumin as a standard.2. As an electron donor, either 1 mM ascorbic acid or 1 μM CcPDH holoenzyme supplemented with 1 mM d-glucosone or l-fucose was supplied, unless otherwise stated. Control reactions were set up in the same manner, leaving out one or more components, as indicated in the figure legends. In the dose-response experiments, the concentrations of NcLPMO9s and CcPDHs were varied between 0.25 and 2.0 μM. Reactions were stopped by boiling for 5 min. All reaction products were analyzed by high-performance anion-exchange chromatography (HPAEC) using a Dionex ICS 3000 system  equipped with a CarboPac PA1 precolumn (2 by 50 mm), a CarboPac PA1 main column (2 by 250 mm), and a detector for pulsed amperometric detection (PAD), applying a 50-min gradient that has been described previously , and the mean of those ratios is presented as relative activity. In the case of NcLPMO9C, which produces large amounts of (partly on column-generated) native oligosaccharides  PASC in 20 mM HEPES buffer (pH 7.0) at 30°C for 10 min. All reaction mixtures contained 100 μM CaCleviously . Chromatcharides , relativ